WO2013102118A2

WO2013102118A2 - Imageable patient tumors in mice

Info

Publication number: WO2013102118A2
Application number: PCT/US2012/072134
Authority: WO
Inventors: Robert M. Hoffman
Original assignee: Anticancer, Inc.
Priority date: 2011-12-30
Filing date: 2012-12-28
Publication date: 2013-07-04
Also published as: WO2013102118A3

Abstract

The present invention relates to a fluorescent animal model for monitoring tumor-stroma interactions during progression, angiogenesis and/or metastasis of tumor cells, methods of producing such animal models, and methods of using such animal models, for example, for assaying the effects of a drug on tumor-stroma interactions during progression, angiogenesis and/or metastasis of tumor cells. The animal model comprises an immunocompromised or syngeneic experimental animal implanted with tumor cells and stromal cells, wherein said stromal cells express one or more fluorescent proteins, such as green fluorescent protein (GFP).

Description

IMAGEABLE PATIENT TUMORS IN MICE

Cross-Reference to Related Application

[0001] This application claims priority from U.S. provisional application 61/581,901 filed 30 December 2011. The contents of this document are incorporated herein by reference.

Field of the Invention

[0002] The invention relates to the study of tumor progression. Specifically, it concerns model animals for studying the tumor-stroma interactions during progression, angiogenesis and/or metastasis of tumor cells and methods for evaluating candidate drugs using the model animals.

Background Art

[0003] The athymic, T-cell-deficient, nude mouse has made a very important contribution to cancer research in that it enabled the systemic serial transplantation of human tumors and cell lines (Rygaard and Povlsen, Acta Pathol Microbiol Scand 77:758-760 (1969)). Our laboratory pioneered surgical orthotopic implantation (SOI) metastatic nude-mouse models of patient tumor specimens in the early 1990s (Fu et al., Proc Natl Acad Sci USA 89: 5645-5649 (1992); Fu et al., Proc Natl Acad Sci USA 88: 9345-9349 (1991)).

[0004] These orthotopic mouse models of patient tumors are more patient-like, especially with regard to metastasis, than ectopic subcutaneous models (Rygaard and Povlsen, Acta Pathol Microbiol Scand 77:758-760 (1969); Pickard et al., Br J Cancer 31:36-45 (1975); Giovanella et al., Cancer 42:2269-2281 (1978); Nowak et al., Br J Cancer 37:576-584 (1978); Bailey et al., Br J Cancer 42:524-529 (1980); Selby et al., Br J Cancer 42:438-447 (1980); Fiebig et al., Dig Surg 1:225-235 (1984), Fiebig et al., Eur J Cancer Clin Oncol 23:937-948 (1987); Sharkey and Fogh, Cancer Metastasis Rev 3:341-360 (1984); Steel, In: Sordat B, editor, Immune-deficient animals, 4th Int. Workshop on Immune-Deficient Animals in Exp. Res. (Chexbres, Oct. 31-Nov 3, 1982), Switzerland: Karger. pp 395-404 (1984); Hwang et al., Clin Cancer Res 9:6534-6544 (2003); Embuscado et al., Cancer Biol Ther 4:548-554 (2005); Rubio-Viqueira et al., Clin Cancer Res 12:4652-4661 (2006); Garber, J Natl Cancer Inst 99: 105-107 (2007); Talmadge et al., Am J Pathol 170:793-804 (2007); Bertotti et al., Cancer Disc 1:508-523 (2011)). In our initial development of SOI nude mouse models of patient tumors, we achieved take rates of 65% for colon cancer (Fu et al., Proc Natl Acad Sci USA 88: 9345-9349 (1991)) and 100% for pancreatic cancer. Subsequently, the NOD/SCID mouse, was developed (Shultz et al., J Immunol 154: 180- 191 (1995)). This mouse is deficient in T-, B- and NK cells and allows for higher take rates of patient tumors including 87% for colorectal cancer liver metastasis (Bertotti et al., Cancer Disc 1:508-523 (2011)). Patient pancreatic tumors have been transplanted to NOD/SCID mice at high frequency (Kim et al., Ann Surg Oncol 19: 395-403 19: 395-403 (2011); Kim et al., Nat Protol 4: 1670-1680 (2009)). However, these models are limited with regard to studying the tumor microenvironment as well as imaging.

[0005] Citation of documents herein is not intended as an admission that any is pertinent prior art. All statements as to the date or representation as to the contents of documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of the documents.

Disclosure of the Invention

[0006] The invention takes advantage of the selective expression of fluorescent proteins in stromal cells acquired by primary tumor cells to use the fluorescence generated as a guide for studying tumor-stroma interactions during progression, angiogenesis and/or metastasis of tumor cells.

[0007] Thus, in one aspect, the invention is directed to a method to produce a fluorescent animal model for monitoring tumor-stroma interactions during progression, angiogenesis and/or metastasis of tumor cells, which method comprises: a) providing tumor cells from a subject; and b) implanting the tumor cells obtained from step a) into a first immunocompromised or syngeneic experimental animal expressing a fluorescent protein.

[0008] In some embodiments, the method may further comprise: c) implanting the tumor cells obtained from step b) into a second immunocompromised or syngeneic experimental animal. In some embodiments, the method may further comprise: d) implanting the tumor cells obtained from step c) into a third immunocompromised or syngeneic experimental animal. In some

embodiments, the immunocompromised or syngeneic experimental animal may be a mouse, wherein the mouse may be a NOD/SCID or nu/nu mouse. In some embodiments, the tumor cells are taken directly from a patient. In some embodiments, the first immunocompromised or syngeneic experimental animal expresses red fluorescent protein (RFP). In some embodiments, the second immunocompromised or syngeneic experimental animal expresses green fluorescent protein (GFP). In some embodiments, the third immunocompromised or syngeneic experimental animal expresses cyan fluorescent protein (CFP). In some embodiments, the third

immunocompromised or syngeneic experimental animal does not express a fluorescent protein. In some embodiments, the tumor cells from step a) are implanted into an immunocompromised or syngeneic experimental animal before step b). In some embodiments, the tumor cells are implanted by subcutaneous injection. In some embodiments, the tumor cells are implanted by surgical orthotopic implantation (SOI).

