Fluorescence-guided surgery with a fluorophore-conjugated antibody to carcinoembryonic antigen (CEA), that highlights the tumor, improves surgical resection and increases survival in orthotopic mouse models of human pancreatic cancer.

BACKGROUND: We have developed a method of distinguishing normal tissue from pancreatic cancer in vivo using fluorophore-conjugated antibody to carcinoembryonic antigen (CEA). The objective of this study was to evaluate whether fluorescence-guided surgery (FGS) with a fluorophore-conjugated antibody to CEA, to highlight the tumor, can improve surgical resection and increase disease-free survival (DFS) and overall survival (OS) in orthotopic mouse models of human pancreatic cancer. METHODS: We established nude-mouse models of human pancreatic cancer with surgical orthotopic implantation of the human BxPC-3 pancreatic cancer. Orthotopic tumors were allowed to develop for 2 weeks. Mice then underwent bright-light surgery (BLS) or FGS 24 h after intravenous injection of anti-CEA-Alexa Fluor 488. Completeness of resection was assessed from postoperative imaging. Mice were followed postoperatively until premorbid to determine DFS and OS. RESULTS: Complete resection was achieved in 92 % of mice in the FGS group compared to 45.5 % in the BLS group (p = 0.001). FGS resulted in a smaller postoperative tumor burden (p = 0.01). Cure rates with FGS compared to BLS improved from 4.5 to 40 %, respectively (p = 0.01), and 1-year postoperative survival rates increased from 0 % with BLS to 28 % with FGS (p = 0.01). Median DFS increased from 5 weeks with BLS to 11 weeks with FGS (p = 0.0003). Median OS increased from 13.5 weeks with BLS to 22 weeks with FGS (p = 0.001). CONCLUSIONS: FGS resulted in greater cure rates and longer DFS and OS using a fluorophore-conjugated anti-CEA antibody. FGS has potential to improve the surgical treatment of pancreatic cancer.

Color-coded imaging of spontaneous vessel anastomosis in vivo.

Vessel anastomosis is important in tumor angiogenesis as well as for vascularization therapy for ischemia and other diseases. We report here the development of a color-coded imaging model that can visualize the anastomosis between blood vessels of red fluorescent protein (RFP)-expressing vessels in vascularized Gelfoam® previously transplanted into RFP transgenic mice and then re-transplanted into nestin-driven green fluorescent protein (ND-GFP) mice where nascent blood vessels express GFP. Gelfoam® was initially transplanted subcutaneously in the flank of transgenic RFP nude mice. Skin flaps were made at 14 days after transplantation of Gelfoam® to allow observation of vascularization of the Gelfoam® using confocal fluorescence imaging. The implanted Gelfoam® became highly vascularized with RFP vessels. Fourteen days after transplantation into RFP transgenic nude mice, the Gelfoam® was removed and re-transplanted into the subcutis on the flank of ND-GFP transgenic nude mice in which nascent blood vessels express GFP. Skin flaps were made and anastomosis between the GFP-expressing nascent blood vessels of ND-GFP transgenic nude mice and RFP blood vessels in the Gelfoam® was imaged 14 and 21 days after re-transplantation. The results presented in this report indicate a possible mechanism for tumor angiogenesis and suggest a new paradigm of therapeutic revascularization of ischemic organs requiring new blood vessels and in other diseases.

Specific route mapping visualized with GFP of single-file streaming contralateral and systemic metastasis of Lewis lung carcinoma cells beginning within hours of orthotopic implantation [correction of implantion].

In this study, we visualized the origin of Lewis lung carcinoma metastasis after transducing tumor cells with green fluorescent protein (GFP) and transplanting them orthotopically in the middle lobe of the right lung of nude mice. Metastasis was visualized in live tissue at single cell resolution by GFP-expression as early as 18 h post-tumor transplant. At this time, single-file streaming lung carcinoma cells already had invaded inferiorly via a tubular lymphatic structure crossing the lower lobes of the lung to the ipsilateral diaphragmatic surface. By post-implantation day 2, the ipsilateral lower lobes of the lung were involved with metastatic cells. By post-implantation day 3, the ipsilateral lower lobes of the lung and the ipsilateral diaphragmatic surface were highly involved with streaming metastatic cells trafficking in single file. By day 4 post-implantation, cancer cells invaded across the diaphragm to the contralateral diaphragmatic surface. Metastatic cells then invaded superiorly through a lymphatic vessel to involve the contralateral mediastinal lymph nodes. In this model of lung cancer, the origin of metastasis was an inferior invasion from the implanted tumor via a lymphatic duct to the ipsilateral diaphragmatic surface. The cancer cells from this site invaded on the surface of the diaphragm to the contralateral diaphragmatic surface and proceeded superiorly through a lymphatic duct to contralateral lymph nodes. Other organs such as the kidneys and the adrenal glands later became involved with metastasis with the contralateral mediastinal lymph nodes as the source. The use of GFP and the highly metastatic orthotopic lung cancer model allowed the visualization of the origin of metastasis at the single-cell level and demonstrated the critical role of lymphatic ducts and the diaphragmatic surface as the path to the contralateral side.

Tumor-educated macrophages promote tumor growth and peritoneal metastasis in an orthotopic nude mouse model of human pancreatic cancer.

BACKGROUND: Macrophages promote tumor growth by stimulating tumor-associated angiogenesis, cancer-cell invasion, migration, intravasation, and suppression of antitumor immune responses. MATERIALS AND METHODS: Ten transgenic nude mice, ubiquitously expressing green fluorescent protein (GFP), were injected subcutaneously with the human pancreatic cancer cell line, BXPC3, stably expressing red fluorescent protein (RFP). GFP-expressing macrophages from the GFP mice with the subcutaneous BxPC3-RFP tumor were harvested and defined as "tumor-educated macrophages". Macrophages were also harvested from transgenic GFP mice (n=10) without tumors and identified as "naïve macrophages." The tumor-educated and naïve macrophages were then implanted into BxPC-3-RFP tumor-bearing non-transgenic nude mice and compared for their ability to enhance tumor progression. RESULTS: In the control group, without macrophage injection, the average primary tumor weighed 668 mg and only three mice (30%) developed peritoneal metastases, which averaged 72 mg. The naïve-macrophage group had an average tumor weight of 823 mg (p=0.51) and 50% developed peritoneal metastases, whose weight averaged 975 mg (p=0.029). The group treated with tumor-educated macrophages had an average primary tumor weight of 2095 mg (p=0.001) and 75% of mice developed peritoneal metastases, whose weight averaged 2135 mg (p=0.008). CONCLUSION: These results suggest that macrophages influence tumors, and tumors influence macrophages, and tumor-educated promote tumor progression. Tumor-educated macrophages may be a target for therapy of metastatic cancer.

Induction of cancer metastasis by cyclophosphamide pretreatment of host mice: an opposite effect of chemotherapy.

Although side effects of cancer chemotherapy are well known, "opposite effects" of chemotherapy that enhance the malignancy of the treated cancer are not well understood. In this report, we describe the induction of intravascular proliferation, extravasation, and colony formation by cancer cells, critical steps of metastasis, by pretreatment of host mice with the commonly used chemotherapy drug cyclophosphamide. In contrast, in the unpretreated mice, most cancer cells remained quiescent in vessels without extravasation. HT1080 human fibrosarcoma cells, labeled in the nucleus with green fluorescent protein and red fluorescent protein in the cytoplasm for imaging, were injected into the epigastric cranialis vein of nude mice. Twenty-four hours before cancer cell injection, cyclophosphamide was given i.p. Double-labeled cancer cells were imaged at the cellular level in live mice with the Olympus OV100 Small Animal Imaging System with variable magnification. Cyclophosphamide seems to interfere with a host process that inhibits intravascular proliferation, extravasation, and extravascular colony formation. Cyclophosphamide does not directly affect the cancer cells because cyclophosphamide has been cleared by the time the cancer cells were injected. This report shows an important unexpected "opposite effect" of chemotherapy that enhances critical steps in malignancy rather than inhibiting them, suggesting that certain current approaches to cancer chemotherapy should be modified.

The integrin-extracellular matrix axis in pancreatic cancer.

Pancreatic cancer is the fifth leading cause of adult cancer death in the United States, with 5-year survival rates of only 1% to 4%. Current therapeutic strategies generally result in only a few months of extended life. Recent evidence from several independent laboratories in vitro and in vivo indicate that integrin-mediated cell attachment to the extracellular matrix (ECM), components of which are highly up-regulated in pancreatic cancer, evokes phenotypes and signaling pathways that regulate tumor cell growth and migration. In this review, we will discuss our current understanding of the role of the ECM in directing pancreatic cancer growth, progression, and metastasis. Topics covered include a survey of the existing literature regarding the in vivo and in vitro expression of the ECM and its cell surface receptors, the integrins, in pancreatic cancer; mechanisms involved in the integrin-ECM-mediated malignant phenotype; and future directions for the study of the integrin-ECM axis and its role in pancreatic cancer progression, including potential therapeutic strategies.

In vivo color-coded imaging of the interaction of colon cancer cells and splenocytes in the formation of liver metastases.

The role of host cells in tumor progression and metastasis is critical. Intrasplenic injection of tumor cells has long been known as an effective method of developing liver metastases in nude mice, whereas portal vein (PV) injection of tumor cells can result in rapid death of the tumor cells. Host cells were thought to play a role in these phenomena. We report here that after splenic injection of tumor cells, splenocytes cotraffic with the tumor cells to the liver and facilitate metastatic colony formation. Human colon cancer cells that express green fluorescent protein (GFP) linked to histone H2B in the nucleus and red fluorescent protein (RFP) in the cytoplasm (HCT-116-GFP-RFP) were injected in either the PV or spleen of nude mice and imaged at the subcellular level in vivo. Extensive clasmocytosis (destruction of the cytoplasm) of the cancer cells occurred within 6 hours after PV injection and essentially all the cancer cells died. In contrast, splenic injection of these tumor cells resulted in the aggressive formation of liver and distant metastasis. GFP spleen cells were found in the liver metastases that resulted from intrasplenic injection of the tumor cells in transgenic nude mice ubiquitously expressing GFP. When GFP spleen cells and the RFP cancer cells were coinjected in the PV, liver metastasis resulted that contained GFP spleen cells. These results suggest a novel tumor-host interaction that enables efficient formation of liver metastasis via intrasplenic injection.

Development of real-time subcellular dynamic multicolor imaging of cancer-cell trafficking in live mice with a variable-magnification whole-mouse imaging system.

With the use of dual-color fluorescent cells and a highly sensitive whole-mouse imaging system with both macro-optics and micro-optics, we report here the development of subcellular real-time imaging of cancer cell trafficking in live mice. To observe cytoplasmic and nuclear dynamics in the living mouse, tumor cells were labeled in the nucleus with green fluorescent protein and with red fluorescent protein in the cytoplasm. Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice. The mice were imaged with an Olympus OV100 whole-mouse imaging system with a sensitive CCD camera and five objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they moved by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. With the dual-color cancer cells and the highly sensitive whole-mouse imaging system described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time. This imaging technology will enable further understanding of the critical steps of metastasis and provide visible targets for antimetastasis drug development.

Common bile duct injection as a novel method for establishing red fluorescent protein (RFP)-expressing human pancreatic cancer in nude mice.

CONTEXT: In our previous pancreatic cancer mouse models, we have used surgical orthotopic implantation of human pancreatic tumors to establish clinically relevant fluorescent mouse models of pancreatic cancer. OBJECTIVE: Since exocrine pancreatic cancer is thought to arise from the cells lining the ducts of the pancreas, we hypothesized that direct injection of tumor cells into the common bile duct would also result in pancreatic tumor formation and metastasis. INTERVENTION: In this study we injected a suspension of the low passage human pancreatic cancer cell line xPA-1 transfected with red fluorescent protein into the common bile duct of nude mice. MAIN OUTCOME MEASURE: Pancreatic tumor growth and metastasis formation was monitored by intravital and whole body fluorescent imaging. Single fluorescent pancreatic cancer cells were imaged in the pancreatic duct shortly after injection using the Olympus OV100 Whole Mouse Imaging System. RESULTS: Five days after tumor cell injection in the common bile duct, tumor colonies could be imaged forming within the pancreatic duct. Metastases in the liver were imaged 14 days post common bile duct injection. By day 28, massive tumors were imaged encompassing the entire pancreas. By day 42, RFP-expressing metastases were imaged in the omentum and liver. CONCLUSION: Common bile duct injection is a novel technique for the development of fluorescent mouse models of metastatic pancreatic cancer.

Dual-color imaging of nuclear-cytoplasmic dynamics, viability, and proliferation of cancer cells in the portal vein area.

We used dual-color in vivo cellular imaging to visualize trafficking, nuclear-cytoplasmic dynamics, and the viability of cancer cells after their injection into the portal vein of mice. For these studies, we used dual-color fluorescent cancer cells that express green fluorescent protein (GFP) linked to histone H2B in the nucleus and retroviral red fluorescent protein (RFP) in the cytoplasm. Human HCT-116-GFP-RFP colon cancer and mouse mammary tumor (MMT) cells were HCT-116-GFP-RFP in the portal vein of nude mice. The cells were observed intravitally in the liver at the single-cell level using the Olympus OV100 whole-mouse imaging system. Most HCT-116-GFP-RFP cells remained in sinusoids near peripheral portal veins. Only a small fraction of the cancer cells invaded the lobular area. Extensive clasmocytosis (destruction of the cytoplasm) of the HCT-116-GFP-RFP cells occurred within 6 hours. The number of apoptotic cells rapidly increased within the portal vein within 12 hours of injection. Apoptosis was readily visualized in the dual-color cells by their altered nuclear morphology. The data suggest rapid death of HCT-116-GFP-RFP cells in the portal vein. In contrast, dual-color MMT-GFP-RFP cells injected into the portal vein mostly survived in the liver of nude mice 24 hours after injection. Many surviving MMT-GFP-RFP cells showed invasive figures with cytoplasmic protrusions. The cells grew aggressively and formed colonies in the liver. However, when the host mice were pretreated with cyclophosphamide, the HCT-116-GFP-RFP cells also survived and formed colonies in the liver after portal vein injection. These results suggest that a cyclophosphamide-sensitive host cellular system attacked the HCT-116-GFP-RFP cells but could not effectively kill the MMT-GFP-RFP cells.

High correlation of whole-body red fluorescent protein imaging and magnetic resonance imaging on an orthotopic model of pancreatic cancer.

We have developed genetically fluorescent orthotopic models of human pancreatic cancer. In these models, noninvasive fluorescent protein imaging (FPI) of internal primary tumors and metastatic deposits has been carried out. Whole-body tumor images are easily and inexpensively obtained using FPI, permitting both detection and quantification of tumor load. In this study, we simultaneously compared single mice with a highly fluorescent, red fluorescent protein-expressing orthotopic pancreatic cancer xenografts with both FPI and high-resolution magnetic resonance imaging (MRI). Images were acquired at multiple time points after tumor implantation in the pancreas. Indwelling pancreatic primary tumors and metastatic foci were detected by both FPI and MRI. Moreover, a strong correlation existed between images taken with these two technologies. FPI permitted rapid, high-throughput imaging without the need for either anesthesia or contrast agents. Both FPI and MRI enabled accurate imaging of tumor growth and metastasis, although MRI enabled tissue structure to be visualized as well. FPI has high resolution and is exceedingly rapid with instant image capture. We suggest a complimentary role for these two imaging modalities.

Nestin-linked green fluorescent protein transgenic nude mouse for imaging human tumor angiogenesis.

We report here a novel transgenic nude mouse for the visualization of human tumor angiogenesis. We have recently shown that the neural stem cell marker nestin is expressed in hair follicle stem cells and blood vessel networks in the skin of C57/B6 transgenic mice with nestin regulatory element-driven green fluorescent protein (ND-GFP). Others have shown ND-GFP is expressed in the brain, pancreas, and testes in these mice. In the present study, the nestin ND-GFP gene was crossed into nude mice on the C57/B6 background to obtain ND-GFP nude mice. ND-GFP was expressed in the brain, spinal cord, pancreas, stomach, esophagus, heart, lung, blood vessels of glomeruli, blood vessels of skeletal muscle, testes, hair follicles, and blood vessel network in the skin of ND-GFP nude mice. Human lung cancer, pancreatic cancer, and colon cancer cell lines as well as a murine melanoma cell line and breast cancer tumor cell line expressing red fluorescent protein were implanted orthotopically, and a red fluorescent protein-expressing human fibrosarcoma was implanted s.c. in the ND-GFP nude mice. These tumors grew extensively in the ND-GFP mice. ND-GFP was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumors, visualized by dual-color fluorescence imaging. Results of immunohistochemical staining showed that CD31 was expressed in the ND-GFP-expressing nascent blood vessels. The ND-GFP transgenic nude mouse model enables the visualization of nascent angiogenesis in human and mouse tumor progression. These results suggest that this model is useful for the imaging of the angiogenesis of human as well as rodent tumors and visualization of the efficacy of angiogenetic inhibitors.

Hair follicle-derived blood vessels vascularize tumors in skin and are inhibited by Doxorubicin.

We have recently shown that the neural-stem cell marker nestin is expressed in hair follicle stem cells and the blood vessel network interconnecting hair follicles in the skin of transgenic mice with nestin regulatory element-driven green fluorescent protein (ND-GFP). The hair follicles were shown to give rise to the nestin-expressing blood vessels in the skin. In the present study, we visualized tumor angiogenesis by dual-color fluorescence imaging in ND-GFP transgenic mice after transplantation of the murine melanoma cell line B16F10 expressing red fluorescent protein. ND-GFP was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumor. Results of immunohistochemical staining showed that the blood vessel-specific antigen CD31 was expressed in ND-GFP-expressing nascent blood vessels. ND-GFP expression was diminished in the vessels with increased blood flow. Progressive angiogenesis during tumor growth was readily visualized during tumor growth by GFP expression. Doxorubicin inhibited the nascent tumor angiogenesis as well as tumor growth in the ND-GFP mice transplanted with B16F10-RFP. This model is useful for direct visualization of tumor angiogenesis and evaluation of angiogenic inhibitors.

Transgenic nude mouse with ubiquitous green fluorescent protein expression as a host for human tumors.

We report here the development of the transgenic green fluorescent protein (GFP) nude mouse with ubiquitous GFP expression. The GFP nude mouse was obtained by crossing nontransgenic nude mice with the transgenic C57/B6 mouse in which the beta-actin promoter drives GFP expression in essentially all tissues. In crosses between nu/nu GFP male mice and nu/+ GFP female mice, the embryos fluoresced green. Approximately 50% of the offspring of these mice were GFP nude mice. Newborn mice and adult mice fluoresced very bright green and could be detected with a simple blue-light-emitting diode flashlight with a central peak of 470 nm and a bypass emission filter. In the adult mice, the organs all brightly expressed GFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, and duodenum. The following systems were dissected out and shown to have brilliant GFP fluorescence: the entire digestive system from tongue to anus; the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart and major arteries and veins. The skinned skeleton highly expressed GFP. Pancreatic islets showed GFP fluorescence. The spleen cells were also GFP positive. Red fluorescent protein (RFP)-expressing human cancer cell lines, including PC-3-RFP prostate cancer, HCT-116-RFP colon cancer, MDA-MB-435-RFP breast cancer, and HT1080-RFP fibrosarcoma were transplanted to the transgenic GFP nude mice. All of these human tumors grew extensively in the transgenic GFP nude mouse. Dual-color fluorescence imaging enabled visualization of human tumor-host interaction by whole-body imaging and at the cellular level in fresh and frozen tissues. The GFP mouse model should greatly expand our knowledge of human tumor-host interaction.

Cellular dynamics visualized in live cells in vitro and in vivo by differential dual-color nuclear-cytoplasmic fluorescent-protein expression.

We report here the genetic engineering of dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed in the cytoplasm of HT-1080 human fibrosarcoma cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Nuclear GFP expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic RFP expression enabled visualization of nuclear cytoplasmic ratios as well as simultaneous cell and nuclear shape changes. Thus, total cellular dynamics can be visualized in the living dual-color cells in real time. The parental HT-1080 and the derived dual-color clones had similar cell proliferation rates, suggesting that expression of GFP and/or RFP does not affect cell cycle progression. The cell cycle position of individual living cells was readily visualized by the nuclear-cytoplasmic ratio and nuclear morphology. Real-time induction of apoptosis was observed by nuclear size changes and progressive nuclear fragmentation. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse brain where extreme elongation of the cell body as well as the nucleus occurred. Dual-color cells in various positions of the cell cycle were visualized in excised mouse lungs after tail-vein injection of the dual-color cells. In the lung, the dual-color cells were observed frequently juxtaposing their nuclei, suggesting a potential novel form of cell-cell communication. The dual-color cells thus are a useful tool for visualizing living-cell dynamics in vivo as well as in vitro. Drugs that could specifically perturb these processes can now be readily screened in real time in vivo.

Survival efficacy of adjuvant cytosine-analogue CS-682 in a fluorescent orthotopic model of human pancreatic cancer.

Adjuvant treatment with the cytosine analogue 1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-N(4)-palmitoylcytosine (CS-682) results in a highly significant increase in survival in the aggressive orthotopic MIA-PaCa-2 human pancreatic cancer mouse model. Seven days after implantation, mice were randomized into eight groups, depending on whether they were to be treated by tumor resection, 5 weeks of CS-682 chemotherapy at 40-60 mg/kg once daily, or both. Throughout the course of treatment, noninvasive optical whole-body imaging based on brilliant red fluorescent protein expression of the tumor permitted visualization and quantification of primary, metastatic, and recurrent disease. Total tumor burden negatively correlated with survival. Untreated mice died of disseminated disