WO2015070197A1 - Detection of malignancy in brain cancer - Google Patents

Abstract

Provided are methods of detecting one or more macrophage markers in cancer cells. Specifically, the macrophage markers are M2 macrophage markers. Also provided are methods of detecting one or more microglial markers in cancer cells. The methods disclosed are useful for research purposes (e.g, labeling malignant cancer cells), and for diagnosis and/or treatment of cancer.

Description

DETECTION OF MALIGNANCY IN BRAIN CANCER

GOVERNMENT FUNDING

This invention was made with government support under grant number R01. CA74145 awarded by the National Cancer institute. The United States government has certain rights in the invention.

BACKGROUND

Glioblastoma multiforme (GBM), or "glioblastoma," remains one of the most aggressive cancers that portends a grim prognosis despite multimodal -therapies. GBM tumor roicroenvironnient may contribute to the disease's irreversible progression due to its complexity and potential role in aiding local tumor infiltration (da Fonseca&Badie, 2013), The main tumor infiltrating leukocytes, tumor associated macrophages (TAMs), have been found to be master orchestrators of solid tumor promotion. TAMs roles in cancer include aiding in invasion., m grat on:, angiogenesis, and metastases formation, as well as attenuating T cell mediated adaptive immune responses (Eljaszewicz et al, 2013; Mantovaal, Germaho, Marches!, Locatelli, & Biswas, 2011) (Pollard 2004),

In GBM, resident myeioid-iineage cells, microglia, peripheral bone-marrow derived myeloid cells, macrophages, comprise TAMs to promote temor growth and invasion (da Fonseea & Badie, 2013} (Hussaifi et al, 2006). I order to promote tumor .growth, TAMs undergo activation and polarization to an anti-inflammatory or M2 alternatively activated phenotvpe. The M2 phenotvpe has been found, to be the predominant macrophage subtype in solid tumors (Mantovani et. al, 2011). In glioma patient samples, the number of infiltrating microglia/macrophages correlated with histological grade, Furthermore, the preponderance of the M2 phenotype In glioma samples also correlated with histologic grade (Y omohara, Oh isbJ, Kuratsu, &. T^'akeya. 2008).

The M2 macrophage phenotype is characterized by expression of cell surface markers such as arginase 1, CD206. and CD 163. M2 macrophages also can direct the immune suppression of T cells and secrete a distinctive cytokine expression pattern, i.e., high expression of IL-l decoy. IL-l Ralpha, IL-6 and IL-l 0, and low expression of tumor necrosis factor and IL- 12 (Gordon & Martinez, 2010) (G. Liu & Yang, 2013) When macrophages were exposed to glioma stem cell supemaiants, they displayed a M2-Iike phenotype (Wu et al., 2010), Glioma cells were found to induce STAT2 expression, down-modulate STAT1 expression, and induce production of immunosuppressive cytokines in macrophages. Macrophages exposed to glioma eel] supematanis also were found to significantly inhibit T cell proliferation (Wu et ah, 2010).

Microglia can undergo phenotypic alteration, as well, which is dependent on the surrounding environment (Boehe, Perry, & Nicoll, 2013). Microglia have a resting phenotype in steady-state conditions which provides constant surveillance and homeostasis. Classically activated, proinflammatory microglia play a critical role in neurodegenerative disease; and alternatively activated microglia are presumed to aid in the immunosupression seen in gliomas (Joseph & Venero, 2013; Saijo & Glass, 201 1 ). Microglia exposed to glioma conditioned media were activated and induced transcription of markers indicative of alternative M2 activation (Ellert-Miklaszewska et ah, 2013),

The presence of M2 activated TAMs has been shown to correlate with poor prognosis in many cancer types such as lung, esophageal, and prostate cancer (Hirayama et a!,, 2012; C.-Y. Liu et al, 2013; Shigeoka et al., 2013), Gene expression profiling of patients with GBM showed that shorter survival correlated with an increased expression of genes related to microglia/macrophage recruitment and activation (Eng!er et al., 2012). Poor clinical prognosis was associated with a higher M2 cell ratio in patients with glioma (YosM ro omohara et al.. 2012). Patients with grade 3 astrocytomas were found to have a survival advantage with lower numbers of CD 163 cells in their tumor specimens (Prosniak et aL, 2013). Also, there is initial evidence that resistance to anti-VEGF therapy and subsequent tumor recurrence is associated with increased infiltration of tumor associated macrophages, which was first demonstrated in an animal mode! (Shojaei et al. 2007 Nature Biotechnology). In confirmation of these results, patients with decreased overall survival following anti-angiogenrc therapy were found to have increased TAMs in tumor bulk and infiltrative tumor (Lu-Emerson et al, 2013). These findings implicate that microglia/macrophages are likely key mediators of developing the tumor mieroenvironment arid ultimately impacting patient prognosis in GBM.

We have recently found that invadopodia are present in GBM cells and that invadopodia marker, a protooncogene Tks5, is expressed in GBM, in accordance with the findings by others (Stylii, aye, and Lock, 2012 J Clin Neurosek data not. shown). TksS appears to be not only important in controlling, the behavior of cancer cells (Seals), but also in the process of diiferentiatioti/polarization of macrophages (Burger 201 l ).We have also demonstrated that lbs- related antigen 1 (Fra-1 ) proto-oncogene is involved in regulating the morphology and migratory nature of glioma cancer cells (Debinski & Gibo, 2005 and 2011). interestingly. Fra-1 has been shown to have a role in macrophages behavior as well. Namely, Fra-1 has been shown to be a regulator of M2 macrophage activation (Wang et al,, 201.0). SUMMARY

Provided herein axe methods of detecting one or more macrophage markers in cancer cells, in some embodiments, the macrophage markers are M2 macrophage markers, Also provided are methods of detecting one or more microglial markers in cancer ceils. The methods taught herein are useful for research purposes (e.g., labeling malignant cancer cells), and for methods of diagnosis and/or treatment of cancer as described herein.

Further provided are methods of detecting malignancy in a solid tumor, comprising providing cancer cells isolated from said tumor; and detecting the expression of one or more macrophage markers by said cancer cells, wherein the expression, of said one or more macrophage markers by said cancer cells indicates malignancy of said tumor. In some embodiments, the malignancy indicates an increased risk of cancer cell invasion. In some embodiments, tire malignancy indicates an increased risk of metastasis.

In some embodiments, the macrophage markers are M2 macrophage markers. In some embodiments, the macrophage markers comprise one or more markers selected from the group consisting of; CD ; 4. CD68_; GDI lb, CD163, CD204, and CD2 6, In some embodiments, the macrophage markers comprise one or more markers selected from the group consisting of: CD68, CDI63, CD204 and CD2G6.

in some embodiments, the method further comprises detecting the presence of one or more microglial markers in said cancer cells, wherein the presence of said one or more macrophage markers and one or more microglial markers in said cancer cells indicates malignancy of said tumor.

in some embodiments, the tumor is a glioma, in some embodiments, the tumor is an ^•astrocytoma, meningioma or oligodendroglioma. In some embodiments, the tumor is glioblastoma.

Also provided are methods of grading a glioma, comprising providing cancer cells isolated from said glioma; and detecting the expression of one or more macrophage markers by said cancer cells, wherein the expression of one or more macrophage markers by said cancer cells indicates an advanced grade of said glioma. In some embodiments, the glioma is glioblastoma mulitforme (GBM),

Further ^'provided, are methods of treating a glioma comprising, providing cancer cells isolated from said giiorna; and detecting the expression of one or more macrophage markers by said cancer cells, and, if said cells express said one or more macrophage markers, treating said glioma. In some embodiments, the treating comprises administering one or more of: radiation therapy, chemotherapy and targeted protein therapeutics, In some embodiments, the glioma is glioblastoma multiforme (GBM).

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A-1G: Macrophage marker expression in GBM. In situ GBM specimens expressed the pan-macrophage marker, CD6_.8 via IHC (A) and IF (B). The microglial marker, CD 14, also showed expression in GBM tumors (image not shown). Cells expressing the macrophage activation marker, CD163, were found to be in vessels (C). CD204 was identified throughout tumor specimen via IHC (D), CD163+/CD206+ cells were found along vessels (E) and in the tumor stroma (F). CD204 expression was present in multiple brain tumor specimen Iysates via immunohlottmg (G).

^'WIG. 2A-2E: Macrophage activation markers are present in glioma cell lines^' and tumor Iysates, In order to provide a comparison of activation markers, a THP-l monocyte activation model was used. M2 macrophages showed expression of. CD 163, CD204, and CD206 as well as the proto-oncogenes Tks5 and fra-1. Ml macrophages showed marked downregulation of these markers (A). CD! 63+ cells isolated from freshly isolated GBM expressed CD206, CD68 and GFAP (B). A cell line derived from GBM tumor (4795) showed increased expression of CD206 and CD68 in comparison to the respective tumor B). Established glioma cancer cells lines showed expression of CD206 and CD68 (C) as well as CD 163 (D), Cell lines derived from GBM tumor express CD204 and CD206 (E).

FIG. 3A-3D: Cells positive for M2 macrophage activation markers + GFAP and M2 macrophage activation markers + proto-oncogenes are found in GBM specimens. Cells expressing CD204 and GFAP were found in GBM tumor via IHC. (A). CD 163 and GFAP were expressed in certain cells in GBM via IF .(B). CD163+/Tks5+ (C) and CD206+ Tks5+ (D) were also present in GBM samples.

FIG. 4A-4C; Behavior of macrophage markers in GBM cells cultured under spheroid conditions (A), hypoxia anoxia (B), or treated with radiation (C).

DETAILED DESCRIPTION OF EMBODIMENTS

"Cancer" or "cancers" that, can be detected and/or treated by the compounds, compositions and methods described herein include, but are not limited to, breast cancer, bladder cancer, pancreatic cancer, colorectal cancer, head and neck cancer, thyroid cancer, prostate cancer, melanoma, and brain cancer such -as gliomas (e.g., GBM), etc. In some embodiments, the cancer is a "brain tumor." The cancer cars, be a primary or secondary brain cancer. Cancers treatable with embodiments of the present invention include, but are not limited to, astrocytoma, oligodendroglioma, .ependymoma, meningiomas, acoustic neurorna/sehwarmomas, and medulloblastoma. Also included is neuroblastoma, In some embodiments, the cancer is a secondary brain cancer which has metastasized from a non-brain cancer.

"Cancer cells" as known in the art are cells thai abnormally grow and divide at an accelerated rate as compared to the corresponding normal cells and/or do not undergo cell death at a normal rate. Their growth often results in a mass of cells called a tumor.

In some embodiments, the cancer ceils are separated or isolated from oilier ceils such as immune and monocytic/macrophage lineage cells before detecting marker expression, in order to not risk or minimize marker contamination by said ceils. This may be performed b methods known in the art, e.g., by immunoiocaii atioa^mmunoisolation such as by fluorescence activated cell sorting (FAGS). Other methods may include culturing said cells for at least 2, 3, or 4 weeks prior to said detecting step, whereby only the cance ceils would survive: freezing cells after collecting from a subject, and then washing said ceils; and/or fixing said cells (e.g., with an aldehyde or permanganate fixative) and then embedding said cells in a wax (e.g., paraffin) support. Another method involves the use of acoustic waves to sort cells. -See Ding et a!., "Cell separation using tilted-angle standing surface acoustic waves." PNAS vol. 111, no, 36: 12992- 12997 (2014).

A tumor may be benign or malignant, A "malignant" tumor is a tumor that causes medical harm, becoming progressively worse. Typically, a malignant tumor shows signs of anaplasia (de-differentiation), invasiveness (spread into surrounding tissues) and/or metastasis,

"Metastasis" is when cancer cells spread to other parts of the body, often via blood circulation after gaining the ability to penetrate into the lymphatic or blood vessels.

All references cited herein are incorporated by reference to the extent they are consistent with the disclosure provided herein.

As used herein, "a," "an" or "the" can mean one or more than one. For example, "a" cell can mean a single cell or a multiplicity of cells.

Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

Furthermore, the term "about," as used herein when referring to a measurable value such as an amoun of a compound or agent, of this invention, dose, time, temperature, and the like, may be variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

A cell "marker" as used herein refers to a biochemical or genetic characteristic that may he used io distinguish between and/or isolate particular ceil types.

A "myeloid" cell refers to a white blood cell derived from a granulocyte precursor ceil in the bone marrow or spinal cord. A monocyte is a type of myeloid cell that may further differentiate into a macrophage or a dendritic cell.

'^'Macrophage" cells are phagocytic cells typically produced by the differentiation of monocytes that have migrated into tissues. Mi macrophages express a killer phenotype with the ability to metabolize arginme to nitric oxide and activate a Thl T~cell response, while M2 macrophages express a repair phenotype with the ability to metabolize arginine to ornithine and activate a Th2 T-cell response (stimulating antibody production).

Many markers are associated with macrophage cells, and include, but are not limited to, specific expression of protein markers including, but not limited to, CD 16 / Fc gamma RHI, CD64 / Fe gamma RI, CD155 / PVR, CD 16-2, CD68 / SR-Dl, GDI 63, GDI 6a, Fc gamma. MIA, CD69, CD200R1, CD 16b / Fc gamma RI!IB, CD74, CD204 / MSRl, CD 18 / Integral beta 2, GD80 / B7-1, CD209/DC-SIGN, CD26 / DPP4, CD84 / SLAMF5, CD20 b / SIGNR! , CD31 i PECAMI, CD85a/LiL 63, CD258 TNFSF14/LIGHT, CD32 / Fc gamma Rll, CD91/LRP1, CD273 / B7-DC / PD-L2, CD32a Fc gamma RIIA, CD93 / CkjR, CD274 / B7- HI, CD32b / Fc gamma RI1B, GD97, CD283 / TLR3, CD33 / Siglec-3, CD 102 / ICAM-2, CD284 / TLR4, CDS 6 /SCARB3, CD 105 / Endog!in, CD300A, CD40 TNFRSF5, GDI 16 / GM-CSFR, CD300C, CD43 / SPN, CD119 IFNGRl, CD301 / CLECIOA, CD45, CD12lb / !L- 1R2, CD302 / CLEC13A, CD49d / Integrin alpha 4, CD 147 / EMMPRIN, CD319 / CRACC / SLAM7, CD61 / integrin heta3, CD! 50 / SLAM, and CD319 / CRACC / SLAM7. These markers may be detected using methods standard in the an, e.g., with antibodies commercially available, such as at Sino Biological, Inc., Beijing, P,R. China.

Macrophage markers further include, but are not limited to, AIF-.I, IFN-gamma Ri/CD119, B220/CD4SR, IFN-gamma R2₅ B7-1/CD80, IGF-Ϊ, B7-H1 PD-L1, IL 1 RII, BLAME/SLA F8, lL-10, Cannabinoid Rl/CBI/CNRl , IL-12, Cannabinoid R2/CB2/CNR2, IL- 12 p70, Carboxypeptidase M, fL-.12/IL~35 p35, CCLl/I-309 TCA-3, IL-27 R alpha WSX- 1/TCCR, CCL6/C10, IL-34, CCL15/MXP-1 delta, ILT2/CD85J, CCL17 TARC, ILT3/CD85k, CCL18/PARC, ILT4/CD85d, CCL20/MIP-3 alpha, ILT5/CD85a, CCL22 MDC, ILT6/CD85e, CD2F- 10/SLAMF9, ILT 11 /LILRA5 , CD31 /PEC AM- 1. Integrin alpha 2b beta 3, CD36/SR-B3, Integrin alpha 4/CD49d, CD40/TNFRSF5, Integrin alpha 4 beta 1, CD43, integrin alpha 4 beta 7/LPAM-l , CD45, integrin alpha X beta 2, CD68/SR-D1, Integrin beta 2/CD18, CD74, Integrin beta. 3/CD6L CD84/SLAMF5, Latexin, CD97, Leukotriene B4 Rl , CD! lc, LiGHT/T FSFl 4, CD155/PVR, LILRCl, CD163, LIMPII/SR-B2, CD200 R L LRP-1, CD300a/LMIRl, LRP-1 Cluster H, CD3G0b/LMIR5, LRP-1 Cluster III, CD3Q0c/LMIR2, LRP-1 Cluster IV, CD300e LMIR6, LRP-IB, CD300ffLMIR3, MARCO, CD302 CLEC13A, MD-1, CD45RO, MD-2, ChemR23, MGLl/CD301a, Chitinase 3-like 3/ECF-L, MGL1/2 (CD301a/b), CLEC10A/CD301, MGL2/CD301b, Common beta Chain, HC class II (I-A/I-E), CRACC/SLAMF7, iNGS, CXCL9/MIG, NRAMPl/SLCUAl, CXCLlO IP-lO/CRG-2, Nucleoporin NUP85, CXCL1 1/I-TAC, Osteoactivin/GP MB, DC-STAMP, Osteopontin/OPN, DC1R/CLEC4A, PD-L2 B7-DC, EMMPRIN/CD147, PPAR gamma NRlC3, EMP/MAEA, RELM alpha, EMR2, Sigiec-1/CD169, EMR3, Siglec-3/CD33, EMR4, Siglec-16, Endoglin/CDIOS, Si GN 1 /CD20 b, F4/80/EMR1, SLAM/CD150, Fc epsiion Rl alpha, Stabilin- I, Fc epsilon Rl beta S4A2, TLR3, Fc gamma RIII (CD16), TLR4, Fe gamma RI CD64, TLR4/MD-2 Complex, Fc gamma RII/CD32, TRA-1-85/CD147, Fc gamma RII/RIII (CD32/CD16), TREM-1, Fc gamma RIIA/CD32a, TREM-2, Fc gamma RIIB/CD32k TREM- 2b, Fc gamma RIIB/C (CD32b/c), TREM-3, Fc gamma RHC/CD32c, TREMLl TLT-1, Fc gamma RfflA/CD16a, TREML2/TLT-2, Fc gamma RIiIB/CD16b₉ TREML4/TLT-4. GM-CSF R alpha, ZBP1 DLM-1 DAI, and ICAM-2/CD102. These markers may be detected using methods standard in the art, e.g., with reagents such, as antibodies commercially available, e.g., at R&D Systems, Minneapolis, M3SL

In some embodiments, the markers comprise one or more of markers selected from the group consisting of CD14, CD4G, CDl lb, CD64, F4/80 in mice or EMR.1 in humans, ysozynie M, MAC-l MAC-3, and CD68. In some embodiments, M2 macrophage markers comprise one or more markers selected from the group consisting of CD 14, CD68, CDl lb, GDI 63, CD204, and CD206, In some embodiments, M2 macrophage markers comprise one or more markers selected from the group consisting of CD la, CDlb, CD93 and CD226. See WO 2013/135775 to Scbu!tze et at

"Microglia!" cells are resident macrophages of the central nervous system, differentiated from monocytes that travel into the brain. Microglial markers include, but are not limited to, CD1 lb, IBA1, CD200, and CX3CR1.

Detection of marker expression may be performed by methods known by those of skill in the art, for example, by nucleic acid amplification and/or restrictio analysis, flow cytometry or immunoassay using antibodies that specifically bind thereto, etc. In some embodiments, detection is performed with a method allowing visualization or localized detection of the markers on the cancer cells In a culture or biopsy. Such methods of detection may include, but are not limited to, immunoassay such as immunohistochemistry (IMC), miniunoQworochemistty (IPC), fluorescence activated cell sorting (FACS), OT .imagining_, techniques. In some embodiments, such detection is carried out in vitro. In some embodiments, imaging of marker expression in vivo may be performed by administering a labeled molecule that will bind to or otherwise indicate the presence of the markers at a site of interest in vivo.

"Express" or "expression" of a protein marker means that a gene encoding the marker protein is transcribed, and preferably, translated. Typically, according to the present invention, expression of a protein coding region will result in production of the encoded polypeptide.

"Brain cancer" o "brain tumor" may be an stage, grade, histon orphological feature, invasiveness, aggressrvity or malignancy of an affected tissue or cell aggregation in any part of the central nervous system (i.e., brain and spina] cord), in some embodiments, the brain tumor is a glioma. In som embodiments, the tumor is an. anaplastic astrocytoma, anaplastic oligoastrocytoma or anaplastic oligodendroglioma, in particular, fibrillary astrocytoma WHO grade II, oligoastrocytoma WHO grade Π, oligodendroglioma grade II, anaplastic astrocytoma WHO grade Hi, anaplastic oligoastrocytoma WHO grade III, anaplastic oligodendroglioma grade ΠΪ or glioblastoma multiforme (see,, e.g., U.S. Patent Application Publication No. 2010/0291590).

Gliomas are tumors occurring in the glial cells, which help support and protect critical areas of the brain. Gliomas are the most common type of brain tumor in adults, responsible for about 42% of all adult brain tumors. Gliomas are further characterized by the types of cells they affect, Into the categories of ^'astrocytoma (affecting astrocytes), oligodendroglioma (affecting oligodendrocytes), ependymoma (affecting ependymal cells), meningiomas (affecting the meninges)_* acoustic neuroma schwannoma (affecting Schwann's cells), and meduiloblastoma (affective cells in the cerebellum). See also U.S. 2013/0012452 to Basile et al.

Astrocytomas are graded from I to IV depending on the speed of progression. Grade I (pilocytic astrocytoma) is slow growing, with little tendency to infiltrate .surrounding brain tissue. Grade II (diffuse astrocytoma) is fairly slow-growing, with some tendency to Infiltrate surrounding brain tissue. Grade III (anapiastic/malignant astrocytoma) tumors grow rather quickly and infiltrate surrounding brain tissue. Grade IV (glioblastoma multiforme, or "GBM") is an extremely aggressive and lethal form of brain cancer. Unfortunately, it is the most common form of brain tumor in adults, accounting for about 67% of all astrocytomas.

Oligodendrogliomas, which make up 4% of brain tumors, mostly affect people over 45 years of age. Some subtypes of this tumor are particularly sensitive to treatment, with radiation therapy and chemotherapy. Half of patients with oligodendrogliomas are still alive after five years.

Ependymomas are rare; about 2% of all brain tumors, but are the most common brain tumor in children. They generally do not affect healthy brain tissue and do not spread beyond the ependyma. Although these tumors respond well to surgery, particularly those on the spine, ependymomas cannot always be completely removed. The .five-year survival rate for patients over age 45 approaches 70%,

Meningiomas affect the meninges, the tissue that forms the protective outer covering of .the brain and spine. One-quarter of all brain and spinal tumors are meningiomas, and up to 85% of them are benign.

Malignant gliomas are a fatal disease with an average life-expectancy following diagnosis of less than one year. The prognosis for patients with high-grade gliomas is very poor, and is especially so for older patients. Of Americans diagnosed each year with malignant gliomas, about half are alive 1 year after diagnosis, and 25% after two years. Those with anaplastic astrocytoma survive about three years. Glioblastoma multiforme has the worst prognosis, with a life expectancy of less than 9-15 months following diagnosis.

An "isolated¹' cancer cell is one that is separated or substantially free from at least some of the other components of the naturally occurring organism or tissue, for example, at least some of the tissue structural components or other proteins commonly found associated with the cells in vivo. As used herein, the "isolated." cells may be provided by biopsy, resection_* or otherwise removal of cells or part, of a tissue/tumor from its natural in vivo environment.

For example, "providing" the cancer cells may be performed by collection of the cells, which may be followed by storing (e.g., freezing) or cuituring the cells for at least 2, 3, or 4 Weeks prior to detecting the markers as taught herein, optionally followed by washing the cells; and/or fixing said cells (e.g., with an aldehyde or permanganate fixative) and then embedding said cells in a wax (e.g., paraffin) support prior to detecting the markers as taught herein,

"Subjects¹" are inclusive of human subjects, as well as animal subjects, particularly mammalian subjects such as canines, for veterinary or research purposes. While subjects may be of any suitable age, the subjects are in .some embodiments neonatal, infant, juvenile, adolescent, adult, or geriatric subjects. In some embodiments, human subjects are at least 50, 60, 65, or 70 years of age. Subjects, ma also include other animal subjects, particularly mammalian subjects such as felines, bo vines,, caprines, equines, ovines, porcmes, rodents (e.g. rats and mice), lagomorphs, primates (including non-human: primates), etc_., screened for veterinary medicine or pharmaceutical drug development purposes. "Treat" as used herein refers to any type of treatment that imparts a benefit to a subject particularly delaying or retarding the progression of the disease or cancer. For example, the treatment may kill or otherwise decrease the .number of cells and/or volume of cancerous tissue in the brain or central nervous system, inhibit or slow ihe progression of ihe cancer, alleviate side effects such as cognitive abnormalities, etc. In some embodiments, treating specifically includes prophylactic treatment to delay or otherwise inhibit tumor growth and/or metastasis.

In some embodiments, the detection of the markers as taught herein indicative of a malignant tumor is followed by or otherwise directive for treatment for the cancer. For example, treatment of a brain cancer such as GMB .may be carried out with methods knows in ihe art, such as surgical removal of .the tumor and/or cheraotherapeutie therapy with a therapeutic agent {e.g., temozoloraide) and/or radiation therapy. Targeted protein therapeutics comprising one or more effector molecules may also he administered. See U.S. Patent Nos. 8,343,461 and 8,362,207 to Debinski et aL which are incorporated by reference herein, See also V.S, Patent Nos. 5,328,984, 6,428,788, 6,456,232, and 7,338,929, which are incorporated by reference herein.

"Effector molecule" as used herein includes therapeutic agents, detectable groups,, targeting Hgands, and delivery vehicles (e.g., antibodies. lipids, liposomes). See, e.g.,. U.S. Patent No, 6,630,576.

'Therapeutic agent" as used herein may be any therapeutic agent useful for the treatment of a cancer, including, hut not limited, to, genetic materials or agents, radionuclides, chemotherapeutic agents, cytotoxic agents (See, e.g., U.S. Patent No, 6.949,245 to S!iwkows ), and amphipathic antimicrobial peptides. Other exemplary therapeutic agents include, but. are not limited to, radiopharmaceuticals, including, but not limited to, auger electrons, chemotherapeutics, and photosensitizers,

"Radionuclide" as described herein includes, but is not limited to, ²²⁷ Ac, ^HAt, ¹³ 'Ba, ⁷⁷Br, ^mCd, ⁵¹Cr, ¾*, ^I65Dy, ^I55Eu, ¹⁵³Gd_s™Au, ¹⁶⁶Ho, ^m¾_? ^m¾, ⁿ\ ^sl, ^mI_f *%, ⁱ⁵¾ ¹⁹²Ir, ¹⁹ Ir, ⁵²Fe, ^S5Fe, ^S9Fe, ^{77Lu, ^m?d, ³²P, ²²⁶Ra, ¹⁸⁶Re, ^I88Re,. ^I53Sm, ⁴⁶Sc, ⁴⁷Sc, ¾e, ⁷⁵Se, ^,05Ag, ^S9Sr, ¾ ^l77Ta_> ^mniSm ^!25Sn, ^I66Yb, ^l69Yb, ⁹⁰Y_S ²ⁿBi, ^{I l9}Sb, ^{; 97})% ⁹⁷Ru, ⁱ⁰⁰Pd, ^m*R and ²¹²Pb.

"Chemotherapeutic agent" as used herein includes, but is not limited to, methotrexate, daunomycin, mitomycin C, cisplaik, vincristine, epirubi n, fiuorouracil, verapamil, cyclophosphamide, cytosine arabinoside, aminopterin, bleomycin, mitomycin C, democolcine, etoposide, mitbramycin,- chlorambucil, melphaian, daimorubiein, doxorubicin, tamosifen, paclitaxei, vincristin. vinblastine, camptothecin,^■aetinomycin D, and cytarabine. Other examples are found in U.S. Patent Application Publication 2006/0121539 (Debinski et al), which is incorporated by reference herein in its entirety,

"Cytotoxic agent" or "toxic agent." as used herein includes, but is not limited to, maytansinoids and maytansmoid analogs, toxoids, CC-1065 and CC-1065 analogs, dolastatin and dolastatin analogs, riein (or more particularly the ricin A chain), aclacinoniycin, Diphtheria toxin, Monensin, Verrucarin A, Abrin, Trkothecenes, and Pseudomonas exotoxin A, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, anti -mitotic agents, such as the vmca alkaloids (e.g., vincristine and vinblastine), colchicin, anthracyclines, such as doxorubicin and daunorabicin, dihydroxy anthracin dione, mitoxantrone, mitnramycin, aclinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidoeaine, propranolol, and puromycin and analogs or homologs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine. 6-thioguanine,■cytarabine, and 5-fluorouracil decafbazine), alkylating agents (e.g., mechiorethamine, .thioepa chlorambucil, melphalan, carmustine (B8NU), loraustine (CCNU), cyclothosphamide, busuifan, dibromomannitol, streptozotoem, mitomycin C, and cis-diehlore iamin platinum (II) (DDP)), and antibiotics, including, but not limited to, dactiuomycin (formerly aetmomycin), bleomycin, mithramyc , calicheamicin, and anthramycin (AMC).

In some embodiments, cytotoxic agents include toxins such as Pseudomonas exotoxin, ricin. abrin, .ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, etc. See, e.g., U.S. Patent No.7,517,964, In some embodiments, Pseudomonas exotoxin or a diphtheria toxin are preferred. See U.S. Patent No. 5,328,984 to Pastau et al. and U.S. Patent No. 6,296,843 to Debinski, which are each incorporated by reference herein in its entirety. Pseudomonas exotoxins can include, but are not limited to, Pseudomonas exotoxin A (PE). The Pseudomonas exotoxin can be modified such that it substantiall lacks domain la, and In some embodiments Pseudomonas exotoxins include PE38QQR and PE4E. Diphtheria toxins can include DT3 0, a diphtheria toxin in which the native binding domain is eliminated. It will be appreciated that in various embodiments, the therapeutic agents can be attached to, e.g., the amino terminus or the carboxyl terminus of a targeting and/or linking protein. See, .g., U.S. 2011/0300099 to Debinski et al, which is incorporated by reference herein,

"Amphipathic antimicrobial peptide" as used herein includes ampliipathic peptides that induce apoptosis of cancer cells, presumably through their abilit to depolarize mitochondrial membranes. See K. Rege et al., Cancer Res. 67, 6368 (July I, 2007). Such peptides are, in general, from 10, 12 or 13 to 20, 30 or 40 amino acids in length, or more, and typically have an ampfaipathic alpha-helical structure. Examples include, but are not limited to, (KLAi LAK)2 (SEQ ID NO: 60); (KLA KLA)?. (SEQ ID NO: 61) (KAAKLKAA)₂ (SEQ ID NO: 62) and (KLGKKLG)₂ (SEQ IB NO: 63) See, e.g., Ruoslahti et al., US Patent Application 20010046498 (November 29, 2001).

EXAMPLES

Glioblastoma (GBM) tumors are infiltrated by Immune and monocytic/macrophage lineage cells; the latter are termed tumor-associated macrophages (TAM). TAM play a permissive role in tumor cells invasion* especially when polarized to an M2 type, it has been suggested that TAMs may contribute up to 30% of GBM tumor mass.

However, when isolating CD 163+ M2-type macrophages or monocytic CD 14+ cells from freshly resected GBM tumors, we could isolate long-term surviving cells in culture demonstrating features of ^'malignant cells. Therefore, we began analyzing whether tumor cells could eventually express M2 macrophage markers.

To our surprise, the lysates of GBM cells., both established and low-passage cells readily expressed a pan-macrophage/microglia marker, such as CD68. Even more .surprising, the CD 163, CD204 and CD206 that; are widely recognized as M2 markers ^' ere all readily detected in GBM cells^' to various extents.

GBM tumor lysates on average were positive for all the tested markers of monocytes/macrophages In western blots, We also performed multi-factorial immuiiohistochemical/immunoilnorescence staining using ^"human, canine and mice GBM specimens. We used GFAP and CD 14, CD68, GDI lb, CD163, CD204, CD206 monocyte/macrophage markers for concomitant detection in situ. Again, to our surprise a population of GBM tumor cells co-stained for M2 macrophage markers and also for a microglia! marker CD! lb,

Thus, we found that GBM malignant cells possess markers typical, of myeloid-derived cells. This is the first time that M2 macrophage markers were readily detected m GBM^' tumor ceils, and this finding has multiple implications. First, the presence, its extent and localization of M2 ^'TAMs in GBM should be revisited. Second, the mechanism behind t!ie appearance of these markers on malignant cells remains to be revealed. Third, it is reasonable that acquiring macrophage markers is related to an increase In GBM tumor cell migration/invasion, and may negatively influence patients' survival. Materials asset Methods

Immunofluorescence. Human tumor specimens were flash frozen immediately after resection. 10 ω sections were thaw- mounted on charged slides and stored at -SO °C until use, Slides were washed in PBS for three changes at room temperature for 5 minutes each. Sections were blocked for ! hour in 10% normal goat serum (LifeTech), Primary antibodies were diluted in PBS/1,5% normal goat serum ami incubated overnight at 4 ^QC. Slides were washed in PBS for three changes at room temperature for 5 minutes each. Secondary antibodies (Anti-mouse A exa F!uor-555, anti-rabbit Alexa Fluor 488) were diluted in PBS/1,5% normal goat serum and incubated on the sections at RT for 1 hour. Nuclei were visualized with DAPI. Slides were washed well in PBS and mounted with FhioreGuard Mounting Media (ScyTek). images at 20X and 40X were acquired on a Olympus 1X70 inverted microscope with a Retiga. 2000R camera system using Image Pro Plus vSTsoftware.

Immunohistochemistty. Human tissue specimens were fixed in 10% formalin and embedded in paraffin. Sections were out at a thickness of 8-10 μη . Slides were heated ^'at 65° C, de-paraffinized in xylene, and re-hydrated. Antigen retrieval was performed with 10 mM sodium c trate buffer, pH 6,0, by microwaving twice for 5 mi, Endogenous peroxidase activity and nonspecific biotin was quenched with Peroxide Blocking Kit and Biotin Blocking Kit respectively (ScyTek Laboratories, Logan, UT). Slides were blocked and incubated with primary antibody or PBS control overnight at 4° C. Slides were washed with PBS followed by incubation with anti- mouse biotinylated secondary antibody for 20 mh¾ then Avidin-HRP for 20 nam (ScyTek). Visualization with NovaRed (Vector Labs) was performed and allowed to develop for 540 min. Slides were counterstained with hematoxylin for 2 minute, dehydrated and mounted with Perniount (ThermoFisher). Images were taken with a 20x or 40 magnification lens.

Western Blot analysis. Cell lines were purchased f om ATCC (Manassas, VA). Cell lysates were prepared from sub-confluent cultures. Cells were washed with PBS and lysed. in radioimmunoprecipitation assay buffer (RIP A) (PBS, 0.5% sodium, deoxyeholate, 0,1% SDS, and 0,5% Igepa!) containing mammalian protease inhibitor cocktail (Sigma). Nonmalignant brain and pathologist-verified GBM tumor tissue were minced into small pieces while frozen and homogenized in RIPA buffer with mammalian protease inhibitor cocktail, Lysates were passed through an 18 -gauge needle to shear the DMA and were incubated on ice for 45 minutes.

Nonsoiub!e debris ^'was pelleted at 10,000g for 10 minutes and the supernatant was collected and stored at -80°C until use. Lysates were separated by SDS-PAGE using 10% or 12% aerylamide.

Proteins were then transferred to a polyvinylidene difluoride membrane (Perkin Elmer, Waltham,

MA) and blocked for at least 1 hour with blotto (5% milk in PBS/0,05% Tween 20), Membranes were incubated- with primary antibody diluted in blotto overnight ^'at 4°C while shaking, β-aetin (1 :50,000) antibody was purchased from Sigma. Following three 5-mmute washes in PBS/0.05% Tween 20, membranes were incubated with secondary antibody conjugated with horseradish peroxidase at a dilution of 1:5,000 in blotto for 1 hour, Membranes were washed thrice for 5 minutes each in PBS/0,05% Tween.20 and detection was done using the ECL2 Western Blotting Substrate (Thermo Scientific, Piscataway, NJ). Membranes were exposed, to autoradiographic ^'film- for various times. Films were scanned at 6Q0¾ dpi and images were compiled using Jasc Paint Shop Pro version 6,0.

Primary Cell Isolation. Human tumor specimens were acquired with 1.5 minutes of resection. Tumors minced in digestion buffer consisting of CoUagenase II, CoUagenase IV, DNAse (Sigma) and 1% FBS were incubated at 37°C with shakin for 45 minutes. Samples were passed through an 8 urn cell strainer (BD Biosciences, San Jose, California) and layered over a ficol gradient (Sigma). After eenirifugation at 300 x g for 35 minutes the interface was washed twice with PBS prior to plating in RPMi-1640 containing 4g L glucose, 10% FBS and Penn/Strep. Cells were allowed to adhere for 48 hours after which the non-attached debris wa removed. For isolation of CD 163+ cells, a portion of the sample from the interface was washed, well with PBS prior to incubation with biotinylated GDI 63 antibody (clone 5C6-FAT). After removal of non-bound antibody, samples were incubated with strepavidin magnetic, beads. CD 163+ cells were recovered using a Dynal magnetic bead system (invitrogen) and plated i RPMI-1640 containing 4g l glucose and 10% FBS).

THP-1 celb^' polarization. THP-1 cells were purchased from ATCC (Manassas, VA). Cells were plated at a density of 500,000 ceils/mi. Phorbol 12-Myristate 1.3-Acetate was added to a final concentration of 40 nM. After 6 hours of incubation Interferon-g (20 mg/mi) (Peprotech Rocky Hill, NJ, and Lipopolysaccbande (100 rng m) were added to generate Mi macrophages and IL-13 and IL-4 were added to generate M2 macrophages. Ceil treated with PMA were designated M0. DMSO treated THP-1 ceils served as controls.

Results

GUobasioma Macrophages/Microglia and M2 macrophages presence in GBM specimens. We first performed staining using antibody against a pan-macrophage marker, CD68, Numerous CD68+ cells were seen using either immunohistochemitry or immuofluorescence (Fig. 1 Α,Β)· There was no predilection for location as these cells were present in the tumor bulk as well as the infiltrating edge of tumor. CD 14 was used to -identify microglia and these cells were also detected in GBM tumor specimens via IHC and IF. Cells with posltivity for CD163, CD204, and CD206 (M2 activation, markers) were also found to he throughout the tumor specimens. There was a characteristic pattern of staining for GDI 63, and other M2 macrophage markers, showing a high content of these cells within the lumen or around of tumor-associated vessels {Fig. 1C), but they were not found on endothelial cells. Human GBM specimens were found to express M2 macrophage activation marker CD204 to a large extent (Fig. I D). Some of these cells have stellate body shape. When specimens were co-stained for CD 163 and D206, the staining overlapped eithe around tumor-associated vessels (Fig. IE) or dispersed in tumor microenvironment (Fig. IF). We then started analyzing the immunoreactivity of M2 macrophage markers in cell/tissue specimens. The established GBM cells, G4Sa, and normal brain lysates were deprived of the CD2Q4 inimtmoreactivity in contrast to all five GBM tumor lysates expressing the marker to various degrees in all of them (Fig, 1 G).

We next looked for M2 markers like CD 163, CD204 and CD2.06 in a model of monocytic cells, THP-1 (Fig, 2A), To confirm activation marker expression in macrophages, we polarized THP-1 monocytes with LPS/PMA or PMA/!L-4/iL-13 to induce Ml or 2 macrophages, respectively. MO denotes monocytes that were treated with PMA alone. Only the CD206 was present in naive TH.P-.1 cells while all three markers were expressed in MO macrophages, but dramatically decreased in MI and then re-expressed to hig levels ki M2 macrophages (Fig, 2A). The same pattern was observed for Tks5 and Fra-1 indicative of the .importance of these factors in macrophage differentiation and polarization to M2 macrophages, With this in mind, we began isolating CD 163 cells from freshly resected tumor specimens of GBM and used one isolate for further investigations.

It was surprising to see thai the CD 163+ cells actually remained in culture for more than fou weeks and they were dividing, contrary to what is expected from differentiated polarized macrophages. We could use the CD! 63 isolates even four weeks afte -separation and performed immunoblot in comparison to two established GBM cell, lines, BTCOE 4795 and G48a. Unexpectedly, these two cell lines expressed CD206 and CD68, but the exhibited very low levels of GFAP seen in GBM cells that are cultured for some time (Fig, 2B). At that time, both + and. - CD163 isolates and the -tumor from which they originated were expressing CD2Q6. and CD68 macrophage markers, but they were positive for GFAP, an astrocytic cell marker,

These intriguing findings prompted us to perform, more studies with both established

GBM cells as. well as with CD! 63 isolates. The A-172, U-2^'51, U-87 and G48a GBM cells all expressed CD206 and CD68 macrophage markers (Fig. 2C). Again, the CD163 isolates had these markers in abundance. The same ceils/tissues also expressed CD163 and only the fresher isolates or normal brain contained GFAP (Fig. 2D). Following this lead, we analyzed the pairs of GBM cells and their tumors of origin and found they readily expressed the marker of M2 macrophages, CD204 (Fig, 2E),

Considering the previousl unlikely scenario of Ml macrophage marker presence on tumors cells, we performed staining for both M2 markers and GFAP in situ. We found multiple cells staining for both using immunobistocheraistxy (Fig. 3A) and irnrmmofiuorescence (Fig. 3B), We also co-stained for CD206, an M2 macrophage marker, and Tks5₃ a protooneogene and found a number of ceils expressing both markers (Fig. 3 C and D). To further analyze cells isolated form GBM., we used magnetic beads for attachment to CD 14 monocytic cells marker. Discuss n

We demonstrated mat glioma cancer cells express M2 macrophage activation markers. The presence of M2 tumor associated macrophages in solid tumors inclusive of glioblastoma has been established previously (Holland's review, Yeshihiro Komohara et ah, 2012; Prosniak et al, 2013). The preponderance of M2 macrophages correlates with histological grade in glioma (Y Komohara et ah, 2008). Specifically there is elevated expression CD 163 and CD204 in high grade glioma (Prosniak et al, 2013). Therefore, it was not surprising to find expression of the macrophage activation markers (CD 163., CD204 and CD206) in. glioblastoma.. WHO grade 4, samples (Fig. 1). However, in an attempt to isolate these myeloid lineage ceils, we used CD163 magnetic bead separation and were perplexed that the cell had extended longevity in in vitro culture and expressed the astrocytic marker GFAP (Fig. 2). These cells thus displayed some features of glioma cancer cells.

Further investigation showed the expression of both macrophage markers (CD68) and M2 macrophage activation markers (CD 163, CD204, and CD206) on established GBM eel! lines via immimobiotting. Ceil lines derived from GBM samples also expressed CD204 and CD206 (Fig, 2). Some previous studies have shown the presence of macrophage markers on astrocytic ceils. However, no study to. date has demonstrated the presence of M2 activation markers on glial cells. Cultured malignant astrocytes were found to b immunoreactlve to the macrophage marker, CD68, and microglial marker, CD! lb (Leenstra, Das, Troost, de Boer, & Bosch, 1 95). GBM eel! lines expressed GDI 4 but this expression was lost after several passages (Pamey, Wa!dron, & Parsa, 2009), CD14 expression, was also found in glioma cell lines by RT-PCR. Cells staining for CD 14 and GFAP were found at the region of infiltrating tumor in both human and rat glioma tumor samples (Deininger, eyermann, & Schluesener, 2003).

The presence of a macrophage activation marker on primary^' central nervous system tumor cells has recently been demonstrated. anno et al. found expression of CD 163 by immunohistocfcemisfry and reverse transcription polymerase chain reaction 1ft meningioma ceils (Kanno ei aL, 2013). CD163 is a receptor of the scavenger receptor cysteitte-rich super family class B family. CD 163 function includes immune response regulation and hemoglobin scavenger activity (Latx, Quia, Weiss, 2004 Am J Clin Pathol), Anti-inflammatory signals induce its expressions and it is a moderator of anti-inflammatory pathways (Akiia, Prashant, Suma, Prasbant, & Chai!ra, 2012), CD 163 has been used as a macrophage marker. In meningioma, CD 163 expression on cancer cells correlated with histological atypia and worse prognosis. CD 163 over-expressing meningioma cells demonstrated accelerated tumor growth in nude mice indicating possible functioned changes of the tumor cells with this macrophage marker expression (Kanno et al,_s 2013). in non-CNS tumors, cancer cells have expressed CD 163. Both breast and rectal cancer cells express CD163. CD 163 expression in both cancer types could, be used as a prognostic biornarker (Shabo, Oisson, Sun, & Svanvik, 20Q9; S-habo, Stil, Olsson, Dote, & Svaavik, 2008). CD 163 expression in glioma also correlated with prognosis. Patients with grade ill gliomas with- increased CD 163 expression had significantly lower percentage survival at five years than, patients with grade III gliomas with low CD 163 expression (Prosniak ei at, .2013). However, in glioma there has not been previously demonstrated co-staining of CD 163 and glial cancer cell marker.

CD204 or scavenger receptor type A (SRA), a pattern recognition receptor, is expressed on antigen presenting cells and is a known attenuator of antitumor immunity by suppressing T cell activation (Yi et ai.,. 2012). Increased expression of CD204 on TAMs has been shown to correlate with increased tumor aggressiveness, the promotion of epifeelial-mesenehymal transition_., and poor prognosis in multiple cancer types (Hirayama et at, 2012; C.-Y, Liu et aL 2013; Shigeoka et ah, 2013). In glioma, CD204 expression is elevated in high grade cancers and correlated with poor prognosis (Yoshihtro Komohara et ah, 2012; Prosniak et al.₅ 2013). In brain, CD204 has been shown to be expressed on microglia, macrophages, and microvessels (Cornejo &. von ^' Bernhardt, 2013). CD204 is also expressed by astrocytes but these experiments were completed using a murine in vitro model (Godoy, Murgas, Tichauer, & Von Bernhardt, 2012). In situ CD204 expression on astrocytic cells is novel

CD206, mannose receptor, is increased in response to IL-4 and XL- 13. and therefore is used as a marker of M2 macrophage activation ( oc et al,_s 2009). In mammary tumors, two distinct subsets of TAMs, perivascular/migratory and sessile, have been identified. Differential expression of CD206 aids in distinguishing these subsets as the sessile TAMs in stromal and hypoxic regions has a more M2 like phenotype (Laoui e ah, 2011). in glioma, CD206 expression was present in only high grade glioma samples and not in grade Π or III tumors. The absence of CD206+ TAMs in lower grade gliomas implies this subset of CD206+ cells has a unique role in tumor progression (Prosniak ei al., 2013). Our study used glioblastoma (grade IV astrocytoma) specimens and therefore CD206 expression was present. In situ we also found CD163+/CD2G6+ cells which may implicate another TAM subpopuiaiion, as well. In the future, we will assess for these macrophage markers in d fferent glioma grades.

The presence of macrophage markers on tumor cells has now been demonstrated in several cancer types inclusive of glioblastoma. However, the mechanisms by which glioma cancer cells can co-express the cell, surface markers from two different lineages remains unknown and warrants further investigation. Several potential mechanisms, such as transdiffereniiatioa, heterotypic cell fusion, and exosomal genetic exchange, may exist Transdifferentiation implies that glioma cells can undergo phenotypic changes that correspond with macrophage properties, Glioma stem-cells have been shown to give rise to pericytes both in vitro and in vivo. Pericytes isolated from human GB.M specimens showed the same genetic alterations present in glioma tumor cells (Cheng et al., 2013), This indicates thai a subset of glioma tumor cells can adapt markedly different functional changes in comparison to the cell of origin. It may be possible for certain selective glioma cells to differentiate into cells with characteristics of macrophages.

Interestingly, proto-oncogenes that regulate cancer cell malignant features have been shown to play integral roles in macrophage activation/polarization. The .proto-oncogene, c-MYC. regulates genes associated with alternative activation in tumor associated macrophages (Pelio et al. 2012 Blood). In a murine model, CDl lb+ cells isolated from glioma tumors showed increased c-MYC gene expression (Gabrusiewicz et al, 201 1). Fra-i is also overexpressed in TAMs from breast tumors in a murine model (Luo, Zhou, Rrueger, Kaplan et al 2009, Oncogene). Interactions between TAMs and breast tumor- cells lead to Fra-1 upregulation in TAMs which, leads to increased invasion and release of pro-angiogenic factors (Luo, Zhou, Rrueger et al, 2010 Oncogene), Tks-5, which is a podosome marker protein, regulates macrophage invasion (Burger, Davis, Is m, Mishra, & Seals, 201 1), Tks-5 is also a known regulator in cancer cell invasion (Courtneidge, Azuceoa, Pass et al. 2005, Cold Spring Harb Sym). We demonstrate Tk S co-staining with the macrophage activation markers, CD206 and CD.16.3-· in GBM tumor samples. Therefore, there is a clear overlap between the genes that regulate both tumor malignancy and macrophage differentiation. Potentially, downstream phenotypic changes in glioma cancer cells induced by proto-oncogenes could result in expressio of differential cell surface markers, such as CD163, CD204, and CD2Q6.

IS Heterotypic cell fusion is another theory on how cancer cells can have markers of myeloid lineage cells. Heterotypic cell fusion can be induced by inflammation, and irradiation. Paweleck hypothesized that heterotypic cell fusion in cancer can lead to cells with the ability to metastasize (La & Kang, 2009). in fee brain, Piirkinje cells and bone-marrow derived stem cells have been shown to fuse in settings of high levels of inflammation. (Diaz, Redo, Weruaga, & Alonso, 2012), In humans Purkinje ceils binueieate heterokaryons have been seen in patients with multiple sclerosis (Kemp, Gray, Wilkins, & Scolding, 2012). in in vitro cancer models, cell fusion between macrophages and cancer ceils has been induced and these cells have stem cell like properties. (Ding, Jin, Chen, Shao, & Wu, 2012). It is theorized that macrophages and cancer cells fuse In order to give rise to ceils that have the. properties of both cells and this: new cell type has the functional ability to metastasize (Huysentruyt, Mukherjee, Banerjee, Shelton, & Seyfried. 2008), However, this remains a theory even in glioma cancer;, and has yet to be proven (Huysentruyt, Akgoc, & Seyfried, 2011).

A third option on how glioma cancer cells can express macrophage activation markers is the potential for transfer of genetic material between TA s and glioma cells. Extracellular membrane vesicles provide an avenue for intercellular communication and can subsequently lead to the induction of new functional properties of the recipient cell, Bxosomes from antigen presenting ceils can carry immune stimulatory and co- stimulatory molecules, such as MHCH and CD80/86, which can ultimately direct the immune response (Cossetti et al., 2012), Glioma cancer cells have been shown to transfer the oncogenic receptor, EGFRvIII between each other and induce phenotypic changes (Al-Nedawi et al, 2008). M2 activated macrophages have been found to secrete microRNAs via microvesicles to breast cancer cells which in turn impacted the invasiveness of the cancer cells (Yang et al, 20 1).

While the mechanism of how glioma cancer cells can express M2 macrophage activation markers remains to be investigated, this finding has many implications. Mainly it may implicate that these cells are a unique subpopu!ation that portend a new function. The increased expression of M2 markers in gliomas of higher grades implicates that M2 TAMs, and possibly 2~like glioma cells, are regulators of disease progression and can in turn determine prognosis. Glioma cells with features of an anti-inflammatory macrophage could potentially be critical in escape during cancer immr oediting (Pel!egafta, Guppini, S Finocchiaro, 2011), As our understanding of the tumor micro-environment expands, we will continue to identify new key mediators of tumor progression, References

Akiia, P., Prashant, V., Suma, M, N._} Prashant, S. N.₅ & Chaitra, T. R. (2012). CD 163 and its expanding functional repertoire. Clinica chimlca acta; international journal of clinical chemistry, 413(7-8 669-74. doi:l .1016 j.cca.2QI2.QI .O28 Al-Nedawi, K„ Meehan, B., Micalief, l.₅ Lhotak, V„ May, I,, Guha, A., &^'Rak, J. (2008).

Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells, Nature cell biology, 10(5), 619-24. doi:10.1Q38/ncbl725

Boehe, D._} Perry* V. H., & eoil, J. a R. (2013). Review: activation patterns of microglia and their identification in the human brain. Neuropathology and applied neurobiology, 39(1), 3- 18. doirlOJ l l l/nan.12011

Burger, , L,₅ Davis, A. L._s Isora, S., Mishra, N._? & Seals, D, F, (2011). The podosome marker protein Tks5 regulates -macrophage invasive behavior. Cytos eleton (Hohok n, N.J.), 68(12% 694-71.1. doi:I0.1002/cm.20545

Cheng, L,_; Huang, Z., Zhou, W., Wu, Q.₅ Donnola, S,_; Liu, J, K., ... Bao, S. (2013),

Glioblastoma, stem cells generate vascular pericytes to support vessel function and tumor growth. Cell 755(1),_.139-52. doi: 10.1016/jxeIL2 13.02.021

Cornejo, F\, & von Bernhardi, , (2013). Role of scavenger receptors in glia-mediaied

nenroinflanimaiory response associated with Alzheimer's disease. Mediators of

inflammation, 2013, 89565.1. doi: 10.1155/2013/895651 Cossetti, C, Smith. J. a, Iraci, „ Leonard!, T., Alfaro-Ceryello, C, & P!uehmo, S. (2012).

Extracellular membrane vesicles and immune regulation in the brain. Frontiers in physiology, J(May), Ϊ 17. doi:10,3389/f hys.2012.00117

Da Fonseea, A, C. C, & Badie. B. (2013). Microglia and macrophages is malignant gliomas: recent discoveries and implications for promising therapies. Clinical ά developmental immunology, 2013, 264124. doi:10.1155/2013/264124

Debinski, W.₅ & Gibo, D. M, (2011), Pos-related antigen 1 (Fra-1) pairing with and

transactivation of JunB in GBM cells, Cancer-Biology & Therapy, 11(2), 254-262, doi:10.4161/cbtl 1.2.13953 Deimnger, M. H„, Meyermann, R., & ScMuesener, H, J. (2003). Expression, and release of CD14 in astrocytic brain tumors. Acta n urop thology 106(3), 271-7. doi:l 0.1007/s00401 -003- 0727-9

Diaz, D.. Recio, J. S., Weraaga, E._s & Alonso, J. . (20.12). Mild cerebellar neurodegeneraiion of aged heterozygous PCD mice increases cell fusion of Puxkinje and bone marrow-derived cells. Cell transplantation, 2/(7), 1595-602. doi: 10.3727/096368912X638900

Ding, J._s Jin, W., Chen, C._} Shao, Z., & Wu, J. (2012). ^'Tumor associated macrophage x cancer cell hybrids may acquire cancer stem cell properties in breast cancer. PloS one, 7(7), e41942, doi: i0,1371/jouniaLpone.0041942

Eljaszewicz, A., Wiese, M,_s Helmin-Basa, A,, Jankowski, M,, Gackowska, L.„ ubiszewska, L, ... Zegarski, V. (2013). Collaborating with the enemy: function of macrophages in the development of neoplastic disease. Mediators of inflammation, 2013, 831387.

doi: 10.1 155/2013/83.1387

EIlert-Miklasze ska, A., Dabrowski, M., Lipko, M., Sliwa, M., Malesze ska. M,, & Kaminska, B. (2013). Molecular definition of file pro-tumorigenic phenoiype of ^'glioma-activated microglia. GU , ^7(7), 1178 -90. doi;.l QJO02/g!ia,22S O

Engler, J. R., Robinson, A, E,, Smimov, L, Hodgson, J. G., Berger, M. S., Gupta, ., ... Phillips, J. J. (2012). Increased microglia macrophage gene expression in a subset of adult and pediatric astrocytomas. PloS one, 7(8), e43339. doi: 10.1371/joamal.pone.0043339

Gabrusiewiez, ., EIlert-Miklaszewska, A., Lipko, M,, Sielska, M,_; Frankowska, M.„ &

Kaminska, B. (2011). Characteristics of die alternative phenoiype of microglia/macrophages, and its modulation in experimental gliomas. PloS one, 6(B), ©23902.

doi; 10.1371/joumaLpone,0Q23902

Godoy, B., urgaSj P., Tichauer. J,, & Von Bernhardi, R. (2012). Scavenger receptor class A Hgands induce secretio oi^'ILlp and exert a modulatory effect on the. inflammatory

.activation of astrocytes in culture. Journal of rte roimmunology, 251(1-2), 6-13.

doi: 10.1016/j jneuroim.2012.06.004 Gordon, S,, & Martinez, F, G, (2010). Alternative activation of macrophages: mechanism and functions. Immunity, 32(5% 593-604, doi:10.1016/j.immuni.2010.05.007

Hirayania, S., Is i, G., Nagai, K», Ono, S._f Kojiraa, M._; Yamauchi, C, ... Ochiai, A, (2012), Prognostic impact of CD204-positive macrophages in lung squamous cell carcinoma: possible contribution of Cd204-pos.itive macrophages to the tumor-promoting

raicroenvironment. Journal of thoracic oncology : official publication of the international Association for the Study of Lung Cancer, 7(12), 1790-7.

dot: 10.1097/JTO.ObOl 3e3182745968

Hussain, S. F.. Yang, IX Suki, IX, Aldape, ., Grimm, E,_s & H mberger, A. B. (2006), The rol of human glioma-infiitrating microglia/macrophages in mediating antitumor immune responses. Neuro-onco gy, 8(5), 261-79, doi:i 0.1215/15228517-2006-008

Huysentruyt, L. C.₅ Akgoc, Z,, & Seyfried, T. N. (2011). Hypothesis: are neoplastic

macrophages/microglia present in glioblastoma multiforme? ASNneuro, 3(4), 183-1 3, doi:10.1042/AN2011001 1

Huysentruyt, L, C, Miikherjee, P., Banerjee, D.. Sheltort, L, M, & Seyfried, T. N, (2008), Metastatic cancer ceils with macrophage properties: evidence from a new murine tumor model. International journal of cancer. Journal international du cancer, 123(1% 73-84. doi:i0,1002/ije.23492

Joseph, B,, & Venero, J, L, (2013), A brief overview of multitalented microglia. Methods in molecular biology (Clifton, NJ.), 1041, 3-8, doi: 10. lO07/978-I-627O3-52O^«O_l

Kanno, ^'Η,, ^'Nishihara, H., Wang, L., Yuzawa, Kobayas , B,₅ Tsuda, M., ... Taisaka, S, (2013). Expression of GDI 63 prevents apoptosis through the production of granulocyte colony-stimulating factor in meningioma. Neuro-oncotogy, 15(7), 853-64.

doi; 10.1 -093/neuonc not028

Kemp, K._? Gray, E., Wilkins, A,, & Scolding, N. (2012). Purkinje ceil fusion and biniicleate heterokaryo formation in multiple sclerosis cerebellum. Brain ; a journal of neurology, I3S(H 10), 2962-72. doi:t0,l093/bram/aws226 Komohara, Y, Orinishi, K., Kuratsu, J.,.& Takeya, M. (2008). Possible involvement of the M2 anti-infl^'aiHniatory macrophage phenotype In growth of human gliomas, (April), 15-24. doi:10.1GG2/path

Komohara, Yoshihiro, Hortad, H,, Ohnishi, K., Fujiwara, . Y„ Bat, B., Nakagawa,.T., ... Takeya, . (2012), importance of direct macrophage-tumor cell interaction on progression of human glioma. Cancer science, 103(12), 2165-72. doi:10.1111/cas.l2015

Laoui, D., Movahedi, K., Oyermeire, E, V. A. V. A. N., Bossche, J. A. N, V, A. N, D. E» R, Schouppe, E., ommer, C._» ... Ginderaehter, J. O. A, V. A. N. (201 !). Tumor-associated macrophages in breast cancer : distinct subsets , distinct functions, (November), 861-867. doi:10,B87/ijdb.

Leenstra, S.. Das, P. ,, Troost, D., de Boer, O. J., & Bosch, D. a. (1 95). Human malignant astrocytes express macrophage phenotype. Journal of neuroimmunology, 56(1% 17-25, Retrieved .from htt ://www , ncbi.nlm.nih. go v/pubnied 7822478

Liu, C.-Y., Xu, J.-Y., Shi, X.-Y., Huang, W., Ruan, T.-Y., Xie, P., & Ding, J.-L. (2013). M2- polarized tumor-asaociated macrophages promoted epithelial-rnesenchymal transition in pancreatic .cancer cells, partially through. TLR4 tL-!0 signaling pathway. Laboratory investigation; a journal of technical methods and pathology, 93(7), 844-54.

doi:10.i03 abmvest2013.69

Liu, G., & Yang, H. (2013). Modulation of macrophage activation and programming in

immunity. Journal of cellular physiology, 228(3), 502- 12. doi: 10.1002/jcp.24157

La, X._s & Kang, Y. (2009). Efficient acquisition of dual metastasis organotropism- to bone and lung .through stable spontaneous fusion between MD A -MB -231 variants. Proceedings of the National Academy of Sciences of the United States of America, 106(23), 9385-90.

doi:l 0.1073/pnas.0900108106 Lu-Emetson, C, Stmderl, M., Kirkpatrick, N. D.„ Goveia J., Davidson, C, Huang, Y,, ... Jain, R, . (2013). increase In tumor-associated macrophages after antiangiogenic therapy is associated with poor survival among patients with recurrent glioblastoma. Neitro-oncology, 75(8), 1079-87. doi:10J093/neuonc/not082

2.3 Mantovani, A,₅ Germane, G., archesi, F._s Locateili, M._s & Biswas, 8. K, {2011}. Cancer- promoting tumor-associated macrophages; new vistas and open questions. European journal of immunology, 41(9% 2522-5. doi: 10.1002/eji.201141894

.Paraey, I F._s Waldron, J. S., & Parsa, A. T, (2009). Flow cytometry and in vitro analysis of human glioma- associated macrophages. Laboratory investigation. Journal of neurosurgery, 110(3% 572-82, doi; 10.3171/2008.7. JNS08475

Pellegatta, S._} Cuppini, L., Sc Finocc aro, G. (20! 1). Brain cancer immunoediting: novel

examples provided by immunotherapy of malignant gliomas. Expert review of anticancer ikerapy, 11(11% 1759-74. doi:10.1S86/era.l.U02

Prosniak, M.₅ Harshyne, L. a, Andrews, Ϊ). W.₅ Kenyon, L. C, Bedelbaeva, ._» Apanasovich, T.

V, ... Hooper, D. C. (2013). Glioma grade is associated with the accumulation and activity of cells bearing ni2 monocyte markers. Clinical cancer research : an official journal of the American Association for Cancer Research, 19(14% 3776-86. doi; 10.1158/1078- 0432.CCR-12 940

Roca, H., Varsos, 2. S., S d, S.,. Craig, M. J,_f Ying, C, & Psenia, K. X (2009). CCL2 and

inter leukin-6 promote survival of human CD! 1 b+ peripheral blood mononuclear cells and induce M2-type macrophage polarization. The Journal of biological chemistry, 284(49), 34342-54. doi: 10.1074/jbc.M10 .G42671

Saijo, K., & Glass, C. K. (2011). Microglial cell origin and pfaenotypes in health, and disease.

Nature reviews. Immunology, 11(11% 775-87. doi:10.1038/nri3086

Shabo, I., Olsson, H._s Sun, X.-F,, Sc Svanvik, J. (2009). Expression of the macrophage antigen CD 163: in rectal cancer cells is associated with early local recurrence and reduced survival time. International journal of cancer. Journal international du cancer, 725(8), 1826-31. doi: 10.1002/ijc.24506

Shabo, I., St l, O., Olsson, H., Dore, S., & Svanvik, J. (2008). Breast cancer expression of

CD163, a macrophage scavenger receptor,, is related to early distant recurrence and reduced patient survival. International journal of cancer. Journal inter national du cancer, 123(4% 780-6, doi; 10,10^'02/ijc23527 Shigeoka, M., Urakawa, N,_; Nakamura, T., Nishio, M., Watajima, T,, Kuroda, D._s ... Yokozaki,

H, (2013). Tumor associated macrophage expressing CD204 is associated with tumor aggressiveness of esophageal squamous cell carcinoma. Cancer science, 104(2), 1 2-9, doi:iOJ i il/cas.I2188 Wang, Q._> Ni, Ή., Lan, L., Wei, X., Xiang, R., & Wang, Y. (2010). Fra-1 protooncogene

regulates IL- 6 expression in macrophages and promotes the generation of M2d

Macrophages. Cell research, 20(6), 701-12. doi:10.1038/cr.20I0.52

Wu_s A., Wei, I, Kong, L.-Y., Wang, Y„ Priebe, W., Qiao, W._? ... Heimberger, A. B. (2010).

Glioma cancer stem cells induce immunosuppressive macrophages/microglia. Neuro- oncology, 12{\ l), 1113-25. doi: 10.1093/neuonc/noq082

Yang, M., Chen, J., Su. F._s Yu, B., Su, F., Lin, L., ... Song, E, (2011). Microvesicles secreted by macrophages shuttle invasion-potentiating rmcroRNAs into breast cancer cells. Molecular cancer, 10, 117. doi:10.1186/1476-4598-10-117

YL H..„ Zuo, D., Yu, X., His, F._? Manjili, M, H., Chen, Z., Wang, X.-Y. (2012). Suppression of^' antigen-specific CD4+ T cell activation by SRA/CD204 through reducing the

ί mm TOO stimulatory capability of antigen-presenting cell. Journal of molecular medicine (Berlin, German}?), 90(4), 4 3-26, doi: 10.1007/s0010.9-01 i -0828-1

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equ alents of the claims to be included therein.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting malignancy in a solid tumor, comprising:

providing cancer cells isolated from said tumor; and

detecting the expression of one or more macrophage markers by said cancer cells, wherein the expression of one or more macrophage markers by said cancer ceils indicates malignancy of said tumor.

2. The method of claim 1, wherein said malignancy Indicates an increased risk of cancer ceil invasion.

3. The method of claim 1, wherein said malignancy indicates an increased risk of metastasis,

4. The method of any one of claims 1-3, wherein said macrophage markers are M2 macrophage markers,

5. The method of any one of claims 1-3, wherein said macrophage markers comprise one or more markers selected from the group consisting of: CD 14, CD68, CD! lb, CD.163, CD204, and CD206.

6. The method of any one of claims 1 -3, wherein said macrophage markers comprise one or more markers selected from the group consisting of: CD68, CD 163, CD204 and CD206,

7. The method of any one of claims I -6, wherein said method further comprises: delecting the presence of one or more microglial markers in said cancer cells, wherein the presence of said one or more macrop age markers and one or more microglial markers in said cancer cells indicates malignancy of said tumor,

8. The method of claim 7, wherein said one or more microglial markers comprise

^'CDl lb.

9. The method of any one of claims 1- 8, wherein said tumor is a glioma.

10. The method of any one of claims 1-8, wherein the tumor is an astrocytoma, meningioma or oligodendroglioma.

11. The method of an one of claims 1 -8, wherein said tumor is glioblastoma ,

12. The method of any one of claims 1-11, wherein said cancer cells are human.

13. The method of any one of claims 1-12, wherein said detecting is carried out by immunolocalization of said cancer cells,

14. The method of any one of claims 1-12, wherein said detecting is carried out by immunohistochemistry (IHC). imm nofluorochemistry (IFC) or fluorescent activated cell sorting (FACS).

15. A method of grading a glioma, comprising;

providing cancer cells isolated from said glioma; and

detecting the expression of one or more macrophage markers by said cancer cells, wherein the expression of one or more macrophage markers by said cancer cells indicates an advanced grade of said glioma.

16. The method of claim 15, wherein said glioma is glioblastoma mulitfomie (GBM).

17. The method of claim 15 or claim 16, wherein said cancer cells are human,

18. The method of any on of claims 15-17, wherein said detecting is carried out by immunoloealization of said cancer cells.

1 . The method of any one of claims 15-17, wherein said detecting is carried out by immunohistochemisiry (IHC), immunofluofochemistry (iFC) or fluorescent activated cell sorting (FACS)-

20. A method of treating a glioma comprising,

providing cancer cells, isolated from said glioma; and detecting the expression of one or more macrophage markers by said cancer cells and, if said cells express said one or more macrophage markers, treating said glioma.

21. The method of claim 20, wherein said treating comprises administering one or more of: radiation therapy, chemotherapy and targeted protein therapeutics,

22. The method of claim 20 or claim 21, wherein said glioma is glioblastoma multiforme (GBM),

23. The method of any one of claims 20-22, wherein said cancer cells are human.

24. The method of any one of claims 20-23, wherein said detecting is carried out by immunoloe&lizaiion of said cancer cells.

25. The method of any one of claims 20-23, wherein, said detecting is carried out by imrauno stochemis ry (IHG), im oBoiluorochemkiry (IFC) or fluorescent activated cell sortin (FACS).

26. The method of any one of claims■ 1-25, wherein said providing step comprises collecting said cells from a subject, and then:

separating said cancer cells from other cells by:

immimoisolati on ;

culturing said ceils for at least 2, 3, or 4 weeks prior to said detecting step;

applying acoustic waves;

freezing said cells after collecting from the subject, and then washing said cells; and/or fixing said cells (e.g., with an aldehyde or permanganate fixative) and then embedding said cells in a wax (e.g., paraffin) support.