MULTI-COLOR PALLET OF FLUORESCENT PROTEINS FOR IMAGING THE TUMOR MICROENVIRONMENT OF ORTHOTOPIC TUMORGRAFT MOUSE MODELS OF CLINICAL PANCREATIC CANCER SPECIMENS
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.
INTRODUCTION<!>Specimen collection<!>GFP, RFP and CFP mice<!>Animal care<!>Establishment of tumorgraft model (F1) of pancreatic cancer patient tumors<!>Orthotopic tumorgraft (F2) of pancreatic cancer patient tumors in transgenic RFP nude mice<!>Orthotopic tumorgraft (F3) of pancreatic cancer patient tumors in transgenic GFP nude mice<!>Orthotopic tumorgraft (F4) of pancreatic cancer patient tumors in transgenic CFP nude mice<!>Tumor imaging<!>Engraftment of patient tumors (F1) in NOD/SCID mouse<!>RFP host stromal cells infiltrate orthotopic pancreatic cancer tumorgraft (F2)<!>GFP host stromal cells infiltrate the orthotopic pancreatic cancer tumorgrafts to form a 2-color stroma model (F3)<!>CFP host stromal cells infiltrate orthotopic pancreatic cancer tumorgrafts to form a 3-color stroma model (F4)
<p>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, 1969]. Our laboratory pioneered surgical orthotopic implantation (SOI) metastatic nude-mouse models using patient tumor specimens in the early 1990's [Fu et al., 1991, 1992]. These orthotopic mouse models of patient tumors are more patient-like than ectopic subcutaneous models [Rygaard and Povlsen, 1969; Fiebig et al., 1984; Fiebig et al., 1987; Steel 1982; Novak et al., 1978; Pickard et al., 1975; Selby et al., 1980; Sharkey and Fogh, 1984; Giovanella et al., 1978; Bailey et al., 1980; Rubio-Viqueira et al., 2006; Embuscado et al., 2005; Talmadge et al., 2007; Hwang et al., 2003; Yokoi et al., 2005; Garber 2007; Bertotti et al., 2011]. However, these models are limited with regard to studying the tumor microenvironment. In our initial development of SOI nude mouse models of patient tumors, we achieved take rates of 65% for colon cancer [Fu et al., 1991] and 100% for pancreatic cancer. Subsequently, the NOD-SCID mouse, was developed [Shultz et al., 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., 2011]. Patient pancreatic tumors can be transplanted to NOD-SCID mice in our laboratory at a frequency approaching 100% (data not shown).</p><p>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, 2006a, 2006b]. 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., 2003, 2004, 2009]. We have previously demonstrated the essential role of tumor-associated host cells in tumor progression and metastasis [Bouvet et al., 2006].</p><p>The present study utilizes a pallet 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 nude mice. This study allows for the first time the visualization and persistence of the TME of patient tumors in mouse models.</p><!><p>All patients provided informed consent and samples were procured and the study was conducted under the approval of the Institutional Review Board of MD Anderson Cancer Center.</p><!><p>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., 2009; Tran Cao et al., 2009; Yamauchi et al., 2006].</p><!><p>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.</p><!><p>Pancreas cancer patient tumor tissue was obtained at surgery and cut into 3-mm3 fragments and transplanted subcutaneously in NOD/SCID mice.</p><!><p>The F1 tumors from NOD/SCID mice were harvested and cut into 3-mm3 fragments and transplanted orthotopically in six-week-old transgenic nude RFP mice (F2 model).</p><!><p>The F2 tumors were harvested from the RFP nude mice and were cut into 3-mm3 fragments and transplanted orthotopically in six-week-old transgenic nude GFP mice (F3 model).</p><!><p>The F3 tumors were harvested from the GFP nude mice and cut into 3-mm3 fragments and transplanted orthotopically in six-week-old transgenic nude CFP mice (F4 model).</p><!><p>The OV100 variable magnification small animal imaging system [Yamauchi et al., 2006], the IV100 scanning laser microscope [Yang et al., 2007], the FV1000 confocal microscope [Uchugonova et al., 2011], and the MVX10 long-working distance fluorescence dissecting microscope [Kimura et al., 2010], all from Olympus Corp. (Tokyo, Japan), were used in this study.</p><!><p>A flow diagram of experimental protocols is shown (Fig. 1A). 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-mm3 fragments.</p><!><p>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. 1B). The RFP stromal cells from the RFP host mouse formed a capsule around the F2 tumor (Fig. 1B) and infiltrated into the central part of the tumor as well (Fig. 1C). RFP-expressing TAMs could be visualized in the tumor.</p><!><p>The F2 tumor was harvested at day-30, cut into 3-mm3 pieces and transplanted orthotopically in six-week-old transgenic GFP nude mice (F3 model). After 14 days, tumors were observed with the OV100 (Fig. 1E). The F2 tumor spread on the host GFP pancreas (Fig. 1F). 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. 1G). The RFP stromal cells were still persisting after passage in the F3 tumorgraft. Under confocal microscopy with the FV1000, RFP and GFP stromal cells were clearly visualized in the tumor (Fig. 1H). GFP and RFP CAFs and TAMs were visualized including the central part of the tumor (Fig. 1H-J).</p><!><p>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. 2A, B). The excised F4 tumor was also observed with the FV1000 confocal microscope (Fig. 2C-F). RFP-, GFP-, and CFP-expressing stromal cells were observed in the human pancreatic cancer patient tumor (Fig. 2C). 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. 2D, F) and GFP blood vessels (Fig. 2D, E) still persisted in the human pancreatic cancer patient tumor after 2 and 1 passages, respectively (Fig. 2F).</p><p>We have demonstrated a new mouse model of cancer-patient tumors whereby stromal elements can be imaged using a pallet of multi-color fluorescent proteins. The fluorescent stroma persisted for at least two passages in the tumors growing in the transgenic mice indicating the intimacy of cancer cells and stroma. The survival of stroma after transplantation was previously suggested [Duda et al., 2004]. The results of the present study suggest the serial transplantability of stroma. For example from the RFP mouse, TAMs and SAFs persisted from F2 to F4, suggesting they may be proliferating along with the cancer cells in the tumor. From the GFP mouse, TAMs and CAFs and blood vessels were found in the tumor and persisted to F4. The CFP mouse contributed mostly CAFs to the tumor. The fluorescent stroma allow the entire tumor to be imaged as well. Both standard and novel cancer- and stroma-targeting agents can be tested in this model and can be used for individualized therapy of cancer patients.</p><p>The new models described in this report offer many opportunities. For example, cell lines can be established from the tumors with fluorescent stroma and the role of stroma in cell line establishment can be imaged longitudinally.</p><p>The stromal cells of each fluorescent color can be isolated by fluorescent-activated cell sorting (FACS) to further characterize them.</p><p>Since the stroma carry genes for fluorescent proteins, it may be possible to observe gene transfer from stromal cells to the cancer cells. We have previously taken advantage of differential fluorescent protein expression in cancer cells to demonstrate gene transfer between cancer cells in vivo [Tome et al., 2009; Glinsky et al., 2006].</p><p>It should also be possible to determine if some of the fluorescent stromal cells are derived from tissue-specific stem cells or mesenchymal stem cells.</p><p>One of the most exciting opportunities directly inspired by observations in the current study enabled by the availability of the color-coded panel of hosts, would be the analysis of the role, requirements, and contribution of host stroma to metastatic initiation and progression.</p>