[0009] In another aspect, the invention is directed to a fluorescent animal model for monitoring tumor-stroma interactions during progression, angiogenesis and/or metastasis of tumor cells, comprising an immunocompromised or syngeneic experimental animal implanted with tumor cells and stromal cells, wherein said stromal cells express one or more fluorescent proteins.

[0010] In some embodiments, the tumor cells are an intact segment of a tumor. In some embodiments, the tumor comprises the stromal cells that express one or more fluorescent proteins. In some embodiments, the stromal cells express RFP. In some embodiments, the stromal cells express GFP. In some embodiments, the stromal cells express CFP. In some embodiments, the stromal cells express both GFP and RFP. In some embodiments, the stromal cells express both GFP and CFP. In some embodiments, the stromal cells express both RFP and CFP. In some embodiments, the stromal cells express GFP, RFP and CFP. In some embodiments, the tumor cells are tissues or of brain, lung, liver, colon, breast, prostate, ovary or pancreas.

[0011] In a third aspect, the invention is directed to a method to monitor tumor-stroma interactions during progression, angiogenesis and/or metastasis of tumor cells using the fluorescent animal model disclosed herein.

[0012] In some embodiments, the fluorescent stromal cells can be imaged either in vivo or ex vivo. In some embodiments, the fluorescent stromal cells can be imaged in the living animal. In some embodiments, the fluorescent stromal cells can be non-invasively imaged in the living animal. In some embodiments, the fluorescent stromal cells are monitored in the third

immunocompromised or syngeneic experimental animal not expressing a fluorescent protein.

[0013] In a fourth aspect, the invention is directed to a method to assay the effects of a drug on tumor-stroma interactions during progression, angiogenesis and/or metastasis of tumor cells using the fluorescent animal model disclosed herein comprising: contacting the animal with said drug, imaging tumor-stroma interactions by observing emissions of said one or more fluorescent proteins, and comparing the resulting images to a control animal not contacted with said drug. [0014] In some embodiments, the fluorescent stromal cells can be imaged either in vivo or ex vivo. In some embodiments, the fluorescent stromal cells can be imaged in the living animal. In some embodiments, the fluorescent stromal cells can be non-invasively imaged in the living animal. In some embodiments, the fluorescent stromal cells are monitored in the third

Brief Description of the Drawings

[0015] Figure 1 shows that GFP host stromal cells infiltrate orthotopic primary pancreatic cancer tumorgrafts. A: Left panel shows host green fluorescent protein (GFP)-expressing mouse pancreas. Right panel shows primary tumor image. Yellow arrows indicate tumor-associated macrophages (TAMs). White arrows indicate cancer-associated fibroblasts (CAFs). Images were taken with an Olympus FVIOOO scanning laser microscope. Bar=20 μιη. B: Excised primary tumor was stained with H&E. Blue arrows indicate pancreatic tubular adenocarcinoma. Bar=100 μιη.

[0016] Figure 2 shows that GFP host stromal cells infiltrate peritoneal disseminated metastases of orthotopic pancreatic cancer tumorgrafts. A: Image of disseminated peritoneal metastasis. Red arrow indicates disseminated peritoneal metastasis with green fluorescent protein (GFP) stroma. Bar=10 mm. B: Image of disseminated peritoneal metastasis. Yellow arrows indicate tumor- associated macrophages (TAMs). White arrows indicate cancer-associated fibroblasts (CAFs). Image was taken with an Olympus FVIOOO confocal laser microscope. Bar=20 μιη. C:

Disseminated peritoneal metastasis stained with H&E. Blue arrows indicate pancreatic tubular adenocarcinoma. Bar=100 μιη.

[0017] Figure 3 shows that GFP host stromal cells infiltrate liver metastases of orthotopic pancreatic cancer tumorgrafts. A: Upper image shows liver metastasis. Red arrows indicate liver metastasis with green fluorescent protein (GFP) stroma. Bar=10 mm. Lower panel shows high- magnification image of liver metastasis. Bar=l mm. Red arrows indicate GFP stroma. B: Image of liver metastasis. Yellow arrows indicate tumor-associated macrophages (TAMs). White arrows indicate cancer-associated fibroblasts (CAFs). Image was taken with an Olympus FVIOOO confocal laser microscope. Bar=20 μιη. C: Liver metastasis stained with H&E. Blue arrows indicate pancreatic tubular adenocarcinoma. Yellow arrows indicate stromal cells. Red arrows indicate hepatocytes. Bar=100 μιη.

[0018] Figure 4 is a graph that shows the effect of UV irradiation on fluorescent dual-labeled tumor cell lines. A: Flow diagram of the experimental protocol. B: Orthotopic tumorgraft model (F2) of human pancreatic-cancer-patient tumor transplanted to RFP transgenic nude mouse. Yellow arrow indicates host RFP nude mouse pancreas. Blue arrow indicates tumor with infiltrating RFP stroma (Bar=10 mm). Image taken with the Olympus OV100. C: Human pancreatic tumor excised from RFP nude mouse with RFP stroma. The image is of a cross-section of the tumor. Blue arrow indicates RFP stroma (Bar=10 mm). Image taken with the Olympus OV100. D: Visualization of RFP tumor-associated macrophages (TAMs) in the human pancreatic cancer patient tumor (F2). High-magnification image taken with the Olympus FV1000 confocal microscope. Yellow arrows indicate RFP TAMs (Bar=50 μιη). E: Orthotopic tumorgraft model (F3) of human pancreatic- cancer patient tumor growing in transgenic GFP nude mice for 30 days. Red arrow indicates host GFP nude mouse pancreas. Blue arrow indicates human pancreatic tumor with RFP stroma (Bar=10 mm). Image taken with the Olympus OV100. F: Pancreatic tumor growing in GFP-host model for 56 days. Red arrow indicates host GFP nude mouse pancreas. Blue arrow indicates human pancreatic tumor with RFP+GFP stroma (Bar=10 mm). Image taken with the Olympus OV100. G: Excised tumor with RFP and GFP stroma. The image is of a cross-section of the tumor. Yellow arrow indicates RFP stroma. Green arrow indicates GFP stroma (Bar=10 mm). Image taken with the Olympus FV1000. H: Human pancreatic-cancer-patient tumor (F3) with RFP and GFP stromal cells. Image was taken with the Olympus FV1000. Green arrows indicate GFP stromal cells from GFP mouse. Red arrows indicate RFP stromal cells from RFP mouse. (Bar=50 μιη) I: Human pancreatic-cancer-patient tumor with RFP stromal cells and GFP TAMs. (Bar=100 μιη). Image taken with the Olympus FV1000. J: High magnification image of (I). RFP stromal cells and GFP-TAMs are readily observed. (Bar=30 μιη). Image taken with the Olympus FV1000.

[0019] Figure 5 shows that CFP host stromal cells infiltrate orthotopic pancreatic cancer tumorgrafts to form a 3-color stroma model. A: Human pancreatic-cancer-patient tumor growing in CFP-host (F4). White arrow indicates host CFP nude mouse pancreas. Blue arrow indicates tumor. Image was taken with the Olympus MVX10 microscope (Bar=10 mm). B: Human pancreatic-cancer-patient tumor (F4) (blue arrow) with RFP, GFP, and CFP stromal cells. Red arrow indicates CFP pancreas. Image was taken with the MVX10 (Bar=10 mm). C: RFP, GFP, and CFP stromal cells were observed. Red arrow indicates RFP stromal cells. Green arrows indicate GFP stromal cells. White arrows indicate CFP stromal cells (Bar=100 μιη). Image taken with the FV1000 confocal microscope. D: RFP TAMs (red arrow) and GFP blood vessel (green arrow) were observed in the F4 tumor (Bar=100 μιη). Image taken with the Olympus FV1000 confocal microscope. E: GFP blood vessels (green arrows) in the F4 tumor. (Bar=100 μιη). Image was taken with the FV1000 confocal microscope. F: RFP CAFs (yellow arrow) and GFP TAMs (green arrows) in the F4 tumor. White arrow indicates CFP CAFs. (Bar=30 μιη). Image was taken with the FV1000 confocal microscope.

[0020] Figure 6 shows non-invasive imaging of fluorescent tumor from (a) patient with pancreatic cancer growing orthotopically in nude mice. A: Flow diagram of the experimental protocol. B: Whole-body non-invasive imaging of human pancreatic cancer orthotopic tumorgraft in non-transgenic nude mice. Mice were non-invasively imaged at day-21 (upper row), day-30 (middle row) and day-74 (lower row). The tumors in the non-transgenic nude mice are in the F4 passage after Fl, in NOD/SCID mice after patient surgery; F2, in transgenic green fluorescent protein (GFP)-expressing nude mice; and F3 in transgenic red fluorescent protein (RFP)- expressing nude mice. The tumor acquired GFP and RFP stroma in the F2 and F3 passages, respectively. Green arrows indicate tumor with RFP stroma. Red arrows indicate tumor with GFP stroma. Images were taken with the Olympus OV100 Small Animal Imaging System. C: Image of human pancreatic cancer tumor tissue resected from the F4 passage with RFP and GFP stroma. Images were taken with an FV1000 confocal laser microscope. Left panel, RFP-expressing and GFP-expressing cancer-associated fibroblast cells (CAFs). Right panel, RFP-expressing blood vessels and GFP-expressing tumor- associated macrophages (TAMs). Yellow arrows indicate RFP- expressing CAFs. Blue arrows indicate GFP-expressing CAFs. White arrows indicate GFP- expressing TAMs. Red arrows indicate RFP-expressing blood vessels.

Modes of Carrying Out the Invention

[0021] The present invention relates to a fluorescent animal model for monitoring tumor- stroma interactions during progression, angiogenesis and/or metastasis of tumor cells, methods of producing such animal models, and methods of using such animal models, for example, for assaying the effects of a drug on tumor-stroma interactions during progression, angiogenesis and/or metastasis of tumor cells. The animal model comprises an immunocompromised or syngeneic experimental animal implanted with tumor cells and stromal cells, wherein said stromal cells express one or more fluorescent proteins, such as green fluorescent protein (GFP).

[0022] As used herein, "GFP" refers to a fluorescent protein of whatever wavelength emitted as well as "enhanced" forms of GFP and the Aequorea victoria green fluorescent protein. The description of transplanted cells or tissues labeled with a visible indicator, such as fluorescent dyes and as generally known in the art, is selected such that a different color fluorescent protein is used in the transplanted cells or tissues in comparison to the fluorescent protein expressed in the host. As a non-limiting example, if green GFP is expressed in the transgenic rodents of the invention, then the transplanted cells or tissues may express red fluorescent protein (RFP).

[0023] Suitable vertebrate subjects for use as models are preferably mammalian subjects, most preferably convenient laboratory animals such as rabbits, rats, mice, and the like. For closer analogy to human subjects, primates could also be used. Particularly useful are subjects that are particularly susceptible to tumor development, such as subjects with impaired immune systems, typically NOD/SCID or nu/nu mice. Any appropriate vertebrate subject can be used, the choice being dictated mainly by convenience and similarity to the system of ultimate interest.

[0024] In some embodiments, the immunocompromised or syngeneic experimental animal may be an immunocompromised rodent, such as a NOD/SCID or nu/nu mouse. The

immunocompromised rodents may be used as a source of cells and/or tissues that express GFP. Non-limiting examples of such tissues include an embryo or embryo tissues; stem cells, and cells or tissues of the brain, liver, kidney, pancreas, adrenal gland, testis (including testicular cells), lung, muscle, heart, intestine, ovary and spleen as well as adipose tissue.

[0025] The fluorescent animal model may be obtained by providing tumor cells from a subject and implanting the tumor cells into an immunocompromised or syngeneic experimental animal expressing a fluorescent protein, wherein the tumor may acquire stromal cells from the host, such as cancer-associated fibroblast cells (CAFs), tumor-associated macrophages (TAMs), etc. In some embodiments, the resulting tumor including the stromal cells acquired from the host may be further implanted to another immunocompromised or syngeneic experimental animal expressing a fluorescent protein. This implantation step may be repeated 2, 3, 4, 5 or more times, resulting in a tumor that contains stromal cells from multiple hosts. In some embodiments, the tumor that contains fluorescent stromal cells may be implanted into a non-fluorescent host for the observation of the fluorescent stromal cells over a period of time.

[0026] Techniques for implantation of the solid tumors into vertebrates include direct implantation by surgical orthotopic implantation (SOI) at the desired site, typically the site from which the tumor cells were derived. Suitable sites include lung, liver, pancreas, stomach, breast, ovary, prostate, bone marrow, brain, and other tissues susceptible to malignancy. Once the solid tumors have been implanted, the vertebrate becomes a model system for studying metastasis. The tumor is thus allowed to progress and develop and the vertebrate is monitored for appearance of the stromal cells that express one or more fluorescent proteins. The monitoring can occur either on the whole vertebrate by opening the animal and observing the organs directly with a fluorescent microscope, or the tissues may be excised and examined microscopically. In some cases the tumors are sufficiently bright that opening the animal is unnecessary— they can be seen directly through the skin. In any case, as GFP is visible to the naked eye, no development systems to stain the tissue samples are required. Tissue samples are simply properly processed as fresh samples in slices of suitable size, typically 1 mm thick, and placed under a microscope for examination. Even colonies of less than 10 cells are thus visible. A variety of microscopic visualization techniques is known in the art and any appropriate method can be used.

[0027] In some embodiments, the stromal cells from the different hosts may express fluorescent proteins that have different colors, allowing the observation of tumor-stroma interaction over a period of time. Non-limiting examples of other fluorescent colors include yellow, blue, and far-red. The expression of other fluorescent indicators may optionally be specific to individual cell types, genes or processes. Non-limiting examples of how to provide such specificity include by operably linking sequences encoding the fluorescent indicators to be under the regulatory control of a promoter that is cell specific, a promoter that is responsive to particular activation events, a promoter that regulates the expression of a particular gene of interest, and a promoter that regulates the expression of a gene product involved in a cell process of interest.

[0028] The use of fluorescent proteins for imaging in vivo was pioneered by out laboratory and has been particularly useful to study tumor growth and progression (Hoffman and Yang, Nat Protoc 1:775-782 (2006); Hoffman and Yang, Nat Protoc 1:928-935 (2006); Hoffman, Nat Rev Cancer 5:796-806 (2005)). With the use of multiple colored fluorescent proteins, we developed imaging of the tumor microenvironment (TME) by color-coding cancer and stromal cells (Yang et al., Proc Natl Acad Sci USA 100: 14259-14262 (2003); Yang et al., Cancer Res 64:8651-8656 (2004); Yang et al., Cancer Res 67:5195-5200 (2007); Yang et al., J Cell Biochem 106:279-284 (2009); Hoffman and Yang, Nat Protoc 1:928-935 (2006); Suetsugu et al., J Cell Biochem

112:949-953 (2011); Suetsugu et al., Anticancer Res 32:31-38 (2012)). With the use of color- coded imaging technology, we have previously demonstrated the essential role of tumor-associated host cells in tumor progression and metastasis (Bouvet et al., Cancer Res 66: 11293-11297 (2006)).

[0029] U.S. Patents 5,726,009; 6,232,523; 6,235,967; 6,235,968; 6,251,384; 6,372,489;

6,649,159; 6,713,273; 7,067,496; 7,625,550; 7,666,675; and 8,148,600, as well as PCT

applications WO9004017; and WO2005057488, and U.S. applications US2002/0132318;

US2003/0031628; US2003/0161788; US2005/0170330; and US2011/0033388, all of which are incorporated herein by reference, describe techniques for labeling tumors and their metastases using fluorescent markers, especially green fluorescent protein (in various colors) as well as detecting angiogenesis using this type of labeling, optionally along with a contrast dye. According to the descriptions of these documents, it is possible to detect tumor progression, angiogenesis, and metastasis by excising the tumor tissue, or, perhaps more benevolently, in real time by following the course of the condition through whole body imaging.

[0030] Turning again to the fluorescent immunocompromised or syngeneic experimental animals of the invention, these may be used to visualize gene expression in the manner taught by Yang et al., Proc. Natl. Acad. Set USA 97: 12278-12282 (2000), which describes visualization, by noninvasive techniques, transgene expression in intact animals. That system permits rapid visualization of transgene expression in major organs of intact live mice which is simple, rapid, and eminently affordable. Against the background of the GFP transgenic, a fluorescent protein of different color is expressed in the cells such as those of brain, liver, pancreas, prostate, and bone, and its fluorescence is encoded in whole-body optical images. As non-limiting examples, higher- magnification imaging may be performed with a trans-illuminated epifluorescence dissecting microscope while low-magnification imaging may be performed atop a fluorescence light box and directly viewed with a thermoelectrically cooled color charge-coupled device camera, or using simpler LED-based devices.

[0031] The transplantation of tissues modified to contain fluorescent protein with a different emission spectrum from that of the host can be practiced to a limited extent with

immunocompetent subjects as well. In order to practice this aspect of the invention in

immunocompetent subjects, the transplanted tissue must be syngeneic or the observations must be limited to short term exploration of an immune response or other response, including rejection of the transplant.

[0032] Since sufficient intensity can be achieved to observe the migration of fluorescent cells in the intact animal, in addition to determining the migration of the cells by excising organs, the progression of metastasis can be observed in the intact subject. Either or both methods may be employed to observe metastasis in evaluating, in model systems, the efficacy of potential anti- metastatic drugs. The success or failure of treatments provided to patients with potentially metastatic cancers can also be followed using the materials and methods of the invention.

[0033] It is particularly convenient to visualize the migration of tumor cells in the intact animal through fluorescent optical tumor imaging (FOTI). This permits real-time observation and monitoring of progression of metastasis on a continuous basis, in particular, in model systems, in evaluation of potential anti-metastatic drugs. Thus, the relative lack of metastasis observed directly in test animals administered a candidate drug in comparison to controls which have not been administered the drugs indicates the efficacy of the candidate and its potential as a treatment. In subjects being treated for cancer, the availability of FOTI permits those devising treatment protocols to be informed on a continuous basis of the advisability of modifying or not modifying the protocol.

[0034] In addition, the development of the tumor can be studied in vitro in histological culture. Suitable systems for such study include solid supported cultures such as those maintained on collagen gels and the like.

[0035] The tools useful in the present invention are described in the publications, U.S. patents, and patent applications incorporated by reference above. Whole body imaging, the nature of fluorescent proteins useful in the invention, and methods to label entire animals have been described in these documents.

[0036] The following examples are intended to illustrate, but not to limit the invention.

Examples

Materials and Methods

[0037] Specimen collection. All patients provided informed consent and samples were procured and the study was conducted under the approval of the Institutional Review Board of the MD Anderson Cancer Center.

[0038] GFP, RFP, and CFP mice. Transgenic nude C57/B6-GFP, RFP, and CFP mice were obtained from Anticancer, Inc. (San Diego, CA). These transgenic nude mice express the fluorescent protein gene under the control of the chicken β-actin promoter and cytomegalovirus enhancer. Most of the tissues from these transgenic mice, with the exception of erythrocytes and hair, fluoresce under proper excitation light (Yang et al., Cancer Res 64:8651-8656 (2004); Tran Cao et al., J Pancreas 10: 152-156 (2009); Yang et al., J Cell Biochem 106:279-284 (2009)).

[0039] Animal care. The transgenic nude mice were bred and maintained in a HEPA filtered environment at Anticancer, Inc. with cages, food, water, and bedding sterilized by autoclaving. All surgical procedures and imaging were performed with the animals anesthetized by

intramuscular injection of a ketamine mixture. All animal studies were conducted in accordance with the principles of and procedures outlined in the NIH guide for the care and use of laboratory animals under assurance number A3873-1.

[0040] Tumor imaging. The OV100 variable magnification Small Animal Imaging System (Yamauchi et al., Cancer Res 66:4208-4214 (2006)), the FV1000 confocal microscope

(Uchugonova et al., J Cell Biochem 112:2046-2050 (2011)), and the MVX10 long-working distance fluorescence dissecting microscope (Kimura et al., J Biomed Optics 15:066029 (2010)), all from Olympus Corp. (Tokyo, Japan), were used in this study.

[0041] Histological analysis. The primary tumor, liver metastases and disseminated peritoneal metastases were sectioned at a thickness of 8 μιη, and stained with hematoxylin and eosin for microscopic analysis. Example 1

Imageable Fluorescent Metastasis Resulting in Transgenic GFP Mice Orthotopically Implanted with Human-patient Primary Pancreatic Cancer Specimens

Tumor models

[0042] Establishment of tumorgraft model (Fl) of pancreatic-cancer patient tumors.

Pancreatic-cancer tumor tissue from patients was obtained at surgery with informed consent and cut into 3- mm fragments and transplanted subcutaneously into NOD/SCID mice (Kim et al., Ann Surg Oncol 19: 395-403 (2011); Kim et al., Nat Protol 4: 1670-1680 (2009)).

[0043] Orthotopic tumorgraft (F2) of pancreatic-cancer patient tumor in transgenic GFP nude mice. The Fl tumors from NOD/SCID mice were harvested and cut into 3 mm fragments and transplanted orthotopically into six-week-old transgenic nude GFP mice (F2 model).

Results

[0044] Engraftment of tumors from pancreatic cancer patients (Fl) in NOD/SCID mouse. Tumors from patients with pancreatic cancer were initially transplanted subcutaneously into NOD/SCID mice within two hours of surgery. Tumors were detected by day 30 (Kim et al., Ann Surg Oncol 19: 395-403 (2011); Kim et al., Nat Protol 4: 1670-1680 (2009)). Tumors were then harvested from the NOD/SCID mice and cut into 3-mm fragments.

[0045] GFP host stromal cells infiltrate orthotopic primary pancreatic cancer tumorgrafts (F2). The harvested human patient tumors from the NOD/SCID mice were transplanted orthotopically into six-week-old transgenic GFP nude mice (F2 model). After 110 days, primary tumors were observed using the OV100 imaging system. The GFP stromal cells from the GFP host mouse had migrated into the orthotopic pancreatic tumor, causing the tumors to fluoresce bright green

(Suetsugu et al., J Cell Biochem 113:2290-2295 (2012)). Both GFP cancer- associated fibroblasts (CAFs) and tumor-associated microphages (TAMs) were observed in the primary tumor (Figure 1). Histological examination at 110 days of tumor growth revealed pancreatic tubular adenocarcinoma (Figure IB).

[0046] GFP host stromal cells infiltrate peritoneal disseminated metastases of orthotopic pancreatic cancer tumorgrafts (F2). Fluorescent peritoneal metastases were examined with the OV100 imaging system. The GFP stromal cells from the GFP host mouse formed a capsule around the F2 disseminated peritoneal metastases (Figure 2A). Both GFP CAFs and TAMs were observed in the disseminated peritoneal metastases (Figure 2B). Histological examination at 110 days of tumor growth revealed pancreatic tubular adenocarcinoma (Figure 2C), similar to the primary tumor.

[0047] GFP host stromal cells infiltrate liver metastases of orthotopic pancreatic cancer tumorgrafts (F2). On day 110 after orthotopic implantation of the patient pancreatic tumor, GFP fluorescence was observed in the experimental liver metastases with the OV100 imaging system (Figure 3A). High-magnification fluorescence imaging showed extensive GFP fluorescence in the liver metastasis (Figure 3A). Host GFP cells extensively accumulated in the liver metastasis. Both GFP CAFs and TAMs were observed in the liver metastasis (Figure 3B). Histological examination of the liver metastasis revealed pancreatic tubular adenocarcinoma (Figure 3C).

Example 2

Multi-color Palette of Fluorescent Proteins for Imaging the Tumor Microenvironment of Orthotopic Tumorgraft Mouse Models of Clinical Pancreatic Cancer Specimens

Tumor models

[0048] Establishment of tumorgraft model (Fl) of pancreatic cancer patient tumors. Pancreas cancer patient tumor tissue was obtained at surgery and cut into 3-mm fragments and transplanted subcutaneously in NOD/SCID mice (Kim et al., Ann Surg Oncol 19: 395-403 (2011); Kim et al., Nat Protol 4: 1670-1680 (2009)).

[0049] Orthotopic tumorgraft (F2) of pancreatic cancer patient tumors in transgenic RFP nude mice. The Fl tumors from NOD/SCID mice were harvested and cut into 3-mm fragments and transplanted orthotopically (Hoffman, Investig New Drugs 17:343-359 (1999)) in 6-week-old transgenic nude RFP mice (Yang et al., J Cell Biochem 106:279-284 (2009)) (F2 model).

[0050] Orthotopic tumorgraft (F3) of pancreatic cancer patient tumors in transgenic GFP nude mice. The F2 tumors were harvested from the RFP nude mice and were cut into 3-mm fragments and transplanted orthotopically (Hoffman, Investig New Drugs 17:343-359 (1999)) in 6-week-old transgenic nude GFP mice (Yang et al., J Cell Biochem 106:279-284 (2009)) (F3 model).

[0051] Orthotopic tumorgraft (F4) of pancreatic cancer patient tumors in transgenic CFP nude mice. The F3 tumors were harvested from the GFP nude mice and cut into 3- mm fragments and transplanted orthotopically (Hoffman, Investig New Drugs 17:343-359 (1999)) in 6-week-old transgenic nude CFP mice (Tran Cao et al., J Pancreas 10: 152-156 (2009)) (F4 model). Results

[0052] Pancreatic-cancer-patient tumor specimens were initially established subcutaneously in SCID-NOD mice immediately after surgery. The patient tumors were then harvested from SCID- NOD mice and passaged orthotopically in transgenic nude mice ubiquitously expressing RFP. The primary patient tumors acquired RFP-expressing stroma. The RFP-expressing stroma included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Further passage to transgenic nude mice ubiquitously expressing GFP resulted in tumors and metastasis that acquired GFP stroma in addition to their RFP stroma, including CAFs and TAMs and blood vessels. The RFP stroma persisted in the tumors growing in the GFP mouse. Further passage to transgenic nude mice ubiquitously expressing CFP resulted in tumors and metastasis acquiring CFP stroma in addition to persisting RFP and GFP stroma including RFP- and GFP-expressing CAFs and TAMs and blood vessels. This model can be used to image primary and metastatic progression of patient pancreatic tumors to visually target stroma as well as cancer cells and individualize therapy.

[0053] The present study utilizes a palette of multicolored fluorescent proteins to image the recruitment over time of stromal cells, including cancer-associated fibroblasts (CAFs) and tumor- associated macrophages (TAMs) by pancreatic-cancer-patient tumors grown orthotopically in three types of transgenic nude mice, each expressing a different color fluorescent protein (Yang et al., Cancer Res 64:8651-8656 (2004); Yang et al., J Cell Biochem 106:279-284 (2009); Tran Cao et al., J Pancreas 10: 152-156 (2009)). This study allows for the first time the visualization and demonstration of the persistence of the TME of patient tumors as well and their fluorescence imaging in mouse models.

[0054] Engraftment of patient tumors (Fl) in NOD/SCID mouse: A flow diagram of experimental protocols is shown (Fig. 4A). Human pancreatic-cancer patient tumors were initially transplanted subcutaneously in NOD/SCID mice. Tumors were detected by day-30. Tumors were harvested from the NOD/SCID mouse and cut into 3-mm fragments.

[0055] RFP host stromal cells infiltrate orthotopic pancreatic cancer tumor graft (F2): The harvested human pancreatic cancer patient tumors from the NOD/SCID mice were transplanted orthotopically in six- week-old transgenic RFP nude mice (F2 model). After 28 days, tumors were observed using the OV100 (Fig. 4B). The RFP stromal cells from the RFP host mouse formed a capsule around the F2 tumor (Fig. 4B) and infiltrated into the central part of the tumor as well (Fig. 4C). RFP-expressing TAMs could be visualized in the tumor. [0056] GFP host stromal cells infiltrate the orthotopic pancreatic cancer tumor grafts to form a 2-color stroma model (F3): The F2 tumor was harvested at day-30, cut into 3-mm pieces and transplanted orthotopically in six-week-old transgenic GFP nude mice (F3 model). After 14 days, tumors were observed with the OV100 (Fig. 4E). The F2 tumor spread on the host GFP pancreas (Fig. 4F). After 56 days, tumors were removed from the GFP nude mice. The human pancreatic- cancer-patient tumors contained both RFP and GFP stromal cells (Fig. 4G). The RFP stromal cells were still persisting after passage in the F3 tumorgraft. Under confocal microscopy with the FVIOOO, RFP and GFP stromal cells were clearly visualized in the tumor (Fig. 4H). GFP and RFP CAFs and TAMs were visualized including the central part of the tumor (Fig. 4H-J).

[0057] CFP host stromal cells infiltrate orthotopic pancreatic cancer tumorgrafts to form a 3- color stroma model (F4): F3 tumors were harvested at day-56 and transplanted orthotopically in six-week-old nude CFP mice (F4 model). After 28 days, F4 tumors were observed with the MVX10 long-working-distance fluorescence microscope (Fig. 5A, B). The excised F4 tumor was also observed with the FVIOOO confocal microscope (Fig. 5C-F). RFP-, GFP-, and CFP- expressing stromal cells were observed in the human pancreatic cancer patient tumor (Fig. 5C). The RFP stroma persisted after two passages and GFP stroma after one passage in the F4 model in CFP mice. RFP TAMs and CAFs (Fig. 5D, F) and GFP blood vessels (Fig. 5D, E) still persisted in the human pancreatic cancer patient tumor after 2 and 1 passages, respectively (Fig. 5F).

Example 3

Non-invasive Fluorescent-protein Imaging of Orthotopic Pancreatic-cancer-patient Tumorgraft

Progression in Nude Mice

Tumor models

[0058] Establishment of tumorgraft model (Fl) of tumor from patients with pancreatic cancer: Tumor tissue from patients with pancreatic cancer obtained at surgery was cut into 3-mm fragments and transplanted subcutaneously into NOD/SCID mice (Kim et al., Ann Surg Oncol 19: 395-403 (2011); Kim et al., Nat Protol 4: 1670-1680 (2009)).

[0059] Orthotopic tumorgraft (F2) of tumor from patients with pancreatic cancer in transgenic GFP -expressing nude mice: The Fl tumors from NOD/SCID mice were harvested and cut into 3- mm fragments and then transplanted orthotopically (Hoffman, Investig New Drugs 17:343-359 (1999)) into six- week old transgenic GFP-expressing nude mice (Yang et al., Cancer Res 64:8651- 8656 (2004)) (F2 model). [0060] Orthotopic tumor graft (F3) of tumors from patients with pancreatic cancer in transgenic RFP-expressing nude mice: The F2 tumors from GFP mice were harvested and cut into 3-mm fragments and then transplanted orthotopically (Hoffman, Investig New Drugs 17:343-359 (1999)) into six-week old transgenic RFP-expressing nude mice (Yang et al., J Cell Biochem 106:279-284 (2009)) (F3 model). For each passage of F2-F3, the tumor grew for 70 days.

[0061] Orthotopic tumorgraft (F4) of pancreatic cancer of patients ' tumors in non-transgenic nude mice: The F3 tumors were harvested from the RFP-expressing nude mice and cut into 3-mm fragments then transplanted orthotopically (Hoffman, Investig New Drugs 17:343-359 (1999)) into six-week-old non-transgenic nude mice (F4 model).

Results

[0062] Tumors from patients with pancreatic cancer were initially transplanted subcutaneously into NOD/SCID mice within two hours of surgery (Figure 6A) (Kim et al., Ann Surg Oncol 19: 395-403 (2011); Kim et al., Nat Protol 4: 1670-1680 (2009)). Tumors were detectable by day 30. The harvested human pancreatic cancer patient tumors from the NOD/SCID mice were

transplanted orthotopically into six-week-old transgenic GFP-expressing nude mice (F2 model) where the tumor acquired GFP-expressing stroma. After 70 days, the F2 tumors were harvested from the GFP nude mice and were transplanted orthotopically into six- week-old transgenic nude RFP-expressing mice (F3 model), where the tumors acquired RFP-expressing stroma in addition to their GFP stroma. After 70 days, the F3 tumors were harvested from the RFP-expressing nude mice and cut into 3-mm fragments and were transplanted orthotopically into six-week-old non- transgenic nude mice (F4 model). The growing orthotopic tumor maintained the very bright GFP and RFP stroma from previous passages. Non-invasive imaging at days 21, 30 and 74

demonstrated extensive orthotopic growth of the pancreatic cancer tumorgraft on the nude mouse pancreas (Figure 6B).

[0063] After resection, the F4 tumor was observed with the FV1000 confocal microscope (Figure 1C). RFP- and GFP-expressing cancer associated fibroblasts (CAFs), tumor associated macrophages (TAMs) and blood vessels from the GFP-expressing and RFP-expressing hosts still persisted in the human pancreatic tumor after the three passages described above (Figure 6C).

[0064] We have demonstrated a new mouse model of patient-derived tumors whereby GFP- and RFP-expressing stromal elements are acquired by passaging the patients' pancreatic-cancer tumorgrafts through transgenic GFP- and RFP-expressing nude mice. The brightly fluorescent stroma enables the patient-derived tumor to be non-invasively and longitudinally imaged after subsequent passage in non-transgenic nude mice. The fluorescent protein-expressing stroma included CAFs and TAMs. The fluorescent stroma persisted for at least three passages in the tumors growing in the transgenic and non-transgenic nude mice. The persistence of the stroma over multiple passages further indicates the intimacy of cancer cells and stroma (Bouvet et al., Cancer Res 66: 11293-11297 (2006)).

[0065] The new non-invasive imaging orthotopic model of cancer patient-derived tumors is superior to the ectopic, non-imageable models currently in use (Fu et al., Proc Natl Acad Sci USA 89: 5645-5649 (1992); Fu et al., Pwc Natl Acad Sci USA 88: 9345-9349 (1991); Rubio-Viqueira et al., Clin Cancer Res 12: 4652-4661 (2006); Embuscado et al., Cancer Biol Ther 4: 548-554 (2005)).

[0066] This nude mouse model, described in this report, can be used to visualize primary and metastatic progression of human-derived tumors over a long period of time and their response to cancer therapy, as well as to stromal therapy. This model will allow standard and experimental drugs to be rapidly screened for patients which should individualize and improve therapy. The model described here will also improve our ability to discover novel effective agents for pancreatic and other cancer types.

[0067] Reports of this work was published by the present applicants in: Suetsugu et al., J Cell Biochem 113:2290-2295 (2012); Suetsugu et al., Anticancer Res 32: 1175-1180 (2012); Suetsugu et al., Anticancer Res 32: 3063-3068 (2012). The contents of these publications are incorporated herein by reference in their entireties.

[0068] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0069] All publications, including patent documents and scientific articles, referred to in this application and the bibliography and attachments are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference.

[0070] While various embodiments of the invention have been described above, it should be understood that they have been presented by way of example only, and not by way of limitation. Likewise, the various diagrams may depict an example architectural or other configuration for the disclosure, which is done to aid in understanding the features and functionality that can be included in the disclosure. The disclosure is not restricted to the illustrated example architectures or configurations, but can be implemented using a variety of alternative architectures and

configurations. Additionally, although the disclosure is described above in terms of various exemplary embodiments and implementations, it should be understood that the various features and functionality described in one or more of the individual embodiments are not limited in their applicability to the particular embodiment with which they are described. They instead can, be applied, alone or in some combination, to one or more of the other embodiments of the disclosure, whether or not such embodiments are described, and whether or not such features are presented as being a part of a described embodiment. Thus the breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments.

Claims

Claims We claim:

1. A method to produce a fluorescent animal model for monitoring tumor- stroma interactions during progression, angiogenesis and/or metastasis of tumor cells, which method comprises:

a) providing tumor cells from a subject; and

b) implanting the tumor cells obtained from step a) into a first immunocompromised or syngeneic experimental animal expressing a fluorescent protein.

2. The method of claim 1, further comprising:

c) implanting the tumor cells obtained from step b) into a second

immunocompromised or syngeneic experimental animal.

3. The method of claim 2, further comprising:

d) implanting the tumor cells obtained from step c) into a third immunocompromised or syngeneic experimental animal.

4. The method according to any one of claims 1-3, wherein the immunocompromised or syngeneic experimental animal is a mouse, such as a NOD/SCID or nu/nu mouse.

5. The method according to any one of claims 1-4, wherein the tumor cells are taken directly from a patient.

6. The method according to any one of claims 1-5, wherein the first

immunocompromised or syngeneic experimental animal expresses red fluorescent protein (RFP).

7. The method according to any one of claims 1-6, wherein the second

immunocompromised or syngeneic experimental animal expresses green fluorescent protein (GFP).

8. The method according to any one of claims 1-7, wherein the third

immunocompromised or syngeneic experimental animal expresses cyan fluorescent protein (CFP).

9. The method according to any one of claims 1-7, wherein the third

immunocompromised or syngeneic experimental animal does not express a fluorescent protein.

10. The method according to any one of claims 1-9, wherein the tumor cells from step a) are implanted into an immunocompromised or syngeneic experimental animal before step b).

11. The method according to any one of claims 1-10, wherein the tumor cells are implanted by subcutaneous injection.

12. The method according to any one of claims 1-10, wherein the tumor cells are implanted by surgical orthotopic implantation (SOI).

13. A fluorescent animal model for monitoring tumor-stroma interactions during progression, angiogenesis and/or metastasis of tumor cells, comprising an immunocompromised or syngeneic experimental animal implanted with tumor cells and stromal cells, wherein said stromal cells express one or more fluorescent proteins.

14. The fluorescent animal model of claim 13, wherein the tumor cells are an intact segment of a tumor.

15. The fluorescent animal model of claim 14, wherein the tumor comprises the stromal cells that express one or more fluorescent proteins.

16. The fluorescent animal model of claim 15, wherein the stromal cells express RFP.

17. The fluorescent animal model of claim 15, wherein the stromal cells express GFP.

18. The fluorescent animal model of claim 15, wherein the stromal cells express CFP.

19. The fluorescent animal model of claim 15, wherein the stromal cells express both GFP and RFP.

20. The fluorescent animal model of claim 15, wherein the stromal cells express both GFP and CFP.

21. The fluorescent animal model of claim 15, wherein the stromal cells express both RFP and CFP.

22. The fluorescent animal model of claim 15, wherein the stromal cells express GFP, RFP and CFP.

23. The fluorescent animal model according to any one of claims 13-22, wherein the tumor cells are tissues or of brain, lung, liver, colon, breast, prostate, ovary or pancreas.

24. A method to monitor tumor-stroma interactions during progression, angiogenesis and/or metastasis of tumor cells using the fluorescent animal model according to any one of claims 13-23.

25. The method of claim 24, wherein the fluorescent stromal cells can be imaged either in vivo or ex vivo.

26. The method of claim 25, wherein the fluorescent stromal cells can be imaged in the living animal.

27. The method of claim 25, wherein the fluorescent stromal cells can be non- invasively imaged in the living animal.

28. The method according to any one of claims 24-27, wherein the fluorescent stromal cells are monitored in the third immunocompromised or syngeneic experimental animal not expressing a fluorescent protein.

29. A method to assay the effects of a drug on tumor-stroma interactions during progression, angiogenesis and/or metastasis of tumor cells using the fluorescent animal model according to any one of claims 13-23 comprising: contacting the animal with said drug, imaging tumor-stroma interactions by observing emissions of said one or more fluorescent proteins, and comparing the resulting images to a control animal not contacted with said drug.

30. The method of claim 29, wherein the fluorescent stromal cells can be imaged either in vivo or ex vivo.

31. The method of claim 30, wherein the fluorescent stromal cells can be imaged in the living animal.

32. The method of claim 30, wherein the fluorescent stromal cells can be non- invasively imaged in the living animal.

33. The method according to any one of claims 29-32, wherein the fluorescent stromal cells are monitored in the third immunocompromised or syngeneic experimental animal not expressing a fluorescent protein.