WO2019018729A1 - Compositions and methods for identifying and treating metastatic small bowel neuroendocrine tumors - Google Patents

Abstract

The present invention relates to compositions and methods for identifying and treating metastatic small bowel neuroendocrine tumors.

Description

COMPOSITIONS AND METHODS FOR IDENTIFYING AND TREATING METASTATIC SMALL BOWEL NEUROENDOCRINE TUMORS

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S.

Provisional Application No: 62/534,764, filed July 20, 2017, which is incorporated herein by reference in its entirety.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under grant number CA151532 awarded by the National Institutes of Health and under grant number P50CA127003 awarded by the National Cancer Institute. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Small bowel neuroendocrine tumors, although rare across the cancer landscape (1 per 100,000), are the most common carcinoid tumor and originate most frequently in the terminal ileum. Tumor growth in SINET is slow and many patients present with advanced disease for which few treatment options exist. Prior to the invention described herein, this slow growing characteristic has meant that the development of in vitro and in vivo models to study this tumor type have been extremely limited. The lack of cell lines and animal models has been a major obstacle in the development of therapeutic agents for advanced carcinoid tumors. As such, prior to the invention described herein, there was a pressing need to identify disease models and therapeutic modalities for small bowel neuroendocrine tumors.

SUMMARY OF THE INVENTION

The invention is based, at least in part, upon the identification of secreted protein biomarkers of metastatic neuroendocrine tumors. Accordingly, provided herein is a method of determining whether a small bowel neuroendocrine tumor (SINET) in a subject, e.g., a human subject, will metastasize. First, a test sample is obtained from a subject having or at risk of developing SINET. The expression level of at least one SINET-associated gene in the test sample is determined. Next, the expression level of the SINET-associated gene in the test sample is compared with the expression level of the SINET-associated gene in a reference sample. Finally, it is determined that the SINET in the subject will metastasize if the expression level of the SINET-associated gene in the test sample is differentially expressed as compared to the level of the SINET-associated gene in the reference sample.

Also provided is a method of determining whether a neuroendocrine tumor (NET) in a subject, e.g., a human subject, will metastasize. First, a test sample is obtained from a subject having or at risk of developing NET. The expression level of at least one NET-associated gene in the test sample is determined. Next, the expression level of the NET-associated gene in the test sample is compared with the expression level of the NET-associated gene in a reference sample. Finally, it is determined that the NET in the subject will metastasize if the expression level of the NET-associated gene in the test sample is differentially expressed as compared to the level of the NET-associated gene in the reference sample.

For example, the expression level of the SINET-associated gene in the test sample is upregulated (i.e., increased) by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 125 fold, at least 150 fold, at least 175 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold or at least 500 fold as compared to the level of the SINET- associated gene in the reference sample.

Alternatively, the expression level of the SINET-associated gene in the test sample is downregulated (i.e., decreased) by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 125 fold, at least 150 fold, at least 175 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold or at least 500 fold as compared to the level of the SINET- associated gene in the reference sample.

In some cases, the test sample is obtained from resected SINET tissue, which is comprised of tumor tissue, stromal tissue, and all tissue encompassing the tumor microenvironment. In another aspect, the test sample is obtained from blood or plasma. For example, the sample comprises circulating tumor cells.

The reference sample is obtained from immortalized healthy normal control fibroblastic cell lines (e.g., normal human fibroblasts), benign SINET tissue, or cancerous SINET tissue. For example, the reference sample is obtained from healthy normal tissue from the same individual as the test sample. Alternatively, the reference sample is obtained from one or more healthy normal tissues from different individuals.

Optionally, the sample comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Exemplary SINET-associated genes comprise interleukin-2 (IL-2), IL-6, IL-12, IL-13, vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein- 1 (MCP-1), urinary plasminogen activator (uPA), hepatocyte growth factor (HGF), RANTES (regulated on activation, normal T cell expressed and secreted, alternatively, chemokine (C-C motif) ligand 5; alternatively CCL5), and Eotaxin. It is determined that the SFNET in the subject will metastasize if the expression level of the SFNET-associated gene in the test sample is higher than the level of the SINET-associated gene in the reference sample.

In other cases, the SINET-associated gene comprises stem cell factor (SCF) and soluble epidermal growth factor receptor (sEGFR), granulocyte macrophage colony stimulating factor (GM-CSF), soluble vascular endothelial growth factor receptor 2 (sVEGFR2), and soluble interleukin 6 receptor, alpha (sIL-6RA). It is determined that the SINET in the subject will metastasize if the expression level of the SINET-associated gene in the test sample is lower than the level of the SINET-associated gene in the reference sample.

For example, the expression level of the SINET-associated gene is detected via an Affymetrix Gene Array hybridization, next generation sequencing, ribonucleic acid sequencing (RNA-seq), a real time reverse transcriptase polymerase chain reaction (real time RT-PCR) assay, immunohistochemistry (IHC), immunofluorescence, methylation-specific PCR, or a bead- based multiplex assay.

In some cases, the subject is treated with a chemotherapeutic agent, radiation therapy, cryotherapy, hormone therapy, or immunotherapy. For example, the chemotherapeutic agent comprises a somatostatin analog. For example, the somatostatin analogue comprises octreotide long-acting repeatable (LAR). In one aspect, the method also includes administering an inhibitor of the SINET- associated gene with a higher level of expression compared to the level of the SINET-associated gene in the reference sample, thereby treating the small bowel neuroendocrine tumor. In some cases, the chemotherapeutic agent is administered in combination with an inhibitor of the SINET-associated gene. For example, tumor-specific inhibitors, e.g., Rapamycin, Cabozantinib, and/or Erlotinib, are combined with stroma inhibition via Withaferin A.

For example, the inhibitor comprises a polypeptide, a small molecule inhibitor, RNA interference (RNAi), an antibody, or any fragment or combination thereof. In one aspect, the antibody or antibody fragment is partially humanized, fully humanized, or chimeric.

Optionally, the antibody or antibody fragment comprises a nanobody, an Fab, an Fab', an (Fab')2, an Fv, a single-chain variable fragment (ScFv), a diabody, a triabody,a tetrabody, a Bis-scFv, a minibody, an Fab2, an Fab3 fragment, or any combination thereof.

An exemplary small molecule inhibitor comprises an inhibitor of NFKB activity. In some cases, the inhibitor of NFKB activity comprises Withaferin A (WFA). Other suitable inhibitors include those that target the j anus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, e.g., Ruxolitinib and Momelotinib. Preferably, expression levels of VEGF, IL- 6, and MCP-1 are increased following administration of WFA.

For example, compositions described herein are administered at a concentration of 1- 1000 nM, e.g., about 1 nM, about 5 nM, about 10 nM, abot 25 nM, about 50 nM, about 75 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, or about 1000 nM.

Also provided is a composition for predicting whether a SINET will metastasize comprising a SINET-associated gene, wherein the SINET-associated gene comprises IL-2, IL-6, IL-12, IL-13, VEGF, MCP-1, uPA, HGF, RANTES, Eotaxin, GM-CSF, sVEGFR2, and sIL- 6RA synthesized complementary deoxyribonucleic acid (cDNA). In some cases, the

composition comprises or further comprises SCF, sEGFR, GM-CSF, sVEGFR2, and/or sIL-6RA synthesized cDNA. Preferably, the SINET-associated gene is immobilized on a solid support. In some cases, the SINET-associated gene is linked to a detectable label. Exemplary detectable labels include a fluorescent label, a luminescent label, a chemiluminescent label, a radiolabel, a SYBR Green label, or a Cy3 -label. Additionally, provided is a composition for predicting whether a SINET will metastasize comprising a SINET-associated gene, wherein the SINET-associated gene comprises a neuroendocrine tumor (NET) specific gene, a cell surface eithelial gene, a stimulant of malignant cell proliferation gene, an epithelial-mesenchymal transition gene, a metastatic transition gene, a mesenchymal-like gene, a matrix-remodeling gene, a hypoxia and angiogenesis gene, a tumor- associated stromal cells and myofibroblasts gene, and a stromal-specific gene.

For example, the NET-specific gene comprises CGA. In another example, the cell surface eithelial gene is selected from the group consisting of CD34, PECAMl, MUC16, KRT5, KRT6A, KRT6B, and KRT17. Exemplary stimulants of malignant cell proliferation genes include EGFR, FGF-2, HGF, and CXCL12. For example, the epithelial-mesenchymal gene is selected from the group consisting of CDH1, CDH2, MMP1, MMP2, ITGAl, ITGA2, ITGA3 ITGA5, ITGA7, ITGAl 1, TGFBl, and TGFBI. Suitable metastatic transition genes are selected from the group consisting of YAP 1, HAS2, LOX, S100A4, GSTPl, and CD81. For example, the mesenchymal-like gene is selected from the group consisting of THY1, NT5E, ENG, and CD44. An exemplary matrix-remodeling gene includes FAP. For example, the hypoxia and

angiogenesis gene is selected from the group consisting of VEGFA, VEGFC, HIFIA, and CAV1. In one aspect, the tumor-associated stromal cells and myofibroblasts gene is selected from the group consisting of PDGFRA, PDGFRB, NRG1, TNC, POSTN, and ACTA2. Finally, in one example, the stromal-specific gene is selected from the group consisting of P4HA1, P4HB, and VIM.

Also provided is a kit comprising a package with a SINET-associated gene, wherein the SF ET-associated gene comprises IL-2, IL-6, IL-12, IL-13, VEGF, MCP-1, uPA, HGF,

RANTES, Eotaxin, GM-CSF, sVEGFR2, and sIL-6RA, and instructions for use thereof in the evaluation of SINET progression and metastasis.

Further provided is a kit comprising a package with a SFNET-associated gene, wherein the SINET-associated gene comprises a neuroendocrine tumor (NET) specific gene, a cell surface eithelial gene, a stimulant of malignant cell proliferation gene, an epithelial- mesenchymal transition gene, a metastatic transition gene, a mesenchymal-like gene, a matrix- remodeling gene, a hypoxia and angiogenesis gene, a tumor-associated stromal cells and myofibroblasts gene, and a stromal-specific gene. For example, the NET-specific gene comprises CGA. In another example, the cell surface eithelial gene is selected from the group consisting of CD34, PECAM1, MUC16, KRT5, KRT6A, KRT6B, and KRT17. In one aspect, the stimulants of malignant cell proliferation gene is selected from the group consisting of EGFR, FGF-2, HGF, and CXCL12. For example, the epithelial-mesenchymal gene is selected from the group consisting of CDH1, CDH2, MMP1, MMP2, ITGAl, ITGA2, ITGA3 ITGA5, ITGA7, ITGAl 1, TGFBl, and TGFBI. In one example, the metastatic transition gene is selected from the group consisting of YAP 1, HAS2, LOX, S100A4, GSTP1, and CD81. In another example, the mesenchymal-like gene is selected from the group consisting of THY1, NT5E, ENG, and CD44. For example, the matrix-remodeling gene comprises FAP. In one example, the hypoxia and angiogenesis gene is selected from the group consisting of VEGFA, VEGFC, HIFIA, and CAV1. In another example, the tumor-associated stromal cells and myofibroblasts gene is selected from the group consisting of PDGFRA, PDGFRB, RG1, TNC, POSTN, and ACTA2. In one aspect, the stromal-specific gene is selected from the group consisting of P4HA1, P4HB, and VFM.

Ex vivo methods of screening therapeutic agents for a neuroendocrine tumor are carried out by establishing an ex vivo organotypic neuroendocrine tumor tissue slice culture;

administering a candidate compound to the culture; and determining that the candidate compound is a therapeutic agent for a neuroendocrine tumor if the candidate compound inhibits the neuroendocrine tumor. Preferably, the proliferation of the neuroendocrine tumor is inhibited. For example, proliferation is evaluated using a bromodeoxyuridine (BrDU) cell proliferation assay. In some cases, the method also includes detecting a level of a candidate compound- specific target. In other cases, the method also provides for detecting a level of a secretory protein in supernatant of the tissue slices prior to and following administration of the candidate compound. In another case, the method includes gene expression measurements via RNA-Seq transcriptional profiling of the tissue slices prior to and following administration of the candidate compound.

An exemplary candidate compound comprises a mechanistic target of rapamycin

(mTOR) inhibitor comprising Rapamycin. Other suitable compounds include Erlotinib

Cabozantinib, a histone deacetylase (HDAC) inhibitor, and a cyclin-dependendt kinase (CDK) 4/6 inhibitor.

In some cases, the compound is administered at a dose of 1-1000 nM, e.g., about 1 nM, about 5 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, or about 1000 nM. For example, the Rapamycin is administered at a dose of 100 nM. In one aspect, the composition is administered at least one time per month, e.g., twice per month, once per week, twice per week, once per day, twice per day, every 8 hours, every 4 hours, every 2 hours, or every hour.

In one aspect, administration of Rapamycin results in a reduction of secretion of osteoponin, IL-16, VEGF, MCP-1, IL-10, macrophage inflammatory protein- la (MIP-la), MIP-lb, interferon-y-inducible protein-10 (IP-10), IL-8, and/or urinary plasminogen activator (uPA) from the culture.

Also provided is a method of generating a neuroendocrine-associated fibroblast culture comprising: obtaining a tissue sample from a patient; dissecting the tissue sample into pieces; enzymatically disaggregating the tissue sample; mechanically disaggregating the tissue sample; filtering and centrifuging the cells; plating the cells on polymer tissue culture plates; and culturing the cells, thereby generating a neuroendocrine-associated fibroblast culture. In one aspect, the tissue sample is enzymatically disaggregated with collagenase II at 2 mg/ml. In some cases, the tissue sample is mechanically disaggregated with an 18-guage needle and syringe. Definitions

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term "about."

The term "antineoplastic agent" is used herein to refer to agents that have the functional property of inhibiting a development or progression of a neoplasm in a human, e.g., a small bowel neuroendocrine tumor. Inhibition of metastasis is frequently a property of antineoplastic agents.

By "agent" is meant any small compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art-known methods such as those described herein. As used herein, an alteration includes at least a 1% change in expression levels, e.g., at least a 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%), 80%), 90%), or 100% change in expression levels. For example, an alteration includes at least a 5%-10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.

By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

The term "antibody" (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

An "isolated antibody" is one that has been separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and γ chains and four CH domains for μ and ε isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71, and Chapter 6.

The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains (CL). Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha (a), delta (δ), epsilon (ε), gamma (y) and mu (μ), respectively. The γ and a classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

The term "variable" refers to the fact that certain segments of the V domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., around about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the V_L, and around about 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the VH when numbered in accordance with the Kabat numbering system; Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and/or those residues from a "hypervariable loop" (e.g., residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the V_L, and 26-32 (HI), 52-56 (H2) and 95-101 (H3) in the V_H when numbered in accordance with the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and/or those residues from a "hypervariable loop'VCDR (e.g., residues 27-38 (LI), 56-65 (L2) and 105-120 (L3) in the V_L, and 27-38 (HI), 56-65 (H2) and 105-120 (H3) in the V_H when numbered in accordance with the EVIGT numbering system; Lefranc, M.P. et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. e al. Nucl. Acids Res. 28:219-221 (2000)). Optionally the antibody has symmetrical insertions at one or more of the following points 28, 36 (LI), 63, 74-75 (L2) and 123 (L3) in the V_L, and 28, 36 (HI), 63, 74-75 (H2) and 123 (H3) in the V_H when numbered in accordance with AHo;

Honneger, A. and Plunkthun, A. J. Mol. Biol. 309:657-670 (2001)).

By "germline nucleic acid residue" is meant the nucleic acid residue that naturally occurs in a germline gene encoding a constant or variable region. "Germline gene" is the DNA found in a germ cell (i.e., a cell destined to become an egg or in the sperm). A "germline mutation" refers to a heritable change in a particular DNA that has occurred in a germ cell or the zygote at the single-cell stage, and when transmitted to offspring, such a mutation is incorporated in every cell of the body. A germline mutation is in contrast to a somatic mutation which is acquired in a single body cell. In some cases, nucleotides in a germline DNA sequence encoding for a variable region are mutated (i.e., a somatic mutation) and replaced with a different nucleotide.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:581-597 (1991), for example.

Monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). Also provided are variable domain antigen-binding sequences derived from human antibodies. Accordingly, chimeric antibodies of primary interest herein include antibodies having one or more human antigen binding sequences {e.g., CDRs) and containing one or more sequences derived from a non-human antibody, e.g., an FR or C region sequence. In addition, chimeric antibodies of primary interest herein include those comprising a human variable domain antigen binding sequence of one antibody class or subclass and another sequence, e.g., FR or C region sequence, derived from another antibody class or subclass. Chimeric antibodies of interest herein also include those containing variable domain antigen-binding sequences related to those described herein or derived from a different species, such as a non-human primate {e.g., Old World Monkey, Ape, etc). Chimeric antibodies also include primatized and humanized antibodies. Furthermore, chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al, Nature 321 :522-525 (1986);

Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

A "humanized antibody" is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization is traditionally performed following the method of Winter and co-workers (Jones et al, Nature, 321 :522-525 (1986); Reichmann et al, Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988)), by substituting import hypervariable region sequences for the corresponding sequences of a human antibody.

Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.

A "human antibody" is an antibody containing only sequences present in an antibody naturally produced by a human. However, as used herein, human antibodies may comprise residues or modifications not found in a naturally occurring human antibody, including those modifications and variant sequences described herein. These are typically made to further refine or enhance antibody performance.

An "intact" antibody is one that comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CH 1, CH 2 and CH 3. The constant domains may be native sequence constant domains {e.g., human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions. An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')₂, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870; Zapata et al, Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The phrase "functional fragment or analog" of an antibody is a compound having qualitative biological activity in common with a full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody is one that can bind to an IgE immunoglobulin in such a manner so as to prevent or substantially reduce the ability of such molecule from having the ability to bind to the high affinity receptor, FceRI.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')₂ fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')₂ antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The "Fc" fragment comprises the carboxy -terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

"Fv" is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

"Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.

Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

As used herein, an antibody that "internalizes" is one that is taken up by {i.e., enters) the cell upon binding to an antigen on a mammalian cell {e.g., a cell surface polypeptide or receptor). The internalizing antibody will of course include antibody fragments, human or chimeric antibody, and antibody conjugates. For certain therapeutic applications, internalization in vivo is contemplated. The number of antibody molecules internalized will be sufficient or adequate to kill a cell or inhibit its growth, especially an infected cell. Depending on the potency of the antibody or antibody conjugate, in some instances, the uptake of a single antibody molecule into the cell is sufficient to kill the target cell to which the antibody binds. For example, certain toxins are highly potent in killing such that internalization of one molecule of the toxin conjugated to the antibody is sufficient to kill the infected cell.

As used herein, an antibody is said to be "immunospecific," "specific for" or to

"specifically bind" an antigen if it reacts at a detectable level with the antigen, preferably with an affinity constant, K_a^ of greater than or equal to about lO^ M^~l, or greater than or equal to about 105 M^~l, greater than or equal to about 10^ M^~l, greater than or equal to about 10^ M^~l, or greater than or equal to 10^ M^"1. Affinity of an antibody for its cognate antigen is also commonly expressed as a dissociation constant KD, and in certain embodiments, HuM2e antibody specifically binds to M2e if it binds with a KD of less than or equal to 10^"4 M, less than or equal to about 10^"5 M, less than or equal to about 10^"6 M, less than or equal to 10^" M, or less than or equal to 10^"8 M. Affinities of antibodies can be readily determined using conventional techniques, for example, those described by Scatchard et al. {Ann. N.Y. Acad. Sci. USA 51 :660 (1949)).

Binding properties of an antibody to antigens, cells or tissues thereof may generally be determined and assessed using immunodetection methods including, for example,

immunofluorescence-based assays, such as immuno-histochemistry (IHC) and/or fluorescence- activated cell sorting (FACS).

An antibody having a "biological characteristic" of a designated antibody is one that possesses one or more of the biological characteristics of that antibody which distinguish it from other antibodies. For example, in certain embodiments, an antibody with a biological characteristic of a designated antibody will bind the same epitope as that bound by the designated antibody and/or have a common effector function as the designated antibody.

The term "antagonist" antibody is used in the broadest sense, and includes an antibody that partially or fully blocks, inhibits, or neutralizes a biological activity of an epitope, polypeptide, or cell that it specifically binds. Methods for identifying antagonist antibodies may comprise contacting a polypeptide or cell specifically bound by a candidate antagonist antibody with the candidate antagonist antibody and measuring a detectable change in one or more biological activities normally associated with the polypeptide or cell.

Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors {e.g., B cell receptor); and B cell activation.

By "binding to" a molecule is meant having a physicochemical affinity for that molecule.

By "control" or "reference" is meant a standard of comparison. In one aspect, as used herein, "changed as compared to a control" sample or subject is understood as having a level that is statistically different than a sample from a normal, untreated, or control sample. Control samples include, for example, cells in culture, one or more laboratory test animals, or one or more human subjects. Methods to select and test control samples are within the ability of those in the art. An analyte can be a naturally occurring substance that is characteristically expressed or produced by the cell or organism (e.g., an antibody, a protein) or a substance produced by a reporter construct (e.g, β-galactosidase or luciferase). Depending on the method used for detection, the amount and measurement of the change can vary. Determination of statistical significance is within the ability of those skilled in the art, e.g., the number of standard deviations from the mean that constitute a positive result.

"Detect" refers to identifying the presence, absence, or amount of the agent (e.g., a nucleic acid molecule, for example deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) to be detected.

By "detectable label" is meant a composition that when linked (e.g., joined - directly or indirectly) to a molecule of interest renders the latter detectable, via, for example, spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Direct labeling can occur through bonds or interactions that link the label to the molecule, and indirect labeling can occur through the use of a linker or bridging moiety which is either directly or indirectly labeled.

Bridging moieties may amplify a detectable signal. For example, useful labels may include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent labeling compounds, electron-dense reagents, enzymes (for example, as commonly used in an enzyme- linked immunosorbent assay (ELISA)), biotin, digoxigenin, or haptens. When the fluorescently labeled molecule is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, p- phthaldehyde and fluorescamine. The molecule can also be detectably labeled using

fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the molecule using such metal chelating groups as diethylenetriaminepentacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA). The molecule also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent- tagged molecule is then determined by detecting the presence of luminescence that arises during the course of chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

A "detection step" may use any of a variety of known methods to detect the presence of nucleic acid (e.g., methylated DNA) or polypeptide. The types of detection methods in which probes can be used include Western blots, Southern blots, dot or slot blots, and Northern blots. As used herein, the term "diagnosing" refers to classifying pathology or a symptom, determining a severity of the pathology (e.g., grade or stage), monitoring pathology progression, forecasting an outcome of pathology, and/or determining prospects of recovery.

By the terms "effective amount" and "therapeutically effective amount" of a formulation or formulation component is meant a sufficient amount of the formulation or component, alone or in a combination, to provide the desired effect. For example, by "an effective amounf'is meant an amount of a compound, alone or in a combination, required to ameliorate the symptoms of a disease, e.g., cancer, relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

The term "expression profile" is used broadly to include a genomic expression profile. Profiles may be generated by any convenient means for determining a level of a nucleic acid sequence, e.g., quantitative hybridization of microRNA, labeled microRNA, amplified microRNA, complementary/synthetic DNA (cDNA), etc., quantitative polymerase chain reaction (PCR), and ELISA for quantitation, and allow the analysis of differential gene expression between two samples. A subject or patient tumor sample is assayed. Samples are collected by any convenient method, as known in the art. According to some embodiments, the term

"expression profile" means measuring the relative abundance of the nucleic acid sequences in the measured samples.

By "FDR" is meant False Discovery Rate. When performing multiple statistical tests, for example, in comparing the signal of two groups in multiple data features, there is an increasingly high probability of obtaining false positive results, by random differences between the groups that can reach levels that would otherwise be considered statistically significant. In some cases, in order to limit the proportion of such false discoveries, statistical significance is defined only for data features in which the differences reached a p-value (by two-sided t-test) below a threshold, which is dependent on the number of tests performed and the distribution of p-values obtained in these tests.

By "fragment" is meant a portion, e.g., a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. For example, a fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. However, the invention also comprises polypeptides and nucleic acid fragments, so long as they exhibit the desired biological activity of the full length polypeptides and nucleic acid, respectively. A nucleic acid fragment of almost any length is employed. For example, illustrative polynucleotide segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length (including all intermediate lengths) are included in many implementations of this invention. Similarly, a polypeptide fragment of almost any length is employed. For example, illustrative polypeptide segments with total lengths of about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 5,000, about 1,000, about 500, about 200, about 100, or about 50 amino acids in length (including all intermediate lengths) are included in many implementations of this invention.

"Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

The terms "isolated," "purified, " or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation.

A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to

modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

Similarly, by "substantially pure" is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it. Typically, the nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%), by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated.

By "isolated nucleic acid" is meant a nucleic acid that is free of the genes which flank it in the naturally-occurring genome of the organism from which the nucleic acid is derived. The term covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule, but is not flanked by both of the nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner, such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a synthetic cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleic acid molecules according to the present invention further include molecules produced synthetically, as well as any nucleic acids that have been altered chemically and/or that have modified backbones. For example, the isolated nucleic acid is a purified cDNA or RNA polynucleotide. Isolated nucleic acid molecules also include messenger ribonucleic acid (mRNA) molecules.

By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

The term "immobilized" or "attached" refers to a probe (e.g., nucleic acid or protein) and a solid support in which the binding between the probe and the solid support is sufficient to be stable under conditions of binding, washing, analysis, and removal. The binding may be covalent or non-covalent. Covalent bonds may be formed directly between the probe and the solid support or may be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Non-covalent binding may be one or more of electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule to the support and the non-covalent binding of a biotinylated probe to the molecule. Immobilization may also involve a combination of covalent and non-covalent interactions.

By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder, e.g., SINET.

By "small bowel neuroendocrine tumor-associated gene" is meant a nucleic acid associated with the pathogenesis of a small bowel neuroendocrine tumor.

By "modulate" is meant alter (increase or decrease). Such alterations are detected by standard art-known methods such as those described herein.

The term, "normal amount" refers to a normal amount of a complex in an individual known not to be diagnosed with SINET. The amount of the molecule can be measured in a test sample and compared to the "normal control level," utilizing techniques such as reference limits, discrimination limits, or risk defining thresholds to define cutoff points and abnormal values (e.g., for SINET). The "normal control level" means the level of one or more proteins (or nucleic acids) or combined protein indices (or combined nucleic acid indices) typically found in a subject known not to be suffering from SINET. Such normal control levels and cutoff points may vary based on whether a molecule is used alone or in a formula combining other proteins into an index. Alternatively, the normal control level can be a database of protein patterns from previously tested subjects who did not convert to SINET over a clinically relevant time horizon. In another aspect, the normal control level can be a level relative to a housekeeping gene. The level that is determined may be the same as a control level or a cut off level or a threshold level, or may be increased or decreased relative to a control level or a cut off level or a threshold level. In some aspects, the control subject is a matched control of the same species, gender, ethnicity, age group, smoking status, body mass index (BMI), current therapeutic regimen status, medical history, or a combination thereof, but differs from the subject being diagnosed in that the control does not suffer from the disease in question or is not at risk for the disease.

Relative to a control level, the level that is determined may be an increased level. As used herein, the term "increased" with respect to level (e.g., expression level, biological activity level, etc.) refers to any % increase above a control level. The increased level may be at least or about a 1% increase, at least or about a 5% increase, at least or about a 10% increase, at least or about a 15% increase, at least or about a 20% increase, at least or about a 25% increase, at least or about a 30% increase, at least or about a 35% increase, at least or about a 40% increase, at least or about a 45% increase, at least or about a 50% increase, at least or about a 55% increase, at least or about a 60% increase, at least or about a 65% increase, at least or about a 70% increase, at least or about a 75% increase, at least or about a 80% increase, at least or about a 85%) increase, at least or about a 90% increase, or at least or about a 95% increase, relative to a control level.

Relative to a control level, the level that is determined may be a decreased level. As used herein, the term "decreased" with respect to level (e.g., expression level, biological activity level, etc.) refers to any % decrease below a control level. The decreased level may be at least or about a 1%) decrease, at least or about a 5% decrease, at least or about a 10% decrease, at least or about a 15%) decrease, at least or about a 20% decrease, at least or about a 25% decrease, at least or about a 30% decrease, at least or about a 35% decrease, at least or about a 40% decrease, at least or about a 45% decrease, at least or about a 50% decrease, at least or about a 55% decrease, at least or about a 60% decrease, at least or about a 65% decrease, at least or about a 70% decrease, at least or about a 75% decrease, at least or about a 80% decrease, at least or about a 85% decrease, at least or about a 90% decrease, or at least or about a 95% decrease, relative to a control level.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity, e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

By "neoplasia" is meant a disease or disorder characterized by excess proliferation or reduced apoptosis. Illustrative neoplasms for which the invention can be used include, but are not limited to pancreatic cancer, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,

endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,

mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

As used herein, in one aspect, "next-generation sequencing" (NGS), also known as high- throughput sequencing, is the catch-all term used to describe a number of different sequencing methodologies including, but not limited to, illumina® sequencing, Roche 454 sequencing™, Ion torrent™: Proton / personal genome machine (PGM) sequencing, and SOLiD sequencing. These recent technologies allow for sequencing DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing. See, LeBlanc et al., 2015 Cancers, 7: 1925-1958, incorporated herein by reference; and Goodwin et al., 2016 Nature Reviews Genetics, 17: 333- 351, incorporated herein by reference.

As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

The phrase "pharmaceutically acceptable carrier" is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose;

starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt;

gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

By "protein" or "polypeptide" or "peptide" is meant any chain of more than two natural or unnatural amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally-occurring or non-naturally occurring polypeptide or peptide, as is described herein.

"Primer set" means a set of oligonucleotides that may be used, for example, for PCR. A primer set would consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers.

The terms "preventing" and "prevention" refer to the administration of an agent or composition to a clinically asymptomatic individual who is at risk of developing, susceptible, or predisposed to a particular adverse condition, disorder, or disease, and thus relates to the prevention of the occurrence of symptoms and/or their underlying cause.

The term "prognosis," "staging," and "determination of aggressiveness" are defined herein as the prediction of the degree of severity of the neoplasia, e.g., small bowel

neuroendocrine tumor, and of its evolution as well as the prospect of recovery as anticipated from usual course of the disease. Once the aggressivenes has been determined, appropriate methods of treatments are chosen.

Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another aspect. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. It is also understood that throughout the application, data are provided in a number of different formats and that this data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal tolO and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, "nested sub-ranges" that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

By "reduces" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

A "reference sequence" is a defined sequence used as a basis for sequence comparison or a gene expression comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 40 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 or about 500 nucleotides or any integer thereabout or there between.

The term "sample" as used herein refers to a biological sample obtained for the purpose of evaluation in vitro. Exemplary tissue samples for the methods described herein include tissue samples from tumors or the surrounding microenvironment (i.e., the stroma). With regard to the methods disclosed herein, the sample or patient sample preferably may comprise any body fluid or tissue. In some embodiments, the bodily fluid includes, but is not limited to, blood, plasma, serum, lymph, breast milk, saliva, mucous, semen, vaginal secretions, cellular extracts, inflammatory fluids, cerebrospinal fluid, feces, vitreous humor, or urine obtained from the subject. In some aspects, the sample is a composite panel of at least two of a blood sample, a plasma sample, a serum sample, and a urine sample. In exemplary aspects, the sample comprises blood or a fraction thereof (e.g., plasma, serum, fraction obtained via leukopheresis). Preferred samples are whole blood, serum, plasma, or urine. A sample can also be a partially purified fraction of a tissue or bodily fluid.

A reference sample can be a "normal" sample, from a donor not having the disease or condition fluid, or from a normal tissue in a subject having the disease or condition. A reference sample can also be from an untreated donor or cell culture not treated with an active agent (e.g., no treatment or administration of vehicle only). A reference sample can also be taken at a "zero time point" prior to contacting the cell or subject with the agent or therapeutic intervention to be tested or at the start of a prospective study.

A "solid support" describes a strip, a polymer, a bead, or a nanoparticle. The strip may be a nucleic acid-probe (or protein) coated porous or non-porous solid support strip comprising linking a nucleic acid probe to a carrier to prepare a conjugate and immobilizing the conjugate on a porous solid support. Well-known supports or carriers include glass, polystyrene,

polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a binding agent (e.g., an antibody or nucleic acid molecule). Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, or test strip, etc. For example, the supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation. In other aspects, the solid support comprises a polymer, to which an agent is chemically bound, immobilized, dispersed, or associated. A polymer support may be a network of polymers, and may be prepared in bead form (e.g., by suspension polymerization). The location of active sites introduced into a polymer support depends on the type of polymer support. For example, in a swollen-gel-bead polymer support the active sites are distributed uniformly throughout the beads, whereas in a macroporous-bead polymer support they are predominantly on the internal surfaces of the macropores. The solid support, e.g., a device contains a binding agent alone or together with a binding agent for at least one, two, three or more other molecules. By "specifically binds" is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.

A "specific binding agent" describes agents having greater than 10-fold, preferably greater than 100-fold, and most preferably, greater than 1000-fold affinity for the target molecule as compared to another molecule. As the skilled artisan will appreciate the term specific is used to indicate that other biomolecules present in the sample do not significantly bind to the binding agent specific for the target molecule. Preferably, the level of binding to a biomolecule other than the target molecule results in a binding affinity which is at most only 10% or less, only 5% or less only 2% or less or only 1%> or less of the affinity to the target molecule, respectively. A preferred specific binding agent will fulfill both the above minimum criteria for affinity as well as for specificity. For example, an antibody has a binding affinity in the low micromolar (10^"6), nanomolar (10^"7-10^"9), with high affinity antibodies in the low nanomolar (10^"9) or pico molar (10^"12) range for its specific target molecule.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50%) identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%>, at least 70%, at least 80%), at least 85%>, at least 90%, at least 95%, or at least 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e^"3 and e^"100 indicating a closely related sequence. The term "subject" as used herein includes all members of the animal kingdom prone to suffering from the indicated disorder. In some aspects, the subject is a mammal, and in some aspects, the subject is a human. The methods are also applicable to companion animals such as dogs and cats as well as livestock such as cows, horses, sheep, goats, pigs, and other

domesticated and wild animals.

A subject "suffering from or suspected of suffering from" a specific disease, condition, or syndrome has a sufficient number of risk factors or presents with a sufficient number or combination of signs or symptoms of the disease, condition, or syndrome such that a competent individual would diagnose or suspect that the subject was suffering from the disease, condition, or syndrome. Methods for identification of subjects suffering from or suspected of suffering from conditions associated with cancer (e.g., small bowel neuroendocrine tumor) is within the ability of those in the art. Subjects suffering from, and suspected of suffering from, a specific disease, condition, or syndrome are not necessarily two distinct groups.

As used herein, "susceptible to" or "prone to" or "predisposed to" or "at risk of developing" a specific disease or condition refers to an individual who based on genetic, environmental, health, and/or other risk factors is more likely to develop a disease or condition than the general population. An increase in likelihood of developing a disease may be an increase of about 10%, 20%, 50%, 100%, 150%, 200%, or more.

The terms "treating" and "treatment" as used herein refer to the administration of an agent or formulation to a clinically symptomatic individual afflicted with an adverse condition, disorder, or disease, so as to effect a reduction in severity and/or frequency of symptoms, eliminate the symptoms and/or their underlying cause, and/or facilitate improvement or remediation of damage. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

As used herein, in one aspect, the "tumor microenvironment" (TME) is the cellular environment in which a tumor exists, e.g., surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix (ECM). The tumor and the surrounding microenvironment are closely related and interact constantly. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of cancerous cells, such as in immuno-editing.

In some cases, a composition of the invention is administered orally or systemically. Other modes of administration include rectal, topical, intraocular, buccal, intravaginal, intracisternal, intracerebroventricular, intratracheal, nasal, transdermal, within/on implants, or parenteral routes. The term "parenteral" includes subcutaneous, intrathecal, intravenous, intramuscular, intraperitoneal, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. They could, however, be preferred in emergency situations. Compositions comprising a composition of the invention can be added to a physiological fluid, such as blood. Oral administration can be preferred for prophylactic treatment because of the convenience to the patient as well as the dosing schedule. Parenteral modalities (subcutaneous or intravenous) may be preferable for more acute illness, or for therapy in patients that are unable to tolerate enteral administration due to gastrointestinal intolerance, ileus, or other concomitants of critical illness. Inhaled therapy may be most appropriate for pulmonary vascular diseases (e.g., pulmonary hypertension).

Pharmaceutical compositions may be assembled into kits or pharmaceutical systems for use in arresting cell cycle in rapidly dividing cells, e.g., cancer cells. Kits or pharmaceutical systems according to this aspect of the invention comprise a carrier means, such as a box, carton, tube, having in close confinement therein one or more container means, such as vials, tubes, ampoules, bottles, syringes, or bags. The kits or pharmaceutical systems of the invention may also comprise associated instructions for using the kit.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

The transitional term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase "consisting of excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference.

Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the workflow from tissue aggregation to target protein quantification.

FIG. 2A-FIG. 21 is a series of photomicrographs with panel A through panel I showing SF ET primary carcinoid (CAR) tumor hematoxylin and eosin (H&E) stain (FIG. 2A), staining of neuroendocrine tumor cells for CgA (FIG. 2B), and staining of adjacent fibroblasts for Vimentin (FIG. 2C). Also shown are patient-derived cancer-associated short-term tumor-CAF "mixed" cell line morphology (FIG. 2D), CAF cells stained for CgA (FIG. 2E), CAF cells stained for Vimentin (FIG. 2F), a xenograft model statined for H&E (FIG. 2G), and fibroblastic cells stained with Hoechst 33358 (FIG. 2H), and tumor cells (FIG. 21) stained by Hoechst 33258.

FIG. 3 A is a series of bar charts showing median secretion levels in normal human fibroblasts (blue) and CAFs (red). FIG. 3B is a Log₂[CAF/Normal] Scatterplot.

FIG. 4A-FIG. 4C shows the results of an experiment, wherein 3 individual multiplex assays comprising cytokines, chemokines, receptors, growth factors, and hormones were utilized to assess NAF supernatants resulting in a total 61 targets being tested (FIG. 4 A), of which a number of detectable targets were duplicated across the assays and 7 were not detected above LLOQ, resulting in a total of 47 detectable targets, the majority of which were detected in greater than 75% of the NAF samples at 24, 48 and 72hr time points (FIG. 4B). FIG. 4C is a pie chart showing low, moderate and high expression levels associated with each of the time points, 24, 48 and 72hr.

FIG. 5A-FIG. 5 J is a heat map and a series of bar charts showing heatmap representation of hierarchical clustering of log₂(fold change) values for comparison of secretion levels in CAF's relative to the normal human fibroblasts, where red indicates over-expressed and green suppressed secretion levels associated with the CAFs (FIG. 5A). A subset of the secreted proteins are plotted as pg/mg (normalized to total protein) at the 48hr timepoint, including IL-6 (FIG. 5B), MCP-1 (FIG. 5C), VEGF (FIG. 5D), IL-12 (FIG. 5E), IFNg (FIG. 5H), IL-lra (FIG. 51) and IL-10 (FIG. 5 J) which are all more highly secreted/over-expressed; and SCF (FIG. 5F) and sEGFR (FIG. 5G) that were suppressed in CAF supernatants collected from SINETs relative to normal human fibroblasts.

FIG. 6A-FIG. 6K is a series of graphs showing upregulated concentrations [pg/ml] of MCP-1 (FIG. 6 A), IL-6 (FIG. 6B), VEGF (FIG. 6H), IL-12 (FIG. 61), IFNg (FIG. 6E), IL-lra (FIG. 6F), IL-10 (FIG. 6G) and downregulated concentrations of sEGFR (FIG. 6C) and SCF (FIG. 6D) that were statistically significant between the healthy control and metastatic SINET plasma samples. FIG. 6J shows that up- and down-regulation of secretion levels were compared and concordance between the supernatants and plasma was calculated. Linear correlation of plasma and NAF supernatants was plotted as log₂(fold change), where concordance was observed (FIG. 6K).

FIG. 7A is a heatmap representation of multiplex secretion profiling results plotted as log₂(Fold Change) comparing expression levels in the CAR24 and CAR26 CAFs treated with increasing doses of WFA over the concentration range 10-lOOOnM relative to DMSO control, where red and green indicates over- and under-expression and (#) in brackets on y-axis after protein label indicates bead encoding reference map. FIG. 7B is a line graph showing cell viability in CAR24 and CAR26 CAFs treated with increasing doses of WFA over the

concentration range 10-lOOOnM. FIG. 7C is a bar chart showing VEGF-A, MCP-1 and IL-6 mPvNA gene expression in CAR24 and CAR26 CAFs treated for 24hr with ΙΟΟηΜ WFA.

FIG. 8 is a series of photomicrographs showing tumor, stroma, and tumor-stroma mix CAR cell lines.

FIG. 9 is a series of photomicrographs showing organotypic slice culture morphology. At 48 hr (right) and 72 hr (left), with hematoxylin and eosin (H&E) staining on top, alpha- methylacyl-CoA racemase (AMACR) immunohistochemistry (AMACR IHC) in the middle row, and mitosis (arrow heads) on the bottom.

FIG. 10 is a schematic for using tissue slice cultures to evaluate response of

neuroendocrine tumors to Rapamycin.

FIG. 11 A-FIG. I ll shows the identification of genes differentially expressed between the WFA-treated and untreated CAF cell lines, CAR24 and CAR26. FIG. 11 A shows a volcano plot illustrating fold change (logbase2) versus adjusted significant P value (-logbaselO), where red and green data points represent a significance level of adj p<0.05 and log2FC>±3.5. FIG. 1 IB and FIG. 11C is a bar chart showing the top 10 MSigDB Hallmark data sets enriched in genes that were upregulated (FIG. 1 IB) and downregulated (FIG. 11C) in response to treatment with ΙΟΟηΜ WFA. FIG. 1 ID is a heatmap representation of hierarchical clustering of FkB signaling genes as defined by the MSigDB Hallmark data set, where red and green indicate up- and downregulated genes in response to treatment with ΙΟΟηΜ WFA. FIG. 1 IE is a heatmap showing gene expression pattern in the CAR24 and CAR26 CAF cell lines that facilitate characterization of tumor-associated stromal fibroblasts. FIG. 1 IF is a volcano plot illustrating fold change (logbase2) versus adjusted significant P value (-logbaselO), where red and green data points represent a significance level of adj p<0.05 and log2FC>±3.5. The top 10 MSigDB Hallmark data sets enriched in genes that were upregulated (FIG. 11G) and downregulated (FIG. 11H) in response to treatment with ΙΟΟηΜ WFA. FIG. 1 II is a heatmap representation of hierarchical clustering of EMT signaling genes as defined by the MSigDB Hallmark data set, where red and green indicate up- and downregulated genes in response to treatment with ΙΟΟηΜ WFA.

FIG. 12 shows images of untreated CAR15 NAFs in vitro in 6-well plates at time Ohr, 72hr and Day 5 (top panel), of CAR 15 NAF's treated with lOOuM uPA inhibitor or ΙΟΟηΜ Survivin at 72hrs and Day 5 (middle panel). Inset: uPA concentration [pg/ml] measured in cell culture supernatant from CAR15 treated with either uPA or survivin inhibitor. Gene expression knockdown and secretion levels relative to untreated control for CAR15 treated with either ΙΟΟηΜ of uPA or survivin inhibitor (bottom panel). DETAILED DESCRIPTION OF THE INVENTION

The invention is based, at least in part, upon the identification of secreted protein biomarkers of metastatic neuroendocrine tumors.

Prior to the invention described herein, the biology of SINETS was poorly understood, as SINETS are a biologically diverse group of well-differentiated tumors, and tumor size is an unreliable predictor of metastatic potential (Vinik et al., Pancreas, 39:713-734 (2010)). While tumor growth is often slower than that of other malignancies, many patients present with advanced disease for which few treatment options currently exist (Gilbert et al., Endocr Relat Cancer, 17:623-36 (2010)). The slow growing characteristics of SINETs has meant that the development of in vitro models to study this tumor type have been limited (Evers et al.,

Gastroenterology, 101 :303-311 (1991); Pfragner et al., Culture of guman neruoendocrine tumor cells. In: culture of human tumor cells. Pfragner R and Reshney RI (eds.). John Wiley & Sons. Hoboken NJ. pp. 373-403 (2004); Pfragner et al., Anticancer Res, 29: 1951-1962 (2009)), and stable human cell lines that recapitulate the NET biology are uncommon. Prior to the invention described herein, the genetic drivers of neuroendocrine tumors were also poorly understood. While pancreatic neuroendocrine tumors are characterized by recurrent mutations in the tumor suppressor gene MENl, as well as ATRX and DAXX, genes implicated in chromatin remodeling (Jiao et al., Science, 331 : 1199-1203 (2011)), whole genome profiling of SINETs revealed recurrent mutations in the cyclin dependent kinase inhibitor CDKN1B, in only 8% of cases (Francis et al., Nat Genet., 45: 1483-1486 (2013)).

Prior to the invention described herein, the lack of targetable mutations, together with a lack of cell lines and animal models has been a major obstacle in the development of innovative therapeutic agents for SINETs (Oberg KE, The management of Neuroendocrine Tumors: Current and Future Medical Therapy Options, 24:382-93 (2012)). With this slow growth and relatively quiet mutational landscape, the tumor microenvironment (TME) may have increased importance. Tumor-promoting inflammation has been established as a hallmark of cancer (Hanahan D and Weinberg RA, Cell, 144:646-674 (2011)) and almost all cancers have an inflammatory TME, irrespective of etiology linked to inflammation (Erez et al., Biochem Biophys Res Comm, 437:397-402 (2013)). Cancer Associated Fibroblasts (CAFs) are a heterogeneous and an activated cell subtype in the TME that promote tumor growth by directly stimulating tumor cell proliferation (Bhomick et al., Nature 432:332-337 (2004)). CAF-tumor interactions in the TME are partially defined by cancer cells inducing a reactive response in stroma and the activated stroma driving cancer cell malignancy in return (De WO and Mareel M. , J Pathol 200:429-447 (2003)). Cancer associated fibroblasts have been shown to have pro-tumorigenic effects (Ostman et al., Curr. Opin. Genet. Devel 19:67-73 (2009); Trimboli et al., Nature, 461 : 1084-1091 (2009)); and a prominent functional role in cancer progression and metastasis (Kalluri R and Zaisberg M., Nat Rev Cancer, 6:392-401 (2006)).

Prior to the invention described herein, a lack of cell lines and animal models had been an obstacle in developing new therapeutic agents for advanced carcinoid tumors. Described herein is a collection of short term carcinoid cell lines, which are used as a model of disease. Carcinoid cells in culture grow slowly and are importantly attached to stromal fibroblasts, referred to as CAF's (Carcinoid Associated Fibroblasts), also known as neuroendocrine-associated fibroblasts (NAFs). NAFs include all cell types that eincompass the entire stromal microenvironment, i.e., all cells except for tumor cells. These "short-term" NAF cultures are derived directly from the tissue from neuroendocrine patients. Described herein is the generation of a large collection of these patient-derived CAF cell lines.

Tumors are complex tissues composed of multiple distinct cell types, including recruited normal cells, which form tumor-associated stroma. These stromal cells are active participants in tumorigenesis and contribute to the development and expression of certain hallmark capabilities (Hanahan D and Weinberg R, 2011 Cell, 144: 646-674, incorporated herein by reference).

Accordingly, the "tumor microenvironment" (TME) is the cellular environment in which a tumor exists, e.g., surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix (ECM). The tumor and the surrounding microenvironment are closely related and interact constantly. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of cancerous cells, such as in immuno- editing.

The tumor's microenvironment is not only useful in elucidating how these tumors progress and become invasive, but is also useful in identifying new therapeutic pathways of disease. As described in detail below, CAF-secreted biomarker signatures are vital in

understanding the neuroendocrine tumor microenvironment. Specifically, described herein are prognostic, diagnostic, and pharmcodynamic biomarkers of metastatic small bowel neuroendocrine tumors. Also provided are rapid screening methods of secreted proteins in patient-derived cultures.

Accordingly, described herein are CAF cell culture supernatants from short-term primary cell lines derived from metastatic small bowel neuroendocrine tumors. As described in detail below, the secreted levels of proteins were measured utilizing a multiplexed bead-based approach, to develop a metastatic-associated secretion signature. Over-expression of secreted proteins (interleukin-6 (IL-6), IL-8, IL-12, IL-13, IL-2, monocyte chemoattractant protein- 1 (MCP-1) and vascular endothelial growth factor (VEGF) associated with immune and

inflammatory response and FkB and janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways was evident, indicating that small molecules that inhibit either or both may be therapeutically important in the context of the CAF cell microenvironment.

As described in detail below, by way of confirming the signature, IL-6, MCP-1, and VEGF were identified as over-expressed in the plasma from matched metastatic patients compared to healthy donors. Finally, CAF short-term cultures were highly sensitive to inhibition of the NFkB pathway by Withaferin A (WFA) at an IC50 of -ΙΟΟηΜ concentration and suppression of the secreted signature was observed in a dose dependent manner.

Neuroendocrine Tumors

Neuroendocrine tumors (NETs), defined as epithelial tumors with predominant neuroendocrine differentiation, are among the most frequent types of small bowel neoplasm (Xavier et al., 2016 World Journal of Gastrointestinal Pathophysiology, 7(1): 117-124, incorporated herein by reference). They represent a rare, slow-growing neoplasm with some characteristics common to all forms and others attributable to the organ of origin.

Traditionally, neuroendocrine tumors have been classified by their anatomic site of origin. NETs can arise in many different areas of the body, and are most often located in the intestine, pancreas or the lungs. The various kinds of cells that can give rise to NETs are present in endocrine glands and are also diffusely distributed throughout the body, e.g., Kulchitsky cells or enterochromaffin-like cells, which are relatively more common in the gastrointestinal and pulmonary systems. NETs include certain tumors of the gastrointestinal tract and of the pancreatic islet cells, certain thymus and lung tumors, and medullary carcinoma of the parafollicular cells of the thyroid. Neuroendocrine tumors, despite differing embryological origin, have common phenotypic characteristics. For example, ETs show tissue immunoreactivity for markers of neuroendocrine differentiation (pan-neuroendocrine tissue markers) and may secrete various peptides and hormones.

Several nomenclatures, grading, and classification systems have emerged for

neuroendocrine tumors. Thus, while the European Neuroendocrine Tumor Society (ENETS) (Eriksson et al., 2008 Neuroendocrinology, 87: 8-19, incorporated herein by reference; Jensen et al., 2006 Neuroendocrinology, 84: 165-172, incorporated herein by reference) favor the use of World Health Organization classification system, the North American Neuroendocrine Tumor Society (NANETS) (Klimstra et al., 2010 Pancreas, 39: 707-712, incorporated herein by reference) propose that some basic data elements (proliferative rate, extent of local spread, immunohistochemical markers) should be specified and documented on pathological reports and that a specified system of nomenclature, grading and staging should be used. This can assure that the basic data are recorded, allowing retrospective comparison of NETs regardless of the specific classification system used.

The World Health Organization (WHO) classification scheme places neuroendocrine tumors into three main categories, which emphasize the tumor grade rather than the anatomical origin:

• well-differentiated neuroendocrine tumours, further subdivided into tumors with benign and those with uncertain behavior

• well-differentiated (low grade) neuroendocrine carcinomas with low-grade

malignant behavior

• poorly differentiated (high grade) neuroendocrine carcinomas, which are the large cell neuroendocrine and small cell carcinomas.

Additionally, the WHO scheme recognizes mixed tumors with both neuroendocrine and epithelial carcinoma features, such as goblet cell cancer, a rare gastrointestinal tract tumor. Placing a given tumor into one of categories depends on well-defined histological features: size, lymphovascular invasion, mitotic counts, Ki-67 labelling index, invasion of adjacent organs, presence of metastases and whether they produce hormones

Symptoms from secreted hormones might prompt measurement of the corresponding hormones in the blood or their associated urinary products, for initial diagnosis or to assess the interval change in the tumor. Secretory activity of the tumor cells is sometimes dissimilar to the tissue immunoreactivity to particular hormones. Given the diverse secretory activity of ETs, there are many other potential markers, but a limited panel is usually sufficient for clinical purposes. Aside from the hormones of secretory tumors, the most important markers are:

chromogranin A (CgA), urine 5-hydroxyindoleacetic acid (5-HIAA), neuron-specific enolase (NSE, gamma-gamma dimer), and synaptophysin (P38). See, e.g., Xavier et al., 2016 World Journal of Gastrointestinal Pathophysiology, 7(1): 117-124, incorporated herein by reference.

CT-scans, MRIs, sonography (ultrasound), and endoscopy (including endoscopic ultrasound) are common diagnostic tools for neuroendocrine tumors. CT-scans using contrast medium can detect 95 percent of tumors over 3 cm in size, but generally not tumors under 1 cm.

Even if the tumor has advanced and metastasized, making curative surgery infeasible, surgery often has a role in neuroendocrine cancers for palliation of symptoms and possibly increased lifespan. Somatostatin analogs such as octreotide are used for treatment of small bowel neuroendocrine tumors (Xavier et al., 2016 World Journal of Gastrointestinal

Pathophysiology, 7(1): 1 17-124, incorporated herein by reference). Cholecystectomy is recommended if there is a consideration of long-term treatment with somatostatin analogs.

Radionuclide therapy, chemotherapy, radiofrequency ablation, and cryoablation are other useful therapies for this condition.

Small intestinal neuroendocrine tumors were first distinguished from other tumors in 1907 (Modlin et al., 2004 Human Pathology, 35 (12): 1440-51; Arnold et al., 2003 "Chapter 15 Neuroendocrine Gastro-Entero-Pancreatic (GEP) Tumors". In Scheppach W, Bresalier RS, Tytgat GN. Gastrointestinal and Liver Tumors. Berlin: Springer, pp. 195-233). They were named carcinoid tumors because their slow growth was considered to be "cancer-like" rather than truly cancerous. However, in 1938 it was recognized that some of these small bowel tumors could be malignant.

Small bowel neuroendocrine tumors, although rare across the cancer landscape (1 per 100,000), are the most common carcinoid tumor and originate most frequently in the terminal ileum (Isac et al., 2003 Acta Oncol., 42: 672-92; Pasieka JL, 2009 Surg Clin N Am., 89: 1123- 37). They are a biologically diverse group of well-differentiated tumors, where tumor size is an unreliable predictor of metastatic potential (Vinik et al., 2010 Pancreas, 39: 713-734; Yao et al., 2008 J Clin Oncol., 26: 3063-72). Tumor growth is slow and many patients present with advanced disease for which few treatment options exist (Moertel CG, 1983 J Clin Oncol., 11 : 727-740; Gilbert et al., 2010 Endocr Relat Cancer, 17: 623-36).

This slow growing characteristic has meant that the development of in vitro and in vivo models to study this tumor type have been extremely limited. Prior to the invention described herein, the lack of cell lines and animal models has been a major obstacle in the development of therapeutic agents for advanced carcinoid tumors (Oberg KE, 2012, Clin. Oncol., 24(4): 282-93). Neuroendocrine tumor cells grow slowly in culture and are importantly attached to fibroblasts, termed Neuroendocrine Associated Fibroblasts (NAFs). These cancer associated fibroblasts have pro-tumorigenic effects (Ostman A and Augsten M, 2009 Curr. Opin. Genet. Devel., 19: 67-73), are attractive targets for cancer therapy, and are valuable clinical biomarkers of prognosis (Rasanen K and Vaheri A, 2010 Exp. Cell Res., 316: 2713-2722).

As described herein, NAF-associated short-term cultures were developed, thereby allowing for the investigation of the carcinoid tumor microenvironment with a view to better understanding the mechanisms that drive tumor progression facilitating improvements in therapeutic intervention. Secreted proteins drive cancer associated fibroblast development within the tumor microenvironment at the crossroads of pro-malignant tumor-stroma interactions (Mishra et al., 2011 J Leuk. Biol., 89: 31-39). Secreted cytokines and chemokines are involved in a diverse array of processes including leukocyte infiltration, angiogenesis, and regulation of malignancy-related functions (Ben-Baruch A, 2006 Cancer Met. Rev., 25: 357-371).

Described herein is the development of a collection of short term NAF cell cultures from patient tumors. As described in detail below, NAF supernatants were collected, and in vitro multiplexed analysis of secreted proteins was peformed to identify a signature profile that characterizes metastatic SF ETs. As described herein, NAF secretion profiles can be replicated in plasma derived from the same patients. Finally, the results presented herein identify secreted biomarkers that are implicated in SINET growth and metastasis.

In a disease where in vitro and in vivo models are limited, generation and

characterization of patient-derived cancer-associated fibroblast (CAF) cultures, described below, elucidated the role CAFs play in the pathogenesis of small intestine neuroendocrine tumors (SINET) biology and insight into the mechanisms by which CAFs drive SINET tumorigenesis. As described in detail below, the small bowel neuroendocrine tumor (SINET) microenvironment was investigated to shed light on tumor-stromal interactions and to identify stromal biomarkers of clinical utility. Experiments established and histologically characterized patient-derived cancer-associated fibroblasts (CAFs) in vitro from disaggregated freshly resected small intestine neuroendocrine tumors. More specifically, short-term Neuroendocrine-Associated Fibroblast (NAF) cell cultures were generated from disaggregated resected tissue. A panel of secreted proteins was screened in the supernatants over the time course, 24-72hr. As described in the examples below, these NAFs have unique secretion profiles with respect to normal human fibroblasts. Accordingly, a panel of up- and downregulated secreted markers that characterize the SF ET patient population was identified. CAFs, in comparison to normal human fibroblasts exhibited pro-oncogenic characteristics, including high secretion of IL-6, MCP-1 and VEGF and other related cytokines and chemokines associated with an NFkB activated state that is distinct from normal human fibroblasts. The secretion profiling was replicated in matched patient plasma, confirming the identification of MCP-1, IL-6, and VEFG as upregulated NAF-specific biomarkers that correlated with the differential expression found in the short-term culture supernatants. In other words, this pro-oncogenic signature was recapitulated in plasma from the originating metastatic SINET patients.

As described in detail below, the NAFs were treated with Withaferin A (WFA), a potent inhibitor of NFkB activation, resulting in significant dose-dependent cell death, suppression of the upregulated secreted proteins, and knockdown of transcriptional activity of their

corresponding genes. Consistent with their NFkB activated state, experiments found that CAFs were highly sensitive to Withaferin A, which resulted in CAFs cell death, suppression of the pro- oncogenic secretion signature/profile, transcriptional downregulation of mRNA levels of the associated/corresponding genes and ultimately blockade of EMT in the treated CAFs. These results provide the first characterization of cancer-associated fibroblasts in small intestine neuroendocrine tumors, and suggest that these fibroblasts have pro-oncogenic properties consistent with an NFkB activated state. Thus, targeting CAFs in small intestine neuroendocrine tumors has therapeutic potential.

As described herein, the short-term NAF cultures were characterized and a panel of upregulated biomarkers including IL-6, MCP-1 and VEGF was identified. Targeting the NFkB pathway in the stromal environment in SINETs has important clinical implications. Finally, NAF short-term cultures are a valuable tool in elucidating the underlying poorly understood SFNET biology. Gene Expression Profiling

In general, methods of gene expression profiling can be divided into two large groups: methods based on hybridization analysis of polynucleotides, and methods based on sequencing of polynucleotides. Methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization, RNAse protection assays, RNA-seq, and reverse transcription polymerase chain reaction (RT-PCR). Alternatively, antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA- RNA hybrid duplexes or DNA-protein duplexes. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS). For example, RT-PCR is used to compare mRNA levels in different sample populations, in normal and tumor tissues, with or without drug treatment, to characterize patterns of gene expression, to discriminate between closely related mRNAs, and/or to analyze RNA structure. As described herein, a bead- based multiplexing approach screens for biomarkers of metastatic SINET disease.

In some cases, a first step in gene expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by amplification in a PCR reaction. For example, extracted RNA is reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, Calif, USA), following the manufacturer's instructions. The cDNA is then used as template in a subsequent PCR amplification and quantitative analysis using, for example, a TaqMan RTM (Life Technologies, Inc., Grand Island, N.Y.) assay.

Microarrays

Differential gene expression can also be identified, or confirmed using a microarray technique. In these methods, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest. Just as in the RT- PCR method, the source of mRNA typically is total RNA isolated from human tumors or tumor cell lines and corresponding normal tissues or cell lines. Thus, RNA is isolated from a variety of primary tumors or tumor cell lines. If the source of mRNA is a primary tumor, mRNA is extracted from frozen or archived tissue samples. In the microarray technique, PCR-amplified inserts of cDNA clones are applied to a substrate in a dense array. The microarrayed genes, immobilized on the microchip, are suitable for hybridization under stringent conditions.

In some cases, fluorescently labeled cDNA probes are generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest (e.g., SINET tissue). Labeled cDNA probes applied to the chip hybridize with specificity to loci of DNA on the array. After washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a charge-coupled device (CCD) camera. Quantification of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance.

In some configurations, dual color fluorescence is used. With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. In various configurations, the miniaturized scale of the hybridization can afford a convenient and rapid evaluation of the expression pattern for large numbers of genes. In various configurations, such methods can have sensitivity required to detect rare transcripts, which are expressed at fewer than 1000, fewer than 100, or fewer than 10 copies per cell. In various configurations, such methods can detect at least approximately two-fold differences in expression levels (Schena et al., Proc. Natl. Acad. Sci. USA 93(2): 106-149 (1996)). In various configurations, microarray analysis is performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Incyte's microarray technology.

RNA-seq

RNA sequencing (RNA-seq), also called whole transcriptome shotgun sequencing (WTSS), uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment in time.

RNA-Seq is used to analyze the continually changing cellular transcriptome. See, e.g., Wang et al., 2009 Nat Rev Genet, 10(1): 57-63, incorporated herein by reference. Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression. In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as miRNA, tRNA, and ribosomal profiling. RNA-Seq can also be used to determine exon/intron boundaries and verify or amend previously annotated 5' and 3' gene boundaries.

Prior to RNA-Seq, gene expression studies were done with hybridization-based microarrays. Issues with microarrays include cross-hybridization artifacts, poor quantification of lowly and highly expressed genes, and needing to know the sequence of interest. Because of these technical issues, transcriptomics transitioned to sequencing-based methods. These progressed from Sanger sequencing of Expressed Sequence Tag libraries, to chemical tag-based methods (e.g., serial analysis of gene expression), and finally to the current technology, NGS of cDNA (notably RNA-Seq).

Gene Signature

Described herein is a gene signature which identifies biomarkers of metastatic small bowel neuroendocrine tumors. Exemplary distinguishing genes are provided below.

An exemplary human Angiopoietin amino acid sequence is set forth below (SEQ ID

An exemplary human epidermal growth factor (EGF) amino acid sequence is set forth below (SEQ ID NO: 3; GenBank Accession No: NP_001954, Version 2, incorporated herein by reference :

An exemplary human EGF nucleic acid sequence is set forth below (SEQ ID NO: 4; GenBank Accession No: NM_001963, Version 3, incorporated herein by reference):

An exemplary human Endoglin amino acid sequence is set forth below (SEQ ID NO: 5;

GenBank Accession No: AAC63386, Version 1, incorporated herein by reference):

An exemplary human Endoglin nucleic acid sequence is set forth below (SEQ ID NO: 6; GenBank Accession No: NM_001114753, Version 2, incorporated herein by reference):

An exemplary human Eotaxin amino acid sequence is set forth below (SEQ ID NO: 7; GenBank Accession No: CAB07027, Version 1, incorporated herein by reference):

An exemplary human Eotaxin nucleic acid sequence is set forth below (SEQ ID NO: 8;

GenBank Accession No: Z92709, Version 1, incorporated herein by reference):

An exemplary human Fibroblast growth factor (FGF) Basic amino acid sequence is set forth below (SEQ ID NO: 9; GenBank Accession No: NP 001997, Version 5, incorporated herein by reference):

An exemplary human FGF Basic nucleic acid sequence is set forth below (SEQ ID NO: 10; GenBank Accession No: NM_002006, Version 4, incorporated herein by reference):

An exemplary human Follistatin amino acid sequence is set forth below (SEQ ID NO: 11; GenBank Accession No: AAH04107, Version 1, incorporated herein by reference):

1 mvrarhqpgg fmedrsaqag ncwlrqakng rcqvlyktel skeeccstgr 61 lstswteedv ndntlfkwmi fnggapncip cketcenvdc gpgkkcrmnk knkprcvcap 121 dcsnitwkgp vcgldgktyr necallkarc keqpelevqy qgrckktcrd vfcpgsstcv

An exemplary human Follistatin nucleic acid sequence is set forth below (SEQ ID

NO: 12; GenBank Accession No: BC004107, Version 2, incor orated herein b reference :

An exemplary human granulocyte-colony stimulating factor (G-CSF) amino acid sequence is set forth below (SEQ ID NO: 13; GenBank Accession No: P09919, Version 1, incorporated herein by reference):

An exemplary human G-CSF nucleic acid sequence is set forth below (SEQ ID NO: 14; GenBank Accession No: M17706, Version 1, incorporated herein by reference):

An exemplary human granulocyte macrophage colony stimulating factor (GM-CSF) amino acid sequence is set forth below (SEQ ID NO: 15; GenBank Accession No: P04141, Version 1, incorporated herein by reference):

An exemplary human GM-CSF nucleic acid sequence is set forth below (SEQ ID NO: 16; GenBank Accession No: M13207, Version 1, incorporated herein by reference):

An exemplary human heparin-binding EGF-like growth factor (HB-EGF) amino acid sequence is set forth below (SEQ ID NO: 17; GenBank Accession No: Q99075, Version 1, incorporated herein by reference):

An exemplary human HB-EGF nucleic acid sequence is set forth below (SEQ ID NO: 18;

GenBank Accession No: NM_001945, Version 2, incorporated herein by reference):

An exemplary hepatocyte growth factor (HGF) amino acid sequence is set forth below (SEQ ID NO: 19; GenBank Accession No: P14210, Version 2, incorporated herein by reference):

An exemplary human HGF nucleic acid sequence is set forth below (SEQ ID NO: 20;

GenBank Accession No: NM_000601, Version 1, incorporated herein by reference):

An exemplary human interferon gamma (IFNg) amino acid sequence is set forth below (SEQ ID NO: 21; GenBank Accession No: AAA53230, Version 1, incorporated herein by reference):

An exemplary human IFNg nucleic acid sequence is set forth below (SEQ ID NO: 22;

GenBank Accession No: U10360, Version 1, incorporated herein by reference):

An exemplary human insulin-like growth factor binding protein 1 (IGFBP-1) amino acid sequence is set forth below (SEQ ID NO: 23; GenBank Accession No: NP_000587, Version 1, incorporated herein by reference):

An exemplary human IGFBP-1 nucleic acid sequence is set forth below (SEQ ID NO: 24; GenBank Accession No: NM 000596, Version 2, incorporated herein by reference):

1

1

1

1

1

1

1

1

1

1

1

An exemplary human interleukin-10 (IL-10) amino acid sequence is set forth below (SEQ

ID NO: 25; GenBank Accession No: AAK38162, Version 1, incorporated herein by reference):

1 mhssallccl vlltgvrasp gqgtqsensc thfpgnlpnm lrdlrdafsr vktffqmkdq 61 ldnlllkesl ledfkgylgc qalsemiqfy leevmpqaen qdpdikahvn slgenlktlr

An exemplary human IL-10 nucleic acid sequence is set forth below (SEQ ID NO: 26; GenBank Accession No: AY029171, Version 1, incorporated herein by reference):

An exemplary human interleukin- 12 (IL-12) amino acid sequence is set forth below (SEQ ID NO: 27; GenBank Accession No: AAD16432, Version 1, incorporated herein by reference):

An exemplary human IL-12 nucleic acid sequence is set forth below (SEQ ID NO: 28;

GenBank Accession No: AF101062, Version 1, incorporated herein by reference):

An exemplary human interleukin-13 (IL-13) amino acid sequence is set forth below (SEQ ID NO: 29; GenBank Accession No: AAH96141, Version 2, incorporated herein by reference):

An exemplary human IL-13 nucleic acid sequence is set forth below (SEQ ID NO: 30;

GenBank Accession No: BC096141, Version 1, incorporated herein by reference):

An exemplary human interleukin-15 (IL-15) amino acid sequence is set forth below (SEQ ID NO: 31; GenBank Accession No: CAA62616, Version 1, incorporated herein by reference):

1 mriskphlrs isiqcylcll lnshflteag ihvfilgcfs aglpkteanw vnvisdlkki 61 edliqsmhid atlytesdvh psckvtamkc fllelqvisl esgdasihdt venliilann 121 slssngnvte sgckeceele eknikeflqs fvhivqmfin ts

An exemplary human IL-15 nucleic acid sequence is set forth below (SEQ ID NO: 32; GenBank Accession No: X91233, Version 1, incorporated herein by reference):

8341 gtggtgatgt gaatctatgc aaatgataaa attgcataaa acttcacaca tatgcaagca 8401 cacacactgt ggcacataaa acttgtgaaa tctgaacaag gtgagtgcat tgtattaatg

11701 gttatttatg aagtccagtg gtgaaaaagg acacagggta ggggcactgg ggctccagtt 11761 agggtaggct gagctaataa gctgccattt gacaaagatg tgagagaatg agccttaagg

An exemplary human interleukein 17 (IL-17) amino acid sequence is set forth below (SEQ ID NO: 33; GenBank Accession No: NP 002181, Version 1, incorporated herein by reference):

An exemplary human IL-17 nucleic acid sequence is set forth below (SEQ ID NO: 34;

GenBank Accession No: NM_002190, Version 2, incorporated herein by reference):

An exemplary human interleukin 18 (IL-18) amino acid sequence is set forth below (SEQ

ID NO: 35; GenBank Accession No: NP_001553, Version 1, incorporated herein by reference):

An exemplary human IL-18 nucleic acid sequence is set forth below (SEQ ID NO: 36;

GenBank Accession No: NM_001562, Version 3, incorporated herein by reference):

An exemplary human interleukin 1 beta (IL-lb) amino acid sequence is set forth below (SEQ ID NO: 37; GenBank Accession No: P01584, Version 2, incorporated herein by reference):

An exemplary human IL-lb nucleic acid sequence is set forth below (SEQ ID NO: 38;

GenBank Accession No: NM_000576, Version 2, incorporated herein by reference):

An exemplary human interleukin 1 receptor agonist (IL-lra) amino acid sequence is set forth below (SEQ ID NO: 39; GenBank Accession No: P18510, Version 1, incorporated herein by reference):

An exemplary human IL-lra nucleic acid sequence is set forth below (SEQ ID NO: 40;

GenBank Accession No: X77090, Version 1, incorporated herein by reference):

An exemplary human interleukin 2 (IL-2) amino acid sequence is set forth below (SEQ ID NO: 41; GenBank Accession No: AAB46883, Version 1, incor orated herein b reference :

An exemplary human IL-2 nucleic acid sequence is set forth below (SEQ ID NO: 42; GenBank Accession No: M13879, Version 1, incorporated herein by reference):

An exemplary human interleukin 4 (IL-4) amino acid sequence is set forth below (SEQ ID NO: 43; GenBank Accession No: NP_000580, Version 1, incorporated herein by reference):

An exemplary human IL-4 nucleic acid sequence is set forth below (SEQ ID NO: 44; GenBank Accession No: NM_000589, Version 3, incorporated herein by reference):

An exemplary human interleukin 5 (IL-5) amino acid sequence is set forth below (SEQ ID NO: 45; GenBank Accession No: NP_000870, Version 1, incorporated herein by reference):

An exemplary human IL-5 nucleic acid sequence is set forth below (SEQ ID NO: 46; GenBank Accession No: NM_000879, Version 2, incorporated herein by reference):

An exemplary human interleukin 6 (IL-6) amino acid sequence is set forth below (SEQ ID NO: 47; GenBank Accession No: NP_000591, Version 1, incorporated herein by reference):

1 mnsfstsafg pvafslglll vlpaafpapv ppgedskdva aphrqpltss eridkqiryi

An exemplary human IL-6 nucleic acid sequence is set forth below (SEQ ID NO: 48;

GenBank Accession No: NM_000600, Version 4, incorporated herein by reference):

An exemplary human interleukin 7 (IL-7) amino acid sequence is set forth below (SEQ ID NO: 49; GenBank Accession No: AAA59156, Version 1, incorporated herein by reference):

An exemplary human IL-7 nucleic acid sequence is set forth below (SEQ ID NO: 50;

GenBank Accession No: J04156, Version 1, incorporated herein by reference):

An exemplary human interleukin 8 (IL-8) amino acid sequence is set forth below (SEQ ID NO: 51; GenBank Accession No: NP_000575, Version 1, incorporated herein by reference):

An exemplary human IL-8 nucleic acid sequence is set forth below (SEQ ID NO: 52; GenBank Accession No: NM_000584, Version 3, incorporated herein by reference):

An exemplary human interleukin 9 (IL-9) amino acid sequence is set forth below (SEQ ID NO: 53; GenBank Accession No: NP_000581, Version 1, incorporated herein by reference):

An exemplary human IL-9 nucleic acid sequence is set forth below (SEQ ID NO: 54;

GenBank Accession No: NM_000590, Version 1, incorporated herein by reference):

An exemplary human interleukin 10 (IP- 10) amino acid sequence is set forth below (SEQ ID NO: 55; GenBank Accession No: NP_000563, Version 1, incorporated herein by reference):

An exemplary human IP-10 nucleic acid sequence is set forth below (SEQ ID NO: 56;

GenBank Accession No: NM_000572, Version 2, incorporated herein by reference):

An exemplary human leptin amino acid sequence is set forth below (SEQ ID NO: 57; GenBank Accession No: NP 000221 Version 1 incor orated herein b reference :

An exemplary human Leptin nucleic acid sequence is set forth below (SEQ ID NO: 58;

GenBank Accession No: NM_000230, Version 2, incorporated herein by reference):

An exemplary human monocyte chemotactic protein 1 (MCP-1 or MCAF) amino acid sequence is set forth below (SEQ ID NO: 59; GenBank Accession No: AAB29926, Version 1, incorporated herein by reference):

An exemplary human MCP-1 nucleic acid sequence is set forth below (SEQ ID NO: 60;

GenBank Accession No: S69738, Version 1, incorporated herein by reference):

An exemplary human macrophage inflammatory protein (MlP-la; also known as chemokine (C-C motif) ligand 3 CCL3) amino acid sequence is set forth below (SEQ ID NO: 61; GenBank Accession No: NP_002974, Version 1, incorporated herein by reference):

An exemplary human MlP-la nucleic acid sequence is set forth below (SEQ ID NO: 62; GenBank Accession No: NM_002983, Version 2, incorporated herein by reference):

An exemplary human macrophage inflammatory protein (MIP-lb; also known as chemokine (C-C motif) ligand 4 CCL4) amino acid sequence is set forth below (SEQ ID NO: 63; GenBank Accession No: P13236, Version 1, incorporated herein by reference):

An exemplary human MIP-lb nucleic acid sequence is set forth below (SEQ ID NO: 64; GenBank Accession No: NM_002984, Version 3, incorporated herein by reference):

An exemplary human osteopontin amino acid sequence is set forth below (SEQ ID NO: 65; GenBank Accession No: AAA86886, Version 1, incorporated herein by reference):

An exemplary human osteopontin nucleic acid sequence is set forth below (SEQ ID NO: 66; GenBank Accession No: J04765, Version 1, incorporated herein by reference):

An exemplary human plasminogen activator inhibitor-1 (PAI-1) amino acid sequence is set forth below (SEQ ID NO: 67; GenBank Accession No: NP 000593, Version 1, incorporated herein by reference):

An exemplary human PAI-1 nucleic acid sequence is set forth below (SEQ ID NO: 68;

GenBank Accession No: NM_000602, Version 4, incorporated herein by reference):

An exemplary human platelet-derived growth factor-AA (PDGF-AA) amino acid sequence is set forth below (SEQ ID NO: 69; GenBank Accession No: NP 148983, Version 1, incorporated herein by reference):

An exemplary human PDGF-AA nucleic acid sequence is set forth below (SEQ ID NO: 70; GenBank Accession No: NM_033023, Version 4, incorporated herein by reference):

An exemplary human platelet-derived growth factor-bb (PDGF-bb) amino acid sequence is set forth below (SEQ ID NO: 71; GenBank Accession No: CAA45383, Version 1, incorporated herein by reference):

An exemplary human PDGF-AA BB nucleic acid sequence is set forth below (SEQ ID NO: 72; GenBank Accession No: X63966, Version 1, incorporated herein by reference):

An exemplary human platelet and endothelial cell adhesion molecule 1 (PECAMl) amino acid sequence is set forth below (SEQ ID NO: 73; GenBank Accession No: NP 000433,

Version 4, incorporated herein by reference):

An exemplary human PECAM1 nucleic acid sequence is set forth below (SEQ ID4; GenBank Accession No: NM_000442, Version 4, incorporated herein by reference):

An exemplary human placental growth factor (PLGF) amino acid sequence is set forth below (SEQ ID NO: 75; GenBank Accession No: NP_002623, Version 2, incorporated herein by reference):

An exemplary human PLGF nucleic acid sequence is set forth below (SEQ ID NO: 76;

GenBank Accession No: NM_002632, Version 5, incorporated herein by reference):

An exemplary human prolactin amino acid sequence is set forth below (SEQ ID NO: 77;

GenBank Accession No: NP_001157030, Version 1, incorporated herein by reference):

An exemplary human prolactin nucleic acid sequence is set forth below (SEQ ID NO: 78; GenBank Accession No: NM_001163558, Version 2, incorporated herein by reference):

An exemplary human RANTES (regulated on activation, normal T cell expressed and secreted, alternatively, chemokine (C-C motif) ligand 5; alternatively CCL5) amino acid sequence is set forth below (SEQ ID NO: 79; GenBank Accession No: NP_001265665, Version 1, incorporated herein by reference):

An exemplary human RANTES (alternatively CCL5) nucleic acid sequence is set forth below (SEQ ID NO: 80; GenBank Accession No: NM_001278736, Version 1, incorporated herein by reference):

An exemplary human soluble CD40 ligand (sCD40L) amino acid sequence is set forth below (SEQ ID NO: 81; GenBank Accession No: NP 000065, Version 1, incorporated herein by reference):

An exemplary human sCD40L nucleic acid sequence is set forth below (SEQ ID NO: 82;

GenBank Accession No: NM_000074, Version 2, incorporated herein by reference):

An exemplary human stem cell factor (SCF) amino acid sequence is set forth below (SEQ

ID NO: 83; GenBank Accession No: P21583, Version 1, incorporated herein by reference):

An exemplary human SCF nucleic acid sequence is set forth below (SEQ ID NO: 84;

GenBank Accession No: M59964, Version 1, incorporated herein by reference):

An exemplary human epidermal growth factor receptor serum (sEGFR) amino acid sequence is set forth below (SEQ ID NO: 85; GenBank Accession No: NP_001171602, Version

1, incorporated herein by reference):

1

1

1

An exemplary human sEGFR nucleic acid sequence is set forth below (SEQ ID NO: 86;

GenBank Accession No: NM_001178131, Version 2, incorporated herein by reference):

An exemplary human Fas soluble ligand (alternatively CD95L or s FASL) amino acid sequence is set forth below (SEQ ID NO: 87; GenBank Accession No: P48023, Version 1, incorporated herein by reference):

An exemplary human sFASL nucleic acid sequence is set forth below (SEQ ID NO: 88;

GenBank Accession No: BC017502, Version 1, incorporated herein by reference):

An exemplary human Receptor tyrosine-protein kinase erbB-2, (also known as CD340

(cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent), or ERBB2 (human), or HER2/neu) amino acid sequence is set forth below (SEQ ID NO: 89; GenBank Accession No:

NP_001276867, Version 1, incorporated herein by reference):

An exemplary human sHER2 neu nucleic acid sequence is set forth below (SEQ ID NO: 90; GenBank Accession No: NM_001289938, Version 1, incorporated herein by reference):

An exemplary human interleukin 6 receptor, alpha (IL-6Ra) amino acid sequence is set forth below (SEQ ID NO: 91; GenBank Accession No: NP 000556, Version 1, incorporated herein by reference):

An exemplary human IL6Ra nucleic acid sequence is set forth below (SEQ ID NO: 92; GenBank Accession No: NM_000565, Version 3, incorporated herein by reference):

An exemplary human angiopoietin-1 receptor (alternatively, CD202B, or TIE -2) amino acid sequence is set forth below (SEQ ID NO: 93; GenBank Accession No: NP_000450, Version 2, incorporated herein by reference):

An exemplary human TIE-2 nucleic acid sequence is set forth below (SEQ ID NO: 94; GenBank Accession No: NM_000459, Version 4, incorporated herein by reference):

1 ttattctcag tttcccgcct atgagaggat acccctattg tttctgaaaa tgctgaccgg

An exemplary human vascular endothelial growth factor receptor 1 (VEGFl) amino acid sequence is set forth below (SEQ ID NO: 95; GenBank Accession No: NP_002010, Version 2, incor orated herein b reference :

An exemplary human VEGFl nucleic acid sequence is set forth below (SEQ ID NO: 96

GenBank Accession No: NM_002019, Version 4, incorporated herein by reference):

An exemplary human vascular endothelial growth factor receptor 2 (VEGFR2) amino acid sequence is set forth below (SEQ ID NO: 97; GenBank Accession No: NP_002244, Version 1, incorporated herein by reference):

An exemplary human VEGFR2 nucleic acid sequence is set forth below (SEQ ID NO: 98;

GenBank Accession No: NM_002253, Version 2, incorporated herein by reference):

An exemplary human transforming growth factor alpha (TGFa) amino acid sequence is set forth below (SEQ ID NO: 99; GenBank Accession No: NP_003227, Version 1, incorporated herein by reference):

An exemplary human TGFa nucleic acid sequence is set forth below (SEQ ID NO: 100;

GenBank Accession No: NM_003236, Version 3, incorporated herein by reference):

An exemplary human tumor necrosis factor alpha (TNFa) amino acid sequence is set forth below (SEQ ID NO: 101; GenBank Accession No: NP_000585, Version 2, incorporated herein by reference):

An exemplary human TNFa nucleic acid sequence is set forth below (SEQ ID NO: 102; GenBank Accession No: NM_000594, Version 3, incorporated herein by reference):

An exemplary human urokinase (urinary plasminogen activator or uPa) amino acid sequence is set forth below (SEQ ID NO: 103; GenBank Accession No: P00749, Version 2, incorporated herein by reference):

An exemplary human uPa nucleic acid sequence is set forth below (SEQ ID NO: 104;

GenBank Accession No: NM_002658, Version 4, incorporated herein by reference):

An exemplary human vascular endothelial growth factor (VEGF) amino acid sequence is set forth below (SEQ ID NO: 105; GenBank Accession No: AAA35789, Version 1, incorporated herein by reference):

An exemplary human VEGF nucleic acid sequence is set forth below (SEQ ID NO: 106;

GenBank Accession No: AY047581, Version 1, incorporated herein by reference):

An exemplary human vascular endothelial growth factor A (VEGFA) amino acid sequence is set forth below (SEQ ID NO: 107; GenBank Accession No: PI 5692, Version 2, incorporated herein by reference):

An exemplary human VEGFA nucleic acid sequence is set forth below (SEQ ID NO: 108; GenBank Accession No: NM_001317010, Version 1, incorporated herein by reference):

An exemplary human vascular endothelial growth factor C (VEGFC) amino acid sequence is set forth below (SEQ ID NO: 109; GenBank Accession No: P49767, Version 1, incorporated herein by reference):

An exemplary human VEGFC nucleic acid sequence is set forth below (SEQ ID

NO: 110; GenBank Accession No: NM_005429, Version 4, incorporated herein by reference):

An exemplary human vascular endothelial growth factor D (VEGFD) amino acid sequence is set forth below (SEQ ID NO: 111; GenBank Accession No: NP_004460, Version 1, incorporated herein by reference):

An exemplary human VEGFD nucleic acid sequence is set forth below (SEQ ID

NO: 112; GenBank Accession No: NM_004469, Version 4, incorporated herein by reference):

An exemplary human chromogranin A (CGA) amino acid sequence is set forth below

(SEQ ID NO: 113; GenBank Accession No: P10645, Version 7, incorporated herein by reference):

An exemplary human CGA nucleic acid sequence is set forth below (SEQ ID NO: 114; GenBank Accession No: NM_001275, Version 3, incorporated herein by reference):

An exemplary human hematopoietic progenitor cell antigen (CD34) amino acid sequence is set forth below (SEQ ID NO: 115; GenBank Accession No: NP_001020280, Version 1, incorporated herein by reference):

An exemplary human CD34 nucleic acid sequence is set forth below (SEQ ID NO: 116; GenBank Accession No: M81104, Version 1, incorporated herein by reference):

An exemplary human mucin 16 (MUC16) amino acid sequence is set forth below (SEQ : 117; GenBank Accession No: Q8WXI7, Version 3, incorporated herein by reference):

An exemplary human keratin 5 (KRT5) amino acid sequence is set forth below (SEQ ID 119; GenBank Accession No: P13647, Version 3, incorporated herein by reference):

An exemplary human KRT5 nucleic acid sequence is set forth below (SEQ ID NO: 120; GenBank Accession No: NM_000424, Version 3, incorporated herein by reference):

An exemplary human keratin 6A (KRT6A) amino acid sequence is set forth below (SEQ

ID NO: 121; GenBank Accession No: P02538, Version 3, incorporated herein by reference):

An exemplary human KRT6A nucleic acid sequence is set forth below (SEQ ID NO: 122;

GenBank Accession No: NM_005554, Version 3, incorporated herein by reference):

An exemplary human keratin 6B (KRT6B) amino acid sequence is set forth below (SEQ ID NO: 123; GenBank Accession No: NP_005546, Version 2, incorporated herein by reference):

An exemplary humanKRT6B nucleic acid sequence is set forth below (SEQ ID NO: 124;

GenBank Accession No: NM_005555, Version 3, incorporated herein by reference):

An exemplary human keratin 17 (KRT17) amino acid sequence is set forth below (SEQ

ID NO: 125; GenBank Accession No: Q04695, Version 2, incorporated herein by reference):

An exemplary humanKRT17 nucleic acid sequence is set forth below (SEQ ID NO: 126;

GenBank Accession No: NM_000422, Version 2, incorporated herein by reference):

An exemplary human fibroblast growth factor 2 (FGF2) amino acid sequence is set forth below (SEQ ID NO: 127; GenBank Accession No: P09038, Version 3, incorporated herein by reference):

An exemplary human FGF2 nucleic acid sequence is set forth below (SEQ ID NO: 128;

GenBank Accession No: NM_002006, Version 4, incorporated herein by reference):

An exemplary human stromal cell-derived factor 1 (CXCL12 or SDF-1) amino acid sequence is set forth below (SEQ ID NO: 129; GenBank Accession No: P48061, Version 1, incorporated herein by reference):

An exemplary human CXCL12 nucleic acid sequence is set forth below (SEQ ID

NO: 130; GenBank Accession No: CR450283, Version 1, incorporated herein by reference):

An exemplary human cadherin 1 (CDH1) amino acid sequence is set forth below (SEQ

ID NO: 131; GenBank Accession No: AAI46663, Version 1, incorporated herein by reference):

An exemplary human CDH1 nucleic acid sequence is set forth below (SEQ ID NO: 132; GenBank Accession No: NM_004360, Version 4, incorporated herein by reference):

An exemplary human cadherin 2 (CDH2) amino acid sequence is set forth below (SEQ

ID NO: 133; GenBank Accession No: P19022, Version 4, incorporated herein by reference):

An exemplary human CDH2 nucleic acid sequence is set forth below (SEQ ID NO: 134; GenBank Accession No: NM_001792, Version 4, incorporated herein by reference):

An exemplary human matrix metalloproteinase 1 (MMP-1) amino acid sequence is set forth below (SEQ ID NO: 135; GenBank Accession No: P03956, Version 3, incorporated herein by reference :

An exemplary human MMP-1 nucleic acid sequence is set forth below (SEQ ID NO: 136; GenBank Accession No: NM_002421, Version 3, incorporated herein by reference):

An exemplary human matrix metalloproteinase (MMP-2) amino acid sequence is set forth below (SEQ ID NO: 137; GenBank Accession No: P08253, Version 2, incorporated herein by reference):

An exemplary human MMP-2 nucleic acid sequence is set forth below (SEQ ID NO: 138; GenBank Accession No: NM_004530, Version 5, incorporated herein by reference):

An exemplary human integrin subunit alpha 1 (INTGA1 or VLA1) amino acid sequence is set forth below (SEQ ID NO: 139; GenBank Accession No: NP_852478, Version 1, incorporated herein by reference):

An exemplary human INTGA1 nucleic acid sequence is set forth below (SEQ ID NO:

140; GenBank Accession No: NM_181501, Version 1, incorporated herein by reference):

An exemplary human integrin subunit alpha 2 (INTGA2) amino acid sequence is set forth below (SEQ ID NO: 141; GenBank Accession No: P17301, Version 1, incorporated herein by reference):

An exemplary human INTGA2 nucleic acid sequence is set forth below (SEQ ID 42; GenBank Accession No: NM_002203, Version 3, incorporated herein by reference):

An exemplary human integrin subunit alpha 3 (INTGA3) amino acid sequence is set forth below (SEQ ID NO: 143; GenBank Accession No: AAI36637, Version AAI36637.1, incorporated herein by reference):

An exemplary human INTGA3 nucleic acid sequence is set forth below (SEQ ID NO: 144; GenBank Accession No: NG 029107, Version NG_029107.2, incorporated herein by reference):

An exemplary human integrin subunit alpha 5 (INTGA5) amino acid sequence is set forth below (SEQ ID NO: 145; GenBank Accession No: NP_002196, Version NP_002196.4, incorporated herein by reference):

An exemplary human INTGA5 nucleic acid sequence is set forth below (SEQ ID

NO: 146; GenBank Accession No: NM_002205, Version NM_002205.4, incorporated herein by reference):

An exemplary human integrin subunit alpha 7 (INTGA7) amino acid sequence is set forth below (SEQ ID NO: 147; GenBank Accession No: AAC18968, Version AAC 18968.1, incorporated herein by reference):

An exemplary human INTGA7 nucleic acid sequence is set forth below (SEQ ID

NO: 148; GenBank Accession No: NG_012343, Version NG_012343.1, incorporated herein by reference):

fort

NP

NO: here

An exemplary human transforming growth factor beta 1 (TGFBl) amino acid sequence is set forth below (SEQ ID NO: 151; GenBank Accession No: P01137, Version 2, incorporated herein by reference):

An exemplary human TGFBl nucleic acid sequence is set forth below (SEQ ID NO: 152; GenBank Accession No: NM_000660, Version 6, incorporated herein by reference):

An exemplary human transforming growth factor beta induced (TGFBI) amino acid sequence is set forth below (SEQ ID NO: 153; GenBank Accession No: Q15582, Version 1, incorporated herein by reference):

An exemplary human TGFBI nucleic acid sequence is set forth below (SEQ ID NO: 154;

GenBank Accession No: NM_000358, Version 2, incorporated herein by reference):

An exemplary human yes-associated protein 1 (YAPl) amino acid sequence is set forth below (SEQ ID NO: 155; GenBank Accession No: P46937, Version 2, incorporated herein by reference):

An exemplary human YAPl nucleic acid sequence is set forth below (SEQ ID NO: 156; GenBank Accession No: NM_001130145, Version 2, incorporated herein by reference):

An exemplary human hyaluronan synthase 2 (HAS2) amino acid sequence is set forth below (SEQ ID NO: 157; GenBank Accession No: Q92819, Version 1, incorporated herein by reference):

An exemplary human HAS2 nucleic acid sequence is set forth below (SEQ ID NO: 158;

GenBank Accession No: NM_005328, Version 2, incorporated herein by reference):

An exemplary human lysyl oxidase (LOX) amino acid sequence is set forth below (SEQ

ID NO

: 159; GenBank Accession No: AAB23549, Version 1, incorporated herein by reference):

An exemplary human LOX nucleic acid sequence is set forth below (SEQ ID NO: 160; GenBank Accession No: AF039291, Version 1, incorporated herein by reference):

An exemplary human SI 00 calcium binding protein A4 (S100A4) amino acid sequence is set forth below (SEQ ID NO: 161; GenBank Accession No: AAH00838, Version 1, incorporated herein by reference):

An exemplary human S100A4 nucleic acid sequence is set forth below (SEQ ID NO: 162;

GenBank Accession No: BC016300, Version 1, incorporated herein by reference):

An exemplary human Glutathione S-transferase PI (GSTP1) amino acid sequence is set forth below (SEQ ID NO: 163; GenBank Accession No: P09211, Version 2, incorporated herein by reference):

An exemplary human GSTPl nucleic acid sequence is set forth below (SEQ ID NO: 164; GenBank Accession No: NM_000852, Version 3, incorporated herein by reference):

An exemplary human Cluster of Differentiation 81 (CD81), also known as Target of the

Antiproliferative Antibody 1 (TAPA-1), amino acid sequence is set forth below (SEQ ID NO:

165; GenBank Accession No: P60033, Version 1, incorporated herein by reference):

An exemplary human CD81 nucleic acid sequence is set forth below (SEQ ID NO: 166; GenBank Accession No: BC093047, Version 1, incorporated herein by reference):

An exemplary human Thymocyte antigen 1 (THYl), also known as Cluster of differentiation 90 (CD90), amino acid sequence is set forth below (SEQ ID NO: 167; GenBank Accession No: P04216, Version 2, incorporated herein by reference):

An exemplary human THYl nucleic acid sequence is set forth below (SEQ ID NO: 168; GenBank Accession No: BC065559, Version 1, incorporated herein by reference):

An exemplary human ecto-5'-nucleotidase (NT5E), also known as Cluster of differentiation 73 (CD73), amino acid sequence is set forth below (SEQ ID NO: 169; GenBank

Accession No: AAH65937, Version 1, incorporated herein by reference):

An exemplary human NT5E nucleic acid sequence is set forth below (SEQ ID NO: 170;

GenBank Accession No: NM_002526, Version 3, incorporated herein by reference):

An exemplary human endoglin (ENG) amino acid sequence is set forth below (SEQ ID

NO: 171; GenBank Accession No: P17813, Version 2, incorporated herein by reference):

An exemplary human ENG nucleic acid sequence is set forth below (SEQ ID NO: 172; GenBank Accession No: BC014271, Version 2, incorporated herein by reference):

An exemplary human Cluster of differentiation 44 (CD44) amino acid sequence is set forth below (SEQ ID NO: 173; GenBank Accession No: ACI46596, Version 1, incorporated herein by reference):

An exemplary human CD44 nucleic acid sequence is set forth below (SEQ ID NO: 174; GenBank Accession No: NM_000610, Version 3, incorporated herein by reference):

An exemplary human Familial adenomatous polyposis (FAP) amino acid sequence is set forth below (SEQ ID NO: 175; GenBank Accession No: Q12884, Version 5, incorporated herein by reference):

661 terfmglptk ddnlehykns tvmaraeyfr nvdyllihgt addnvhfqns aqiakalvna 721 qvdfqamwys dqnhglsgls tnhlythmth flkqcfslsd

An exemplary human FAP nucleic acid sequence is set forth below (SEQ ID NO: 176;

GenBank Accession No: NM 004460, Version 4, incor orated herein b reference :

An exemplary human Hypoxia-inducible factor 1 -alpha (HIFIA) amino acid sequence is set forth below (SEQ ID NO: 177; GenBank Accession No: Q16665, Version 1, incorporated herein by reference):

An exemplary human HIFIA nucleic acid sequence is set forth below (SEQ ID NO: 178;

GenBank Accession No: NM_001530, Version 3, incorporated herein by reference):

An exemplary human Caveolin-1 (CAVI) amino acid sequence is set forth below (SEQ

ID NO: 179; GenBank Accession No: Q03135, Version 4, incorporated herein by reference):

An exemplary human CAVI nucleic acid sequence is set forth below (SEQ ID NO: 180; GenBank Accession No: NM_001753, Version 4, incorporated herein by reference):

An exemplary human Platelet-derived growth factor receptor alpha (PDGFRA) amino acid sequence is set forth below (SEQ ID NO: 181; GenBank Accession No: P16234, Version 1, incorporated herein by reference):

An exemplary human PDGFRA nucleic acid sequence is set forth below (SEQ ID 82; GenBank Accession No: NM_006206, Version 5, incorporated herein by reference):

An exemplary human Platelet-derived growth factor receptor beta (PDGFRB) amino acid sequence is set forth below (SEQ ID NO: 183; GenBank Accession No: P09619, Version 1, incorporated herein by reference):

An exemplary human PDGFRB nucleic acid sequence is set forth below (SEQ ID

NO: 184; GenBank Accession No: NM_002609, Version 3, incorporated herein by reference):

An exemplary human Neuregulin 1 (NRGl) amino acid sequence is set forth below (SEQ

ID NO: 185; GenBank Accession No: Q02297, Version 3, incor orated herein b reference :

An exemplary humanNRGl nucleic acid sequence is set forth below (SEQ ID NO: 186;

GenBank Accession No: NM_013960, Version 4, incorporated herein by reference):

An exemplary human Tenascin C (TNC) amino acid sequence is set forth below (SEQ ID 87; GenBank Accession No: P24821, Version 3, incorporated herein by reference):

An exemplary human TNC nucleic acid sequence is set forth below (SEQ ID NO: 188; GenBank Accession No: NM_002160, Version 3, incorporated herein by reference):

An exemplary human Periostin (POSTN) amino acid sequence is set forth below (SEQ

ID NO: 189; GenBank Accession No: AAI06710, Version 1, incorporated herein by reference):

An exemplary human POSTN nucleic acid sequence is set forth below (SEQ ID NO: 190;

GenBank Accession No: NM_006475, Version 2, incorporated herein by reference):

An exemplary human Alpha-actin-2 (ACTA2) amino acid sequence is set forth below

(SEQ ID NO: 191; GenBank Accession No: P62736, Version 1, incorporated herein by reference):

An exemplary human ACTA2 nucleic acid sequence is set forth below (SEQ ID NO: 192; GenBank Accession No: NM_001141945, Version 2, incorporated herein by reference):

An exemplary human Prolyl 4-hydroxylase subunit alpha-1 (P4HA1) amino acid sequence is set forth below (SEQ ID NO: 193; GenBank Accession No: P13674, Version 2, incorporated herein by reference):

An exemplary human P4HA1 nucleic acid sequence is set forth below (SEQ ID NO: 194; GenBank Accession No: NM_000917, Version 3, incorporated herein by reference):

An exemplary human Prolyl 4-hydroxylase subunit beta (P4HB) amino acid sequence is set forth below (SEQ ID NO: 195; GenBank Accession No: P07237, Version 3, incorporated herein by reference):

An exemplary human P4HB nucleic acid sequence is set forth below (SEQ ID NO: 196;

GenBank Accession No: NM_000918, Version 3, incorporated herein by reference):

An exemplary human vimentin (VIM) amino acid sequence is set forth below (SEQ ID

NO: 197; GenBank Accession No: NP_003371, Version 2, incorporated herein by reference):

An exemplary human VIM nucleic acid sequence is set forth below (SEQ ID NO: 198;

GenBank Accession No: XM_006717500, Version 1, incorporated herein by reference):

Pharmaceutical Therapeutics

For therapeutic uses, the compositions or agents described herein may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the drug in the patient. Treatment of human patients or other animals will be carried out using a therapeutically effective amount of a therapeutic identified herein in a physiologically-acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of the neoplasia, i.e., the SINET. Generally, amounts will be in the range of those used for other agents used in the treatment of other diseases associated with neoplasia, although in certain instances lower amounts will be needed because of the increased specificity of the compound. For example, a therapeutic compound is administered at a dosage that is cytotoxic to a neoplastic cell. Formulation of Pharmaceutical Compositions

The administration of a compound or a combination of compounds for the treatment of a neoplasia, e.g., a SINET, may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing a neoplasia. The compound may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Human dosage amounts can initially be determined by extrapolating from the amount of compound used in mice, as a skilled artisan recognizes it is routine in the art to modify the dosage for humans compared to animal models. In certain embodiments it is envisioned that the dosage may vary from between about 1 μg compound/Kg body weight to about 5000 mg compound/Kg body weight; or from about 5 mg/Kg body weight to about 4000 mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kg body weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg body weight; or from about 100 mg/Kg body weight to about 1000 mg/Kg body weight; or from about 150 mg/Kg body weight to about 500 mg/Kg body weight. In other cases, this dose may be about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 mg/Kg body weight. In other aspects, it is envisaged that doses may be in the range of about 5 mg compound/Kg body to about 20 mg compound/Kg body. In other embodiments, the doses may be about 8, 10, 12, 14, 16 or 18 mg/Kg body weight. Of course, this dosage amount may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of the initial clinical trials and the needs of a particular patient. Pharmaceutical compositions according to the invention may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with the thymus; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a neoplasia by using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type (e.g., neoplastic cell). For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner.

Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

Parenteral Compositions

The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, nontoxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms (e.g., in single- dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that reduces or ameliorates a neoplasia, the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.

As indicated above, the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable active antineoplastic therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the compounds is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol.

Controlled Release Parenteral Compositions

Controlled release parenteral compositions may be in form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, or emulsions. Alternatively, the active drug may be incorporated in biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices.

Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactin, poly-(isobutyl cyanoacrylate), poly(2- hy droxy ethyl -L-glutam- nine) and, poly(lactic acid). Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in implants can be nonbiodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters) or combinations thereof).

Kits or Pharmaceutical Systems

The present compositions may be assembled into kits or pharmaceutical systems for use in ameliorating a neoplasia (e.g., SINET). Kits or pharmaceutical systems according to this aspect of the invention comprise a carrier means, such as a box, carton, tube or the like, having in close confinement therein one or more container means, such as vials, tubes, ampoules, or bottles. The kits or pharmaceutical systems of the invention may also comprise associated instructions for using the agents of the invention.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1 : Understanding the Role of Carcinoid Associated Fibroblasts in the Neuroendocrine Tumor Microenvironment

Prior to the invention described herein, a lack of cell lines and animal models had been an obstacle in developing therapeutic agents for advanced carcinoid tumors. Described herein is the development of a collection of short term carcinoid cell lines. Carcinoid cells in culture grow slowly and are importantly attached to stromal fibroblasts, referred to as CAF (Carcinoid Associated Fibroblasts). As described herein, CAF cell culture supernatants have been collected from 22 of the short-term primary cell lines, and the secreted levels of proteins have been measured utilizing a multiplexed bead-based approach to develop a secretion gene signature.

The carcinoid turn orj -stroma microenvironment was investigated to shed light on tumor- stromal interactions that might point to potential biomarkers of clinical utility. Described herein is (1) the development of short-term primary CAF cell cultures from carcinoid patient tumors; (2) the optimization of a culturing protocol for collecting CAF supernatants, whereby sufficient concentration of secreted proteins would be detectable in vitro; and (3) the performance of multiplexed analysis of secreted proteins on supernatants to investigate whether there is a secreted gene signature profile for well-differentiated tumors.

As shown in FIG. 1, primary tumor tissue was minced in culture medium and

disaggregated by overnight incubation in collagenase (lOOU/ml at 37°C). 50,000 cells were seeded and cultured in 2 ml Dulbecco's modified eagle's / F12 (50:50) medium containing 10% fetal bovine serum and antibiotic-antimycotic and glutamax.

Cell culture supernatants were collected after 24, 48, and 72 hours and centrifuged at 12,300 rpm for 10 min (4°C). A Bioplex 200 platform was utilized for multiplex analysis. Cell culture supernatant concentration levels [pg/ml] of each protein were derived from 5-parameter curve fitting models. All concentrations were normalized to total protein concentration. Fold changes relative to normal human fibroblasts (BJ and FIFF1 lines) were calculated.

Table 1 : Primary tumor banked an short-term cell lines developed

Short-term culures were generated for all but 1 primary tumor received. The collection comprises mostly mixed tumor/CAF cultures of varying tumor composition and pure CAF cultures, of which there are 22 (Table 1). Carcinoid cells stain positively for Chromogranin A, CgA, a marker of neuroendocrine phenotype. Carcinoid-associated fibroblasts stain positively for Vimentin, a mesenchymal marker. A unique library of frozen and FFPE primary tumors, short-term cell cultures and exnograft models, was developed (Figure 2). In particular, note the CAF cells in the xenograft models were of human and not host origin (2H).

56 target proteins were measured across 3 multiplex assays utilizing a Bioplex platform, including IL-Ιβ, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, FGF Basic, G-CSF, GM-CSF, IFN-γ, IP- 10, MCP-1 (MCAF), MIP-la,

PDGF-BB, ΜΙΡ-Ιβ, RANTES, TNF-a, VEGF, sEGFR, Folastatin, sHER2 neu, HGF, sIL-6Ra, Leptin, Osteopontin, PDGF-AA BB, PECAM1, Prolactin, SCF, sTIE-2, sVEGFRl, sVEGFR2, Angiopoietin, sCD40L, EGF, Endoglin, sFASL, HB-EGF, IGFBP-1, IL-18, PAI-1, PLGF, TGFa, uPA, VEGF-A, VEGF-C and VEGF-D. Target proteins with a concentration below the lower limit of quantitation (LLOQ) or which were detected in less than 50% of the supernatants were eliminated from the data set. Fold changes for all target proteins remaining where calculated for each time point and trends evaluated.

Fold expression trends across the time course, 24 - 72hr were calculated. Within the CAF cohort, the secretion levels varied widely. This underlines the complexity of not only protein secretion within each tumor microenvironment but also the timing and mechanistic process by which these proteins are shed or secreted from CAF's. However an 8 marker signature, comprised of IL-6, IL-12, IL-13, RANTES, sEGFR, sVEGFR2, sTIE-2 and SCF, was identified (3B).

A multiplexed bead-based suspension assay was conducted with the cell library to investigate the protein secretion profile of carcinoid associated fibroblasts in cell supernatants derived from short-term primary cultures. Next, the 8 marker signature is validated in the plasma from patients with metastatic disease to establish their value in early diagnosis and/or assessing progression and prognosis.

Example 2: Secretion Profiling Identifies Biomarkers of Metastatic Small Bowel Neuroendocrine Tumors in Short-Term Neuroendocrnie-Associatd Fibroblast Primary Cultures

The following materials and methods were utilized in this example.

Clinical specimens

Neuroendocrine tumor tissue and plasma samples were obtained from the Dana-Farber Cancer Institute Neuroendocrine Tumor Biospecimen database, under an IRB-approved protocol (TRB# 02-314) in which patients provide informed consent for collection of tissue specimens and blood obtained during routine clinical care. Blood/plasma was also collected from consenting spousal/friend as healthy controls. Briefly, blood (up to 10ml) was collected into tubes containing EDTA, and the separation procedure was carried out within 3hr of venipuncture. The samples were spun at 3000rpm for 15min resulting in approximately 3 -6ml plasma. The plasma was aliquoted and stored at -80°C for preservation.

Patient-derived CAF cultures and supernatant collection

A portion of each tumor tissue specimen was minced in culture medium and

disaggregated by overnight incubation in collagenase (100 U/ml at 37°C). Cells were initially cultured in Dulbecco's modified eagle's / F12 (50:50) medium containing 10% fetal bovine serum, antibiotic-antimycotic and glutamax. 50k cells were seeded in 2ml media in 6-well plates. Cell culture supernatants and cell pellets were collected at 24, 48 and 72hr. 1.5 ml of cell culture supernatant was centrifuged at 12300rpm for 10 min at 4°C and stored at -80°C. Cell were trypsinized, washed in PBS, and lysed for total protein extraction. Total proteins were quantitated by Bradford assay (Biorad cat #500-0002). Normal human fibroblast cell lines, BJ and HFF1 (ATCC cat# CRL-2522 and SCRC-1041) were cultured under the same conditions. Clinical characteristics of patient-derived CAFs

Tumors from 20 SINET patients (55%/45% female to male ration) were collected for the generation of short-term cultures of patient-derived fibroblasts (Table 2). The median age of the patients was 58.5 years of age. 50% were of grade 1 and 25% of grade 2. 65% of patients were treated with Octreotide, of which almost half of these patients also had additional treatment(s) at the time of surgery.

Table 2: Clinical characteristics of SINET specimens utilized in creation of short-term cultures

Short-term NAF cultures

A portion of each tissue sample was frozen and evaluated to confirm histological features and rule out the presence of significant hemorrhagic/necrotic areas, not suitable for experimentation. Additional portions of each tissue were snap frozen and banked, and also formalin-fixed and processed for paraffin-embedding (FFPE). Tumor tissue was then minced in culture medium and disaggregated by overnight incubation in collagenase (100 U/ml at 37°C). Cells were initially cultured in Dulbecco's modified eagle's / F12 (50:50) medium containing 10% fetal bovine serum, antibiotic-antimycotic and glutamax.

Cell Culture Supernatant Collection

50k cells were seeded in 2ml DMEM F12 supplemented with 10% FBS and 1% P/S in 6- well plates. Cell culture supernatants and cell pellets were collected at 24, 48, and 72hr. 1.5 ml of cell culture supernatant was centrifuged at 12,300rpm for 10 min at 4°C and aliquoted (3 x 500 μΐ aliquots) for storage at -80 °C. The remaining cells were then washed in PBS, and lysed for total proteins extraction. Total Proteins were quantitated by Bradford assay (Biorad cat #500-0002).

Multiplex Secretion profiling

Multiplex assays were performed utilizing a bead-based immunoassay approach, namely the Bio-Plex Pro™ Human Cytokine 27-plex Assay (Cat# M500KNAF0Y), Bio-Plex Pro™ Human Cancer Biomarker Panel 1, 16-plex (Cat#171 AC500M) and Bio-Plex Pro™ Human Cancer Biomarker Panel 2, 18-plex (Cat# 171 AC600M) on a Bio-plex 200 system (Cat#

171000201). Manufacturer guidelines were followed without deviation or amendments. Briefly, capture antibodies for each protein were coupled to a uniquely encoded bead. A sandwich ELISA in suspension ensues incorporating a dual detection system that identifies the encoded bead and measures the fluorescent intensity of the associated protein in the cell culture supernatant. Plasma and cell culture supernatant concentration levels [pg/ml] of each protein were derived from 5 -parameter curve fitting models. All concentrations were normalized to total protein concentration. Normalized secreted protein levels in the carcinoid associated fibroblasts NAF's supernatant were compared to normal human fibroblast cell lines, BJ and HFF1 (ATCC cat# CRL-2522 and SCRC-1041). Fold changes relative to the mean of these normal human fibroblasts (NHF's) were calculated and two-tailed student t-tests performed, where p-values <0.05 were considered significant. Hierarchical clustering utilized Gene Cluster 3.0 employing Euclidean distance and average linkage for clustering of secreted proteins and time points. Java Treeview (Version 1.1.5) software was used to plot the heatmap from hierarchical clustering results. RNA Extraction and Quantification

CAF/NAF and normal human fibroblasts (NHF) cells were trypsinized, scraped and transferred to an RNase-/DNase-free 2ml eppendorf tube. Cells were pelleted by centrifugation and washed in 1 ml PBS in triplicate. RNA was extracted from the cell pellets utilizing the AllPrep mini kit [Qiagen cat# 80204], as described by the manufacturers protocol. RNA isolates were eluted in a 14μ1 volume of RNase/DNase-free H20. All RNA was stored at -80°C. RNA isolates were quantified utilizing the Quant-iT RiboGreen assay (Life Technologies - cat# R11490). Ι μΐ of RNA is required for quantification. Concentration was measured as ng/μΐ. For RNA quantification, isolates were excited at 485 ± 10 nm and the fluorescence emission intensity was measured at 530 ± 12 nm using a Victor X3 spectrophotometer (Perkin Elmer cat# 2030-0030). Fluorescence intensity was plotted versus RNA concentration over the calibration range, 0 -100 ng/μΐ.

qRT-PCR

RT-PCR was employed to interrogate list all genes studied (Life Technologies, High Capacity cDNA Reverse Transcription Kit [Cat# 4368814], TaqMan® Gene Expression Master Mix [Cat# 4369016], see Table 3 for probe id information) 20ng RNA input was utilized in the reverse transcription reaction (High-Capacity cDNA Reverse Transcription Kit Life

Technologies) and 2μ1 of the subsequent cDNA product was transferred to the PCR assay (Taqman Gene Expression Mastermix, Cat # 4369510, Life Technologies). Triplicate reactions were prepared using Taqman assay probes for for IL-6 (Hs00174131_ml), MCP-1 (CCL2; Hs00234140_ml) and VEGFA (Hs00900055_ml) for each sample. GAPDH (Taqman cat #: Hs 02758991_gl, Life Technologies) was utilized as the reference housekeeping gene and subsequent differential expression was assessed by comparative CT analysis as previously described (Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method (Schmittgen TD and Livak KJ, Nature Protocols, 3 : 1101-1108 (2008)). Table 3

Cell Viability Assay

The ATP activity was assessed by CellTiter-Glo® Luminescent Cell Viability Assay

(Promega cat # G7571) to determine cell viability in NAF cell cultures. Briefly, 200μ1 of

CellTiter-Glo® Reagent was added directly to cells, resulting in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. Luminescent intensity was measured using a Victor X3 spectrophotometer (Perkin Elmer cat# 2030-0030) after lOmin incubation at room temperature and 2min shaking at 600rpm on an orbital shaker (IKA, MTS 2/4 digital microtiter shaker, cat # 0003208001).

Automated Library Preparation and RNA Sequencing

A total of 50ng of RNA was utilized as input for RNA-Seq library preparation utilizing the TruSeq Stranded RNA Access Library Prep Kit (cat# RS-301-2001). The method was automated on the Biomek® FXP Laboratory Automation Workstation (Beckman Coulter). This method facilitates enrichment of the coding regions of the transcriptome that are captured using sequence-specific probes to create the final library. cDNA libraries were quantified utilizing the

Quanti-iT PicoGreen assay (Life Technologies - cat# P7589). 1 μΐ of cDNA was required for quantification. Concentration is measured as ng/ul. Libraries were excited at 480 nm and the fluorescence emission intensity was measured at 520 nm using a Victor X3 spectrophotometer

(Perkin Elmer cat# 2030-0030). Fluorescence intensity was plotted versus concentration over the low calibration range, O-SOng/μΙ. Libraries were also quality checked by Agilent Bioanalyzer using the High Sensitivity DNA kit (cat# 5067-4626). cDNA libraries were then sequenced on the Dlumina NextSeq500 platform as 75bp paired end reads. Data was streamed in real-time to the Illumina BaseSpace cloud tool. The STAR RNA sequencing alignment tool (Spliced Transcripts Alignment to a Reference) aligner [STAR_2.5.0a]) was utilized to align the data to the genome, Homo sapiens UCSC hgl9 (RefSeq gene annotations). DeSeq2 (Love et al., Genome Biol, 15:550 (2014)) was utilized to perform differential expression analysis (±2-fold, P value<0.05). Overlaps between the DE gene set and the annotated Hallmark gene sets in the Molecular Signature DataBase, MSigDB (FDR q-value<0.05, Subramanian et al., Proc Natl Acad Sci USA, 102: 15545-50 (2005)), were computed. Gene Cluster 3.0 was utilized to perform hierarchical clustering and subsequent plotting of the resulting heatmap was completed in Java Tree View.

Immunohi stochemi stry

A portion of each primary tissue was formalin-fixed and paraffin-embedded (FFPE) to confirm histological features and assess hemorrhagic / necrotic areas. Single

immunohistochemical staining for Chromogranin A, CgA, and Vimentin, Vim, was performed on 4μπι sections cut from primary tissue FFPE blocks. Immunostaining was performed on tissue sections following deparaffinization in for 5 min in xylene and rehydration through graded alcohols to distilled water. After blocking endogenous peroxidase activity, sections were subjected to heat-induced epitope retrieval in citrate buffer (pH 6.1) for 5 min. Following heat- induced epitope retrieval, the primary mouse monoclonal antibody for Chromogranin A (Ms mAb, Clone LK2H10, MS-324-PABX, NeoMarkers) and the primary mouse monoclonal antibody Vim (Ms mAb, Clone V9, MU074-UC, Biogenex) was applied to the sections. Protein levels were examined using a dilution of 1 :4000 for CgA and 1 :500 for Vim respectively for 1 hour at room temperature. Incubation with the biotinylated universal secondary antibody was then performed. Visualization was performed with 3,3'-diaminobenzidine (DAB) as the chromogenic substrate.

Exclusion Criteria

In experiments, 61 target proteins were measured across 3 multiplex assays utilizing a Bioplex platform (See FIG. 18A- FIG. 18C as a referece), including IL-lb, IL-lra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, FGF Basic, G-CSF, GM- CSF, IFNg, IP-10, MCP-1 (MCAF), MIP-la, PDGF-bb, MIP-lb, RANTES, TNFa, VEGF, sEGFR, Folastatin, sHER2 neu, HGF, sIL-6Ra, Leptin, Osteopontin, PDGF-AA BB, PEC AMI, Prolactin, SCF, sTIE-2, sVEGFRl, sVEGFR2, Angiopoietin, sCD40L, EGF, Endoglin, sFASL, HB-EGF, IGFBP-1, IL-18, PAI-1, PLGF, TGFa, uPA, VEGF-A, VEGF-C and VEGF-D. Target proteins with a concentration below the lower limit of quantitation (LLOQ) or which were detected in less than 75% of the supernatants were eliminated from the data set. Secretion levels were assessed as being low (<100pg/ml), moderate (100-lOOOpg/ml) and high (>1000pg/ml) expressing. To account for increasing levels of secretion associated with cell growth all protein concentrations were normalized to total protein levels in the cell lysate from which the supernatant was collected. These normalized levels of secreted protein were utilized for all subsequent analysis.

The results of the experiments described above are presented below.

Basic characterization of SINET tissue and short-term NAF cultures

A portion of each primary SINET tumor was fixed and embedded in paraffin for histopathological confirmation of the presence of tumor cells by H&E and IHC (FIG. 2A).

SINET cells stain positively for Chromogranin A, CgA, a marker of neuroendocrine phenotype (FIG. 2B). NAFs stain positively for Vimentin, a mesenchymal marker in the stroma indicative of an activated fibroblastic phenotype (FIG. 2C).

The short-term cultures comprise mixed tumor/NAF cultures of varying tumor composition (FIG. 2D), and pure NAF cultures, where tumor cells stain for CgA (FIG. 2E) and NAFs for VEVI (FIG. 2F). SINET patient-derived xenografts (PDX) in mice remain viable for months; however, their growth rate is almost negligible, which means that even though they replicate the tumor biology, they are unsuitable as surrogate in vivo models to study the disease biology. Basic histological characterization of PDX' s generated herein revealed morphology that is almost identical to the primary tumors (FIG. 2G). In the PDX models, it was identified that SFNET cells grow in close association with fibroblasts (termed NAFs), and that the fibroblasts from the original tumor specimen are preserved (FIG. 2G) and grow in association with tumors in implanted xenografts. In particular, it was noteworthy that the NAF cells in the xenograft models where of human and not host origin (FIG. 2H and FIG. 21). These

observations suggest that tumor-stromal interactions play a critical, underappreciated role in driving and maintaining neuroendocrine tumor growth.

Stromal cells form a significant component of the xenografts. This is consistent with observations in primary tumors, and suggests that tumor-stromal interactions may play a key role in SFNET growth, particularly in light of the relatively silent mutational landscape of these tumors (Francis et al., 2013 Nat Genet., 45(12): 1483-6; Jiao Y et al., 2011 Science, 331(6021): 1199-203). With the development of this resource of short-term primary cell lines and control normal cultured fibroblast cell lines, teasing apart the tumor-stroma relationship is feasible via collection of cell culture supernatants and in vitro screening of secreted proteins. A single protein is insufficient to fully reflect this complex relationship. Accordingly, it was envisaged that a panel of secreted proteins would be more informative. Because the commonly used single ELISA assays in standardized 96-well plates are cumbersome and both sample exhaustive and labor-intensive, a bead-based multiplexing approach was utlized to screen for potential biomarkers of metastatic SINET disease. It is increasingly evident that biomarker signatures have a higher probability of being prognostic or predictive than any single biomarker on its own.

Establishment of cancer-associated fibroblast cultures from SINET tissue

The basic histopathology of SINETs comprise islands of neuroendocrine tumors cells (FIG. 2A), which stain positively for chromogranin A, CgA, the neuroendocrine tumor-specific markers (FIG. 2B), with surrounding dense regions of stroma that stain positively for the fibroblast-specific marker, Vimentin, VEVI (FIG. 2C). A portion of each tumor was

disaggregated in culture. Experiments allowed for in vitro observation that SINET cells grow in clusters and appear to adhere to a scaffold of adjacent fibroblasts (FIG. 2D). In culture tumor cells were quickly overgrown by fibroblasts and the prohibitive rate of tumor to fibroblast cell growth resulted in loss of tumor cells completely from the culture within several passages. As in the primary tumor, the neuroendocrine tumor cells stain for CgA (FIG. 2E) and fibroblasts for VEVI (FIG. 2F). Experiments created a total of 20 CAF cultures, and then utilized these patient- derived CAF cultures for subsequent studies. Table 4 displays secretion levels as measured by multiplex assays for 24 healthy donors versus 16 metastatic SINET patients. Differences in expression were considered significant, where p-value < 0.05. For plasma analysis, log₂(fold change) differences between the metastatic patients and healthy controls were reported and two- tailed student t-tests were performed, where p-values <0.05 were considered significant. Table 4: Secretion levels as measured by multiplex assays for 24 healthy donors versus 16 metastatic SINET patients

In-vitro NAFs/CAFs have a distinct secretion profile relative to normal human fibroblasts

First the secreted protein profiles of CAFs were examined, and it was determined whether these profiles differed from normal human fibroblasts. A total of 61 target proteins were measured across 3 multiplex assays utilizing a Bioplex platform (FIG. 4A), including IL-lb, IL- lra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, FGF Basic, G-CSF, GM-CSF, IFNg, IP-10, MCP-1 (MNAF), MIP-la, PDGF-bb, MIP-lb, RANTES, TNFa, VEGF, sEGFR, Folastatin, sHER2 neu, HGF, sIL-6Ra, Leptin, Osteopontin, PDGF-AA BB, PEC AMI, Prolactin, SCF, sTIE-2, sVEGFRl, sVEGFR2, Angiopoietin, sCD40L, EGF, Endoglin, sFASL, HB-EGF, IGFBP-1, IL-18, PAI-1, PLGF, TGFa, uPA, VEGF- A, VEGF-C and VEGF-D. Target proteins with a concentration below the lower limit of quantitation

(LLOQ) or which were detected in less than 75% of the supematants were eliminated from the data set (FIG. 4B), resulting in a total of 47 secreted proteins of interest: Angiopoietin, EGF, Endoglin, Eotaxin, FGF Basic, Follistatin, GM-CSF, HB-EGF, HGF, IFNg, IGFBP-1, IL-10, IL- 12 (p70), IL-13, IL-18, IL-lra, IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IP-10, Leptin, MCP-1 (MCAF), MIP-lb, Osteopontin, PAI-1, PDGF-AA BB, PDGF-bb, PLGF, RANTES, sCD40L, SCF, sEGFR, sFASL, sHER2 neu, sIL-6Ra, sTIE-2, sVEGFRl, sVEGFR2, TGFa, uPA, VEGF, VEGF-A, VEGF-C and VEGF-D. Secretion levels were assessed as being low (<100pg/ml), moderate (100-lOOOpg/ml) and high (>1000pg/ml) expressing, the distribution for each time point is shown by pie chart (FIG. 4C), where increasing expression of secreted proteins are observed over the time course 24-72hr, resulting in a doubling of the number of high expressing proteins being detected after 72hr. To account for increasing levels of secretion associated with cell growth all protein concentrations were normalized to total protein levels in the cell lysate from which the supernatant was collected. These normalized levels of secreted protein were utilized for all subsequent analysis.

Within the NAF cohort, the secretion levels varied widely. This underscores the biological diversity of these well differentiated tumors and the complexity of secretion feedback loops within the tumor microenvironment with respect to the kinetics of activation and inactivation and mechanistic process by which these proteins are shed or secreted from NAF's. NAF supematants were compared to supematants collected from 2 normal human fibroblast cell lines, BJ and HFF1 (NHF's). Mean expression levels for NAF versus NHF were utilized to calculate fold changes for each target protein for each time point and trends were evaluated across the time course, 24 - 72hr. Multiplex bead-based evaluation of a panel of cytokines, chemokines, hormones and growth factors in culture supernatants collected at 24hr, 48hr and 72hr time points from the patient-derived CAF cells grown in vitro were performed. Fold changes were log₂ transformed and plotted versus time point. Hierarchical clustering of the secreted proteins versus time point, are represented by the heatmap shown in FIG. 5 A. Fold changes that were greater than ±1.5 fold and/or where the differential expression levels were sustained over the entire time course, where noted. At the mean expression level, either sustained trends of higher secretion levels over the entire time course or increasing secretion over the time course were observed for the majority of secreted proteins evaluated in the CAFs relative to NHFs. For example, a group of 16 proteins had sustained higher secretion levels over the time course of 72hr. However, when evaluating the individual tumors, it is evident that not all SINET samples secrete similar profiles. Among the secreted proteins that were profiled, IL-6, MCP-1, VEGF, IL-12, IFNg, IL-lra and IL-10 (FIG. 5B-FIG. 5E and FIG. 5H- FIG. 5 J), showed marked trends of overexpression (>2-fold induction of the mean expression level) in the CAF's relative to the NHFs. Representative bar charts of NAF's and NHF's plotting secreted levels of over- expressing uPA, IL-6, MCP-1 and VEGF are shown in FIG. 5B-FIG. 5E. IL-6, MCP-1, IL-12 and VEGF, along with IL-2 and IL-13 share a more common pattern across the majority of NAF's showing substantially higher secretion levels than the NHF's. Inversely, there were several protein secretion levels that were suppressed in CAFs/NAF's relative to the NHF's, such as SCF and sEGFR (>1.5-fold suppression of the mean expression level, FIG. 5F-FIG. 5G). FIG. 5F is a graph of SCF concentration for CAF and NHG, and FIG. 5G is a graph of sEGFR concentration in CAF and NHG further supporting the aforementioned results.

Secreted proteins measured in plasma from matched SINET patients with metastatic disease correlate with the short-term NAF supernatants

40 plasma samples (16 matched tumors and 24 healthy controls) were screened for the 61 proteins across the 3 multiplex assays as described for the cell culture supernatants. 59/61 proteins were measured in >75% of both the healthy donor and metastatic patient groups. The results of which are shown in Table 5, where mean expression, standard deviation, lower and upper confidence levels are provided for the control and CAR groups. The control and tumor groups were compared and statistical significance was determined to be a p-value < 0.05. 35 targets, i.e., sEGFR, IL-lb, Angiopoietin, IL-5, IFNg, sVEGFR2, SCF, MIP-la, sHER2 neu, IL-lra, MCP-1 (MCAF), IL-6, VEGF-A, IL-4, Endoglin, IL-17, IL-2, IL-7, IL- 12(p70), IL-10, VEGF-D, IL-9, PLGF, Leptin, VEGF-C, sFASL, HB-EGF, Follistatin, sIL-6Ra, TNFa, sCD40L, HGF, EGF, TGFa and IGFBP-1, across the 3 panels were differentially expressed between the control and tumor group, which suggest that there is a diagnostic, prognostic, and/or predictive association of clinical utility.

Table 5: Secretion levels as measured by multiplex assay for 24 healthy donors versus 16 metastatic patients. Differences in expression were considered significant, where p-value < 0.05.

The CAF secretion signature is recapitulated in plasma from matched SINET patients with metastatic disease

To validate the findings in vivo, evaluation of whether the CAF secreted protein signatures identified in vitro would also be present in the plasma of the matched patients was performed. Plasma samples from the same 16 patients from whom the cultures had been derived were compared to 24 control samples derived from spousal and friend controls. Using the same assay utilized to evaluate the CAF supernatants, it was found that in the patient plasma samples IL-6, MCP-1, VEGF, IL-12, IFNg, IL-lra and IL-10 (FIG. 6 A- FIG. 6B and FIG. 6E- FIG. 61) were over-expressed and SCF (FIG. 6D) and sEGFR (FIG. 6C) were suppressed in plasma derived from the SINET patients relative to the normal controls, mirroring the secretion patterns observed in the CAFs relative to the NHFs. Specifically, this result mimicked the short-term culture supernatant findings (FIG. 5B, FIG. 5D, FIG. 5F and FIG. 5G). Global comparison of the mean log2[mets/ctrl] in the metastatic plasma samples to the supernatant results found high concordance (FIG. 6J). Comparing the global log₂ transformed fold differences in expression in the metastatic plasma samples relative to the normal controls, to those in the CAF's/NAF's relative to the NHF's, resulted in 75% concordance of the secreted proteins in terms of trend observed, as shown in FIG. 6J. The correlation is shown in FIG. 6K as a scatterplot, where the Pearson correlation coefficient was calculated to be 0.5427 (P<0.001).

Genes expressed in CAFs harbor characteristics of an aggressive tumor-associated stromal cell phenotype

Prior to the invention described herein, no single marker of tumor-associated stromal cells has been identified, rather a combination of a panel of markers aid CAF characterization. Studies found that, consistent with the IHC results (FIG. 12), CAR24 and CAR26, two representative CAFs, did not express CgA but strongly expressed VEVI. Tumor-associated stromal cells do not have epithelial or endothelial characteristics and therefore do not express cell surface markers such as CD34, CD31 (PECAM1) and MUC16, as well as cytokeratins, such as KRT5, KRT6 and others, which is observed replicated in the CAF gene expression data

(FIG. 1 IE, Table 6). In contrast, aSmooth muscle actin (ACTA2) and fibroblast activation protein (FAP), along with a panel of general markers of tumor-associated stromal cells, including tenascin-C (TNC), Periostin (POSTN), Neuron-glial antigen-2 (NRG2) and platelet-derived growth factor receptors (PDGFRA,-B) were highly expressed in CAFs. E-cadherin (CDH1) expression was very low in the CAFs, a feature known to be associated with tumor invasiveness and transformation via epithelial mesenchymal transition (EMT). EMT is also regulated by exogenous stimulating factors released by the microenvironment including Matrix

Metallopeptidase (MMP-1,-2), integrin (ITGA-1,-2,-3,-5,-7,-11) and TGF-β (-1,-induced) factors, which were found to be highly expressed in the representative CAFs, CAR24 and CAR26.

Table 6: Genes that aid characterization of stromal -associated stromal fibroblasts

NAF/CAFs short term cultures are highly sensitive to inhibition of the NFkB pathway

From the initial NAF screening, a set of secreted proteins including, IL-6, MCP-1, VEGF, IL-2, IL-12 and IL-13 that were upregulated in the supernatants was identified. Since all of these proteins are related to NFKB signaling, the NAF short term cultures were treated with a small molecule inhibitor, Withaferin A (WFA) that potently prevents NFKB activation by inhibiting activation of ΙΚΚβ, displaying anti -inflammatory, antitumor and anti angiogenic activity. Withaferin A is also known to bind directly to vimentin and disrupts the cytoskeleton (Stevens et at., 2013 Gut, 62(5):695-707; Bargagna-Mohan et al., 2015 PLoS One,

10(7):e0133399).

CAR24 and CAR26 were selected as representative NAF cultures. Since it was anticipated that NAF's would be sensitive to NFKB inactivation, it was determined to treat CAR24 and CAR26 for 24hr with Withaferin A (WFA), a potent inhibitor of NFKB activation, over the concentration range, 10 - ΙΟΟΟηΜ (10, 25, 50, 100, 250, 500 and ΙΟΟΟηΜ), to establish a dose curve response and evaluate dysregulation of the secreted protein profile.

Secretion profiling fold changes between the WFA treated supernatants and those treated with dimethyl sulfoxide (DMSO) as the vehicle were converted to log₂(Fold Change) for generating the heatmap as shown in FIG. 7 A. A dose dependent suppression of the pro- oncogenic secretion profile (approximately 30 of the soluble proteins measured) was observed in the CAFs/NAFs treated with increasing concentrations of WFA (FIG. 17 A) over the

concentration range, 50-1000nM. The WFA-treated NAF secretion profiles for CAR24 and CAR26 were compared at the 50uM dose resulting in a Pearson correlation coefficient of 0.7217 (P<0.0001). This suggests that even though secretion profiles vary across NAFs derived from SINET patient tumors as demonstrated in FIG. 7A-FIG. 7G, NFkfi inactivation with a 50nM concentration of WFA induces a common suppression signature of secreted proteins as measured in supernatants derived from treated short-term NAF cultures. Inversely, sEGFR was induced in the NAFs in response to increasing concentration of WFA, which mirrors the statistically significant higher plasma concentration of sEGFR observed in controls (FIG. 6C, P=0.002) and the observation that normal human fibroblasts secrete larger amounts of sEGFR with respect to NAFs derived from SINET tumors (FIG. 5G).

Cell viability decreased in a dose dependent manner (FIG. 7B), where both CAR24 and CAR26 NAF were sensitive to WFA inhibition with greater than 70% cell death observed at 24hr post-treatment with a 250nM single dose. The suppression of secreted proteins associated with NFkfi signaling closely trends with the decrease in cell viability with increasing WFA concentration.

In parallel, mRNA was extracted to assess transcriptional regulation of VEGF-A, MCP-1 and IL-6 in response to a single potent dose of lOOuM WFA treatment, as shown in FIG. 7C. Fold change due to WFA treatment was plotted as

The mRNA expression of

VEGF-A, IL-6 and MCP-1 in CAR24 NAF was reduced 1.45-fold, 16.82-fold and 36.92-fold, respectively, due to treatment with lOOnM WFA for 24hr. The mRNA expression of VEGF-A, IL-6 and MCP-1 in CAR26 NAF was reduced 2.18-fold, 9.01-fold and 3.32-fold, respectively, due to treatment with lOOnM WFA for 24hr. The transcriptional downregulation of mRNA levels of VEGF-A, IL-6 and MCP-1 in response to NFkfi inactivation by lOOnM WFA treatment (FIG. 7C) was observed and mirrored the suppression observed in the supernatant secretion levels measured for VEGF-A, IL-6 and MCP-1 (2.57-fold, 6.99-fold and 4.95-fold for CAR24; 1.72-fold, 4.13-fold and 1.57-fold for CAR26), respectively. In FIG. 7C, fold change due to WFA treatment was plotted as Overall, trends show that these 3 genes were

transcriptionally downregulated and correlate with the suppression of VEGF-a, IL-6 and MCP-1 secretion levels in response to inhibition of FKB activation by WFA.

Transcriptional changes in response to NFKB inactivation results in blockade of EMT

Whole transcriptome sequencing on the representative CAFs, CAR24 and CAR26 evaluated transcriptional differences in gene expression in response to a single lOOnM dose of WFA. RNA-Seq was utilized for transcriptional profiling on total RNA extracted from both CAR24 and CAR26 NAFs treated with lOOnM WFA or DMSO as the control. In total, 3825 genes were differentially expressed between the WFA treated CAFs and the untreated controls (AdjP<0.05, log₂FC±0.6). A total of 156 genes (Table 7) in the NAFs showed a log₂(FC) of 3.5 increase or reduction compared with the control, as shown in the Volcano plot (FIG. 11 A). Of these, 106 genes had a biological significance of log₂(FC) of ±3.5, as shown in the Volcano plot (FIG. 1 IE, full DE genes list, see Table 8). Gene sets were evaluated using the MSigDB tool (software.broadinstitute.org/gsea/msigdb/annotate.jsp) to determine which were most associated with the differentially expressed genes. Separating the differentially expressed genes into 2 groups; those that were upregulated (n=1832) and those that were downregulated (n=1993)„ the top 10 Hallmark gene sets for each (FIG. 1 IB/FIG. 11G and FIG. 1 IC/FIG. 11H, respectively, full enrichment results, see Table 9 and Table 10) were identified. The upregulated genes were enriched for MTOR signaling, a pathway known to be active in NET biology and hypoxia pathways, a stress response typical of the TME. The downregulated genes were predominantly enriched for proliferation-related gene sets, mostly cell cycle-related and notably EMT

(FIG. 1 IC/FIG. 11H). NFkB-related signaling was enriched in both groups, indicated as the blue bar with black border on both plots.

Specifically, the 10 hallmark gene sets include a NET specific marker set: CGA, a cell surface speithelial specific markers set: CD34, PECAMl, MUC16, KRT5, KRT6A, KRT6B, and KRT17, a stimulants of malignant cell proliferation set: EGFR, FGF-2, HGF, and CXCL12, an epithelial-mesenchymal transition set: CDH1, CDH2, MMP1, MMP2, ITGA1, ITGA2, ITGA3 ITGA5, ITGA7, ITGA11, TGFB1, and TGFBI, a metastatic transition set: YAPl, HAS2, LOX, S100A4, GSTP1, and CD81, a mesenchymal-like set: THY1, NT5E, ENG, and CD44; a matrix- remodeling set: FAP, a hypoxia and angiogenesis set: VEGFA, VEGFC, HTFIA, and CAV1, a tumor-associated stromal cells and myofibroblasts set: PDGFRA, PDGFRB, NRGl, TNC, POSTN, and ACTA2, and a stromal-specific set: P4HA1, P4HB, and VFM. Unsurprisingly, NFkB signaling was identified in both groups as being one of the most statistically significant enriched data sets, indicated as white shaded bar on both plots. Next, the MSigDB Hallmark set of genes regulated by NF-kB in response to TNF in CAR24 and CAR26 NAFs data [HALLM ARK TNF A S IGN ALING VI A NFKB - M5890] was explored. Genes comprising the EMT signature were selected and plotted as a heatmap (FIG. 11I/FIG. 1 ID), evaluating transcriptional changes pre- and post-treatment with Withaferin. In FIG. 1 ID, the heatmap representing supervised hierarchical clustering shows clear distinction between the treated and untreated NAFs. The saturation of either color (scale from green to red) reflects the magnitude of the difference on gene expression level. As noted above, VEGF-A and IL-6, were downregulated post-treatment with lOOnM WFA, consistent with the secretion profiling and qRT-PCR results (FIG. 7A and FIG. 7C). Many other genes in the EMT signature are known to characterize tumor-associated stromal fibroblasts (FIG. HE), including NT5E, CXCL12, POSTN, PDGFRB, LOX, ACTA2, FGF2, ITGA2, VEGFC, CHD2 and TNC. These were also downregulated in response to WFA treatment, consistent with a role of NFkB pathway activation in inducing EMT in the CAFs.

Table 7: 156 Genes in the NAFs analyzed by RNA-Seq

Table 8: 106 differentially expressed genes associated with WFA treatment on CAR24 and CAR26, where biological and statistical significance thresholds were log2FC+/-3.5 and

AdjP<0.05, respectivel

Table 9: Hallmark enrichment gent set analysis associated with upregulated genes in the WFA- treated CAFs using MSigDB tool

Table 10: Hallmark enrichment gent set analysis associated with downreeulated genes in the WFA-treated CAFs using MSigDB tool

Differential gene expression analysis

The focus on NAF's was initially influenced by the fact that SINET patient-derived xenografts (PDX) do not grow. During initial histological characterization of the PDX tissue, the NAF cells were observed to be of human and not host origin, which led to full consideration of the importance and role of fibroblasts in neuroendocrine tumor biology. As described herein, short-term NAF cultures were developed to study secretion patterns that differentiated these activated fibroblasts from quiescent normal human fibroblasts. An enhanced secretory signature comprised of induced IL-6, IL-10, VEGF and MCP-1 was identified. HGF, uPA and RANTES were also found to be enhanced in the NAFs. These 3 secreted proteins coupled with positive vimentin expression have been implicated as markers of an activated fibroblast phenotype (Kalluri R., 2016 Nat Rev Cancer, 16:582-98).

In 2011, Hanahan and Weinberg included tumor-promoting inflammation and avoidance of immune destruction in their reassessment of the hallmarks of cancer (Hanahan D, Weinberg RA., 2011 Cell., 144: 646-674). Hanahan and Weinberg described the inadvertent corruption of the innate immune cell inflammatory response being consequential in promoting tumor growth. IL-6, IL-10, and MCP-1 are key components of a CAF-mediated pro-inflammatory signature (Harper J and Sainson RCA, 2014 Semin Cancer Biol, 25:69-77). MCP-1 is a well -characterized chemokine involved in attracting macrophages into the tumor microenvironment driving invasion and metastasis (Roca H et al. 2009 J Biol Chem, 284:34342-54). MCP-1 has also been shown to be involved in the recruitment of macrophages and monocytes to the site of

inflammation IL-6, IL-10 VEGF (angiogenesis).

Crosstalk between various cell components within the tumor microenvironment facilitates the development of an activated fibroblastic phenotype. Under these conditions, an NFkB-mediated release of chemokines and cytokines into the stromal environment is initated. Plasma matched studies and potential

Additionally, mechanistic elucidation of the corresponding signaling pathways involved in neuroendocrine tumor microenvironments and functional characterization of these

mechanisms could lead to a more targeted approach to current patient treatment options. Current trends indicate that a more thorough investigation of the tumor microenvironment results in more sophisticated combinatorial drug approaches. As described herein, it is determined whether combining tumor targeting drugs in conjunction with repressing cytokine signaling in the stromal environment provides the narrative for a new paradigm in the treatment of advanced small intestinal neuroendocrine tumors.

The identification and validation of biomarkers for neuroendocrine tumors enhances the use of currently available therapies and provides a path to identify therapeutic targets.

Targeting NFkB suppresses secretion and gene expression signatures in cancer-associated fibroblasts derived from metastatic small intestine neuroendocrine tumors

The focus on cancer-associated fibroblasts was based on observations that SINET patient-derived xenografts do not grow in vivo, coupled with the lack of tumor cells and the prevalence of fibroblast growth in vitro. CAFs have been established as playing a prominent role in cancer progression and metastasis, as well in tumor-stroma crosstalk (Huang et al., World J Gastroenterol, 20: 17804-17818 (2014)). This study demonstrates for the first time that CAFs from SINETS are capable of being independently cultured and have pro-oncogenic properties as measured by both secretion profiling and transcriptional expression. Studies further demonstrate that these properties are inhibited with Withaferin A, a potent inhibitor of the NFkB pathway.

In other malignancies, CAFs have been shown to contribute to tumor proliferation, invasion, and metastasis via secretion of growth factors, cytokines, chemokines and degradation of extracellular matrix (ECM) proteins (Yamamura et al., Cancer Res, 75:813-823 (2015), Ben- Baruch A, Cancer Met. Rev, 25:357-371 (2006). Observations that IL-6, MCP-1 and VEGF were upregulated in the CAFs relative to normal human fibroblast, and moreover that these proteins were also identified in the plasma of patients compared to normal controls, is consistent with the hypothesis that CAFs may play a key role in promoting the growth of SINETS. MCP-1 is involved in attracting macrophages into the tumor microenvironment driving invasion and metastasis (Roca et al., J Biol Chem, 284:34342-54 (2009)) and also has been shown to recruiting the macrophages and monocytes to sites of inflammation (Roca et al., J Biol Chem, 284:34342-54 (2009)). Observation that CAFs in SINETS also secrete VEGF is consistent with an established role CAFs in other settings in modulating angiogenesis to expand the tumor mass and facilitate metastasis (Vong S and Kalluri R, Genes Cancer 2: 1139-1145 (2011)). VEGF is secreted by many cancers and is associated with poor outcome, such as, gastric and esophageal cancers (Salvatore et al., Oncotarget, 8:9608-9616 (2017); Chen et al., Tumour Biol, 35:2513- 2519 (2014); Kozlowski et al., Adv Med Sci, 58:227-234 (2013)). VEGF correlated with increased angiogenesis and decreased progression-free survival among patients with low-grade NETs (Zhang et al., Cancer, 190: 1478-1486 (2007)). VEGF secretion by CAFs has, in turn, been shown to be induced by IL-6 (Adachi et al., Int J Cancer, 119: 1303-11(2006); Zhu et al., World J Gastroenterol, 17:2315-2325 (2011), Nagasaki et al., Br J Cancer, 110:469-478 (2014)). IL-6 is a multifunctional cytokine integral to inflammation and immune regulatory responses that is known to promote migration and EMT in the TME (Osuala et al., BMC Cancer, 15:584 (2015); Giannoni et al., Cancer Res, 70:6945-6956 (2010)). High IL-6 expression in many cancers, such as bladder cancer, cervical cancer and NSCLC has been associated with a worse outcome (Chen et al., PLoS ONE, 8:e61901 (2013); Song et al., Inter Med J Exp Clin Res, 22:4475-4481 (2016); Silva et al., PLoS One, 12:e0181125 (2017)).

Observations are further suggestive that CAFs in SINETs are driven by activation of the NFkB pathway. This observation is consistent with studies in other malignancies that CAFs are activated by tumor and immune-cell derived factors in the early stages of tumorigenesis to express pro-inflammatory genes such as IL-6, in an NFKB-dependent manner (Subramanian et al., Proc Natl Acad Sci USA, 102: 15545-50 (2005)), Kozlowski et al., Adv Med Sci, 58:227-234 (2013); Bhowmick et al., Nature, 432:332-337 (2004)). Along with IL-6, MCP-1 and VEGF, RANTES, IL-2, IFNg, IL-2, IL-10, IL-12 and IL-13 were found to have higher secretion levels in the CAFs relative to normal human fibroblasts. These secreted proteins form part of the cytokine signaling that is associated with the NFkB-dependent inflammatory response in the TME (Katanov et al., Stem Cell & Ther, 6:87 (2015); Schauer et al., Neoplasia, 15:409-420 (2013)). CAFs were treated with Withaferin A, an agent that prevents NFkB activation by inhibiting activation of ΙΚΚβ (Kaileh et al., J Biol Chem, 282:4253-64 (2007)). WFA also had a potent effect on cell viability at low concentrations (FIG. 4B) and suppressed mRNA expression of IL-6, MCP-1 and VEGF.

A role for NFkB signaling in SINET CAFs is further supported by the Enrichment analysis, which revealed that WFA treatment suppressed transcription of genes associated with MTOR signaling, a pathway known to be activated in NET, and EMT, a process related to NFkB signaling and a more invasive tumor microenvironment. EMT is co-opted by tumor cells to facilitate an invasive-metastatic cascade that relies on the plasticity of the stromal fibroblasts in the microenvironment (Wu et al., Oncotarget, 8:20741-20750 (2017)).

As part of this study, patient-derived cancer-associated fibroblast cultures were developed and characterized. CAFs harbor a signature that is different from normal human fibroblasts and are shown to express genes that characterize an aggressive tumor-associated stromal fibroblast phenotype. CAFs exhibit a pro-malignant secretion profile that is distinct from normal human fibroblasts, where IL-6, MCP-1 and VEGF are upregulated. SINET associated CAFs are further characterized by a gene expression profile that reflects an aggressive tumor- associated stromal fibroblast phenotype associated with EMT. Treatment with Withaferin A, resulted in downregulation of secreted IL-6, MCP-1 and VEGF, and suppressed transcription of genes associated with EMT. Further studies evaluating the therapeutic potential of targeting the tumor microenvironment in SF ETS are warranted.

Example 3 : Development of an Ex-Vivo Model of Neuroendocrine Tumors and Exploration of Therapeutics

Prior to the invention described herein, a lack of cell lines and animal models of neuroendocrine tumors has been a challenge in the development of new therapeutic agents for this disease. As described herein, in the past two years, a program has been initiated to develop such models. Initial attempts have focused on the development of short-term cell lines and xenografts. Over 50 fresh tumor specimens have been collected from consented patients undergoing surgical resection of neuroendocrine tumors at Brigham and Women's Hospital. In an unexpected observation, it was identified that carcinoid cells grow in close association with fibroblasts (termed CAFs), and that carcinoid associated fibroblasts from the original tumor specimen are preserved and grow in association with carcinoid tumors in implanted xenografts (FIG. 8). These findings suggest that tumor-stromal interactions may play a critical role in driving neuroendocrine tumor growth.

The Center for Molecular Pathology at Dana-Farber Cancer Institute has recently developed an ex -vivo tissue slice model appropriate for evaluating the biological effects of therapeutics on specific malignancies (Vairo et al, 2010 PNAS, 107: 8352-8356, incorporated herein by reference). The ability to rapidly section and culture tissue from specimens immediately after surgical excision allows for the preservation of morphological tissue integrity, including general epithelial architecture, which can be retained up to 72-96 hours. This technique has been widely used in a range of tumor types at Dana-Farber since the initially reported by the Dana-Farber group in 2010. Representative organotypic human prostate tissue culture H&E and immunohistochemical (IHC) images are shown in FIG. 9. The preservation of tissue antigenicity is demonstrated through AMACR IHC. Mitotic activity is preserved through 72 hours in this experiment.

As described herein, this technique is extended to neuroendocrine tumors.

Approximately one neuroendocrine tumor surgical specimens are collected per month, through an IRB approved protocol. As described in detail below, ex -vivo organotypic neuroendocrine tumor tissue slice cultures are be established. At baseline (TO), cultures are processed for histology and IHC staining, while additional PCa tissue slice cultures are treated with therapeutic agents of interest.

Study of neuroendocrine tumor tissue slices

First, the feasibility of using NET slices for evaluation of therapeutics is assessed and conditions are optimized for these experiments.

As an initial proof of principle, the effect of Rapamycin (100 nM, demonstrated effective in NIH/3T3 cells and known to be active in neuroendocrine tumors) is evaluated on tissue slices. For each of the treatments, two neuroendocrine tumor slices are used. Each culture is subjected to drug treatment and corresponding vehicle control on alternate slices, harvested 48 or 72 hours and fixed in FFPE. For each time point, pathology review is conducted using H&E slides of FFPE material to assess the morphological integrity of tissue samples and to evaluate

neuroendocrine tumor morphology. Incorporation of BrDU is evaluated to assess cell proliferation following exposure to rapamycin. Expression of downstream targets of mTOR is evaluated using IHC. Targets include normal and phosphorylated 4EBP1 and s6Kl .

Use of tissue slices to evaluate potential therapeutic effects of agents

Following optimization of the protocol above, tissue slices are utilized to explore the potential antitumor and antisecretory effects of therapeutic agents, in collaboration with Ipsen. Potential antitumor effects are evaluated using BrDU cell proliferation assays, together with measurement of drug-specific targets in treated and untreated tumor tissue. Potential antisecretory effects are evaluated by measuring levels of the validated secretory markers chromogranin A (CGA) and neuron specific enolase (NSE) in the supernatant of the tissue slices prior to and after treatment. Example 4: Patient-derived ex vivo tissue slice cultures as models to characterize treatment response in GI malignancies

Prior to the invention described herein, there was a great need for models that replicate the tumor environment and are predictive of in vivo efficacy in patients with neuroendocrine tumors. The use of tumor tissue slices that incorporate not only tumor cells, but also 3D tissue architecture and the tumor microenvironment, provides a new opportunity to identify drugs and treatment targets (Grosso et al., 2013 Cell Tissue Res., 352(3):671-84, incorporated herein by reference). As described herein, this technology is developed for neuroendocrine tumors, where prior to the invention described herein, a lack of cell lines and animal models has been a particularly acute problem in identifying new therapeutic agents. mTOR inhibitors are known to be active in neuroendocrine tumors, and as an initial proof of principle, the response to treatment in neuroendocrine tumors treated with the mTOR inhibitor rapamycin was evaluated.

Tissue Collection

To date 12 tumor specimens have been collected to conduct ex vivo tissue slice culture [xTSC] studies to investigate their response to MTOR inhibition. Patients are identified pre- operatively in clinic, and consented to the tissue banking protocol. Clinical information including demographics, diagnosis, staging, treatment and survival data is also collected.

xTSC

This is an organotypic culture method where fresh tissues are rapidly sectioned and cultured immediately after surgical excision. Morphological tissue integrity, including general epithelial architecture is preserved. As schematically represented in FIG. 10, briefly, tissues are mounted and cut into 300 μΜ thick slices on a vibrotome instrument. Single slices are placed in individual wells in a 6-well plate format and supplemented with with modified eagle's / F12 (50:50) medium supplemented with 10% FBS and antibiotics/antimycotics and Glutamax. Slices are treated with DMSO, as control or 100 nM rapamycin for 24hr. The xTSC supernatant is collected. Tissue is preserved in OCT and/or FFPE.

The histologic response to mTOR inhibitors in neuroendocrine tumors has been evaluated using a tissue slice model. Differentially expressed secreted proteins have been identified before and after mTOR inhibition. IL-6, IL-8, VEGF, MCP-1, Osteopontin, Angiopoietin-2, sTIE-2, uPA, PAI-1, Follistatin, PECAM-1 are strongly secreted from SINET tissue slices in culture for 24hr. The secretion profile from xTSC is not dissimilar from the NAF observations. Post- treatment with a single 100 nM dose of rapamycin, significant reduction in secretion of

Osteopontin, IL-6, VEGF, MCP-1, IL-10, MIP-la, MIP-lb, IP-10, IL-8 and uPA is observed. Library preparation and RNA-Seq on RNA isolated from sections derived from the tissue slice model have been completed to identify differentially expressed genes and pathways enriched for these differentially expressed genes in neuroendocrine tumors prior to and following MTOR inhibition.

These methods are utilized to evaluate drugs targeting the neuroendocrine tumors and neuroendocrine associated fibroblasts (NAFs).

Example 5: Protocol for NAF models and short-term culture generation

A protocol for NAF models and short-term culture generation is provided below.

1. A portion of each tissue sample is frozen in OCT and evaluated to confirm histological features and rule out the presence of significant hemorrhagic / necrotic areas, not suitable for experimentation.

2. Additional portions of each tissue are snap frozen and banked, and also processed for FFPE.

3. Remaining tissue is cut in to small pieces.

4. Add medium and collagenase II at a final concentration 2mg/ml.

5. Overnight incubation in 5% C0₂ at 37°C.

6. Mechanical disaggregation with 18-gauge needle and syringe.

7. Cells are filtered through a cell strainer, pore size 70 microns.

8. Cells are spun in centrifuge at lOOOrpm for 5min.

9. The supernatant is removed and cells are washed with 10ml lx PBS.

10. The cells are spun a second time, same centrifuge settings.

11. If the pellet is bloody the cells are hemolyzed using ACK lysis buffer.

12. Then, the cells are plated into 5-15cm tissue culture plates (polymer, not glass) with modified eagle's / F12 (50:50) medium supplemented with 10% FBS and

antibiotics/antimycotics and Glutamax. This particular culture is referred to as the "cells" population.

13. Remaining tissue pieces from the strainer are added to the dish, where collagenization took place. 14. This population is refered to as "cuts and pieces", which is supplemented with the same media as described above.

15. After 3 - 10 days of culture, when cells have attached and start to grow (varies from

tumor to tumor), a fraction of the cells are cryopreserved as PO and the rest is passaged and re-plated.

16. PO is the first short-term culture population that most resembles the cell composition of the tumor and includes both tumor and stromal cell populations.

17. Subsequently, cells are passage 2-4 times and cryopreserved each passage.

18. For each cryopreserved passage, the composition of tumor to stroma cells present is

annotated.

19. Pure NAF cultures are generated for all fresh tumors received. For the majority of

tumors, a mixed tumor/NAF culture is also generated.

20. Cells are seeded at 50k per well in 6-well plate format for 24hr. Then, the media is

replenished, and 24hr later cell culture supematants are collected.

21. Cell culture supematants are centrifuged at 1200rpm for lOmin (at RT) to remove debris and aliquoted in 65ul vials and stored at -80°C.

22. The remaining cells are then washed in PBS and scraped.

23. Cells are lyzed to collect total proteins extracted using the ELB lysis buffer with a

proteinase inhibitor.

Example 6: uPA secretion in a subset of short term NAF cultures

Additionally, in a subset of short term NAF cultures, uPA secretion was extremely high. Preliminary analysis was performed on an example NAF cell line, CAR15. Specifically, an invasion migration assay was performed in vitro in culture over a 5 day time period. CAR15 was treated with an anti-uPA inhibitor, which did not result in cell killing; however, migration of cells was impeded in the short-term cultures as compared with the untreated CAR15 culture. uPA secretion levels were not shut down, since no cell death occured. However, uPA is important for migration of NAFs. This study was repeated with an alternate compound also known to impair uPA activity, an anti-Survivin inhibitor. The same impeded migration was observed in the treated short term NAF culture. The results are presented in FIG. 12, which shows images of untreated CAR15 NAFs in vitro in 6-well plates at time Ohr, 72hr and Day 5 (top panel), of CAR15 NAF's treated with lOOuM uPA inhibitor or lOOnM Survivin at 72hrs and Day 5 (middle panel). Inset: uPA concentration [pg/ml] measured in cell culture supernatant from CAR15 treated with either uPA or survivin inhibitor. Gene expression knockdown and secretion levels relative to untreated control for CAR15 treated with either lOOnM of uPA or survivin inhibitor (bottom panel).

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and

modifications are within the scope of the following claims.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference.

Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

What is claimed is:

1. A method of determining whether a small bowel neuroendocrine tumor (SINET) in a subject will metastasize comprising:

obtaining a test sample from a subject having or at risk of developing SINET;

determining the expression level of at least one SINET-associated gene in the test sample;

comparing the expression level of the SINET-associated gene in the test sample with the expression level of the SINET-associated gene in a reference sample; and

determining that the SINET in the subject will metastasize if the expression level of the SINET-associated gene in the test sample is differentially expressed as compared to the level of the SINET-associated gene in the reference sample.

2. The method of claim 1, wherein the test sample is obtained from resected SINET tissue.

3. The method of claim 1, wherein the test sample is obtained from blood or plasma.

4. The method of claim 1, wherein said sample comprises circulating tumor cells.

5. The method of claim 1, wherein the reference sample is obtained from immortalized healthy normal control fibroblastic cell lines or cancerous SINET tissue.

6. The method of claim 1, wherein the reference sample is obtained from healthy normal tissue from the same individual as the test sample or one or more healthy normal tissues from different individuals.

7. The method of claim 1, wherein said sample comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

8. The method of claim 1, wherein the SINET-associated gene comprises interleukin-2 (IL-2), IL-6, IL-12, IL-13, vascular endothelial growth factor (VEGF), monocyte chemoattractant protein- 1 (MCP-1), urinary plasminogen activator (uPA), hepatocyte growth factor (HGF), RANTES (regulated on activation, normal T cell expressed and secreted, alternatively, chemokine (C-C motif) ligand 5; alternatively CCL5), and Eotaxin; and

determining that the SINET in the subject will metastasize if the expression level of the SF ET -associated gene in the test sample is higher than the level of the SINET-associated gene in the reference sample.

9. The method of claim 1, wherein the SINET-associated gene comprises stem cell factor (SCF), soluble epidermal growth factor receptor (sEGFR), granulocyte macrophage colony stimulating factor (GM-CSF), soluble vascular endothelial growth factor receptor 2 (sVEGFR2), and soluble interleukin 6 receptor, alpha (sIL-6RA); and

determining that the SINET in the subject will metastasize if the expression level of the SINET-associated gene in the test sample is lower than the level of the SINET-associated gene in the reference sample.

10. The method of claim 1, wherein the SINET-associated gene comprises a NET-specific gene set where the gene comprises CGA; and

determining that the SINET in the subject will metastasize if the expression level of the SINET-associated gene in the test sample is lower than the level of the SINET-associated gene in the reference sample.

11. The method of claim 1, wherein the SINET-associated gene comprises a cell surface epithelial-specific gene set selected from the group consisting of CD34, PEC AMI, MUC16, KRT5, KRT6A, KRT6B, and KRT17; and

determining that the SINET in the subject will metastasize if the expression level of the SINET-associated gene in the test sample is lower than the level of the SINET-associated gene in the reference sample.

12. The method of claim 1, wherein the SINET-associated gene comprises a stimulants of malignant cell proliferation gene set selected from the group consisting of EGFR, FGF-2, HGF, and CXCL12; and determining that the SINET in the subject will metastasize if the expression level of the SINET-associated gene in the test sample is higher than the level of the SINET-associated gene in the reference sample.

13. The method of claim 1, wherein the SINET-associated gene comprises an epithelial - mesenchymal transition gene set where the gene comprises CDH1; and

determining that the SINET in the subject will metastasize if the expression level of the SINET-associated gene in the test sample is lower than the level of the SINET-associated gene in the reference sample.

14. The method of claim 1, wherein the SINET-associated gene comprises an epithelial- mesenchymal transition gene set selected from the group consisting of CDH2, MMP1, MMP2, ITGA1, ITGA2, ITGA3 ITGA5, ITGA7, ITGA11, TGFB1, and TGFBI; and

determining that the SINET in the subject will metastasize if the expression level of the SINET-associated gene in the test sample is higher than the level of the SINET-associated gene in the reference sample.

15. The method of claim 1, wherein the SINET-associated gene comprises a metastatic transition gene set selected from the group consisting of YAP 1, HAS2, LOX, S100A4, GSTPl, and CD81; and

determining that the SINET in the subject will metastasize if the expression level of the SINET-associated gene in the test sample is higher than the level of the SINET-associated gene in the reference sample.

16. The method of claim 1, wherein the SINET-associated gene comprises a mesenchymal - like gene set selected from the group consisting of THY1, NT5E, ENG, and CD44; and

determining that the SINET in the subject will metastasize if the expression level of the SINET-associated gene in the test sample is higher than the level of the SINET-associated gene in the reference sample.

17. The method of claim 1, wherein the SINET-associated gene comprises a matrix- remodeling gene set where the gene comprises FAP; and

determining that the SINET in the subject will metastasize if the expression level of the SINET-associated gene in the test sample is higher than the level of the SINET-associated gene in the reference sample.

18. The method of claim 1, wherein the SINET-associated gene comprises a hypoxia and angiogenesis gene set selected from the group consisting of VEGFA, VEGFC, HIF1A, and CAV1; and

determining that the SINET in the subject will metastasize if the expression level of the SF ET -associated gene in the test sample is higher than the level of the SINET-associated gene in the reference sample.

19. The method of claim 1, wherein the SINET-associated gene comprises a tumor- associated stromal cells and myofibroblasts gene set selected from the group consisting of PDGFRA, PDGFRB, NRG1, TNC, POSTN, and ACTA2; and

determining that the SINET in the subject will metastasize if the expression level of the SINET-associated gene in the test sample is higher than the level of the SINET-associated gene in the reference sample.

20. The method of claim 1, wherein the SINET-associated gene comprises a stromal-specific gene set selected from the group consisting of P4HA1, P4HB, and VIM; and

determining that the SINET in the subject will metastasize if the expression level of the SINET-associated gene in the test sample is higher than the level of the SINET-associated gene in the reference sample.

21. The method of claim 1, wherein the expression level of the SINET-associated gene is detected via an Affymetrix Gene Array hybridization, next generation sequencing, ribonucleic acid sequencing (RNA-seq), a real time reverse transcriptase polymerase chain reaction (real time RT-PCR) assay, immunohistochemistry (IHC), immunofluorescence, methylation-specific PCR, or a bead-based multiplex assay.

22. The method of claim 1, wherein said subject is a human.

23. The method of claim 1, further comprising treating the subject with a chemotherapeutic agent, radiation therapy, cryotherapy, hormone therapy, or immunotherapy.

24. The method of claim 23, wherein the chemotherapeutic agent comprises a somatostatin analog, wherein the somatostatin analogue comprises octreotide long-acting repeatable (LAR).

25. The method of claim 24, further comprising administering an inhibitor of the SINET- associated gene with a higher level of expression compared to the level of the SINET-associated gene in the reference sample, thereby treating the SF ET.

26. The method of claim 25, wherein the inhibitor comprises a tumor-specific inhibitor selected from the group consisting of Rapamycin, Cabozantinib, and Erlotinib.

27. The method of claim 25, wherein the inhibitor comprises a polypeptide, a small molecule inhibitor, RNA interference (RNAi), an antibody, or any fragment or combination thereof.

28. The method of claim 27, wherein the antibody or antibody fragment is partially humanized, fully humanized, or chimeric.

29. The method of claim 27, wherein the antibody or antibody fragment comprises a nanobody, an Fab, an Fab', an (Fab')2, an Fv, a single-chain variable fragment (ScFv), a diabody, a triabody,a tetrabody, a Bis-scFv, a minibody, an Fab2, an Fab3 fragment, or any combination thereof.

30. The method of claim 27, wherein the small molecule inhibitor comprises an inhibitor of NFKB activity.

31. The method of claim 30, wherein the inhibitor of FKB activity comprises Withaferin A (WFA).

32. The method of claim 25, wherein the inhibitor comprises Ruxolitinib or Momelotinib.

33. The method of claim 31, wherein WFA is administered at a concentration of 10- 1000 nM.

34. The method of claim 33, wherein expression levels of VEGF, IL-6, and MCP-1 are increased following administration of WFA.

35. The method of claim 1, further comprising administering an agonist of the SF ET- associated gene with a lower level of expression compared to the level of the SINET-associated gene in the reference sample, thereby treating the SINET.

36. A composition for predicting whether a SINET will metastasize comprising a SINET- associated gene, wherein the SINET-associated gene comprises IL-2, IL-6, IL-12, IL-13, VEGF, MCP-1, uPA, HGF, RANTES, Eotaxin, GM-CSF, sVEGFR2, and sIL-6RA synthesized complementary deoxyribonucleic acid (cDNA).

37. A composition for predicting whether a SINET will metastasize comprising a SINET- associated gene, wherein the SINET-associated gene comprises a neuroendocrine tumor (NET) specific gene, a cell surface eithelial gene, a stimulants of malignant cell proliferation gene, an epithelial-mesenchymal transition gene, a metastatic transition gene, a mesenchymal-like gene, a matrix-remodeling gene, a hypoxia and angiogenesis gene, a tumor-associated stromal cells and myofibroblasts gene, and a stromal-specific gene, wherein the NET-specific gene comprises CGA, the cell surface eithelial gene is selected from the group consisting of CD34, PECAMl, MUC16, KRT5, KRT6A, KRT6B, and KRT17, the stimulants of malignant cell proliferation gene is selected from the group consisting of EGFR, FGF-2, HGF, and CXCL12, the epithelial- mesenchymal gene is selected from the group consisting of CDH1, CDH2, MMP1, MMP2, ITGAl, ITGA2, ITGA3 ITGA5, ITGA7, ITGAl l, TGFBl, and TGFBI, the metastatic transition gene is selected from the group consisting of YAP 1, HAS2, LOX, S100A4, GSTPl, and CD81, the mesenchymal-like gene is selected from the group consisting of THY1, NT5E, ENG, and CD44; the matrix-remodeling gene comprising FAP, the hypoxia and angiogenesis gene is selected from the group consisting of VEGFA, VEGFC, HTF1A, and CAV1, the tumor- associated stromal cells and myofibroblasts gene is selected from the group consisting of PDGFRA, PDGFRB, RG1, TNC, POSTN, and ACTA2, and the stromal-specific gene is selected from the group consisting of P4HA1, P4FIB, and VFM.

38. The composition of claim 36, wherein said composition further comprises SCF and sEGFR, GM-CSF, sVEGFR2, and sIL-6RA synthesized cDNA.

39. The composition of claim 36, wherein the SINET-associated gene is immobilized on a solid support.

40. The composition of claim 36, wherein the SINET-associated gene is linked to a detectable label.

41. The composition of claim 40, wherein the detectable label comprises a fluorescent label, a luminescent label, a chemiluminescent label, a radiolabel, a SYBR Green label, or a Cy3 -label.

42. A kit comprising a package with a SINET-associated gene, wherein the SINET- associated gene comprises IL-2, IL-6, IL-12, IL-13, VEGF, MCP-1, uPA, HGF, RANTES, Eotaxin, GM-CSF, sVEGFR2, and sIL-6RA and instructions for use thereof in the evaluation of SINET progression and metastasis.

43. A kit comprising a package with a SINET-associated gene, wherein the SINET- associated gene comprises a neuroendocrine tumor (NET) specific gene, a cell surface eithelial gene, a stimulants of malignant cell proliferation gene, an epithelial-mesenchymal transition gene, a metastatic transition gene, a mesenchymal-like gene, a matrix-remodeling gene, a hypoxia and angiogenesis gene, a tumor-associated stromal cells and myofibroblasts gene, and a stromal-specific gene, wherein the NET-specific gene comprises CGA, the cell surface eithelial gene is selected from the group consisting of CD34, PECAM1, MUC16, KRT5, KRT6A, KRT6B, and KRT17, the stimulants of malignant cell proliferation gene is selected from the group consisting of EGFR, FGF-2, HGF, and CXCL12, the epithelial-mesenchymal gene is selected from the group consisting of CDHl, CDH2, MMPl, MMP2, ITGAl, ITGA2, ITGA3 ITGA5, ITGA7, ITGAl 1, TGFBl, and TGFBI, the metastatic transition gene is selected from the group consisting of YAP 1, HAS2, LOX, S100A4, GSTPl, and CD81, the mesenchymal -like gene is selected from the group consisting of THY1, NT5E, ENG, and CD44; the matrix- remodeling gene comprising FAP, the hypoxia and angiogenesis gene is selected from the group consisting of VEGFA, VEGFC, HIFIA, and CAV1, the tumor-associated stromal cells and myofibroblasts gene is selected from the group consisting of PDGFRA, PDGFRB, RG1, TNC, POSTN, and ACTA2, and the stromal-specific gene is selected from the group consisting of P4HA1, P4HB, and VFM.

44. An ex vivo method of screening therapeutic agents for a neuroendocrine tumor comprising:

establishing an ex vivo organotypic neuroendocrine tumor tissue slice culture;

administering a candidate compound to the culture; and

determining that the candidate compound is a therapeutic agent for a neuroendocrine tumor if the candidate compound inhibits the neuroendocrine tumor.

45. The ex vivo method of claim 44, wherein proliferation of the neuroendocrine tumor is inhibited.

46. The ex vivo method of claim 45, wherein proliferation is evaluated using a

bromodeoxyuridine (BrDU) cell proliferation assay.

47. The ex vivo method of claim 44, further comprising detecting a level of a candidate compound-specific target.

48. The ex vivo method of claim 44, further comprising detecting a level of a secretory protein in supernatant of the tissue slices prior to and following administration of the candidate compound.

49. The ex vivo method of claim 48, wherein the secretory protein comprises chromogranin A (CGA) or neuron specific enolsase (NSE).

50. The ex vivo method of claim 44, wherein said candidate compound comprises a mechanistic target of rapamycin (mTOR) inhibitor comprising Rapamycin.

51. The ex vivo method of claim 44, wherein said candidate compound comprises

Cabozantinib, Erlotinib, a histone deacetylase (HDAC) inhibitor, or a cyclin-dependendt kinase (CDK) 4/6 inhibitor.

52. The ex vivo method of claim 49, wherein the Rapamycin is administered at a dose of 100 nM.

53. The ex vivo method of claim 50, wherein administration of Rapamycin results in a reduction of secretion of osteoponin, IL-16, VEGF, MCP-1, IL-10, macrophage inflammatory protein- la (MlP-la), MTP-lb, interferon-y-inducible protein- 10 (IP- 10), IL-8, and urinary plasminogen activator (uPA) from the culture.

54. A method of generating a neuroendocrine-associated fibroblast culture comprising:

obtaining a tissue sample from a patient;

dissecting the tissue sample into pieces;

enzymatically disaggregating the tissue sample;

mechanically disaggregating the tissue sample;

filtering and centrifuging the cells;

plating the cells on polymer tissue culture plates; and

culturing the cells,

thereby generating a neuroendocrine-associated fibroblast culture.

55. The method of claim 54, wherein the tissue sample is enzymatically disaggregated with collagenase II at 2 mg/ml.

56. The method of claim 54, wherein the tissue sample is mechanically disaggregated with an 18-guage needle and syringe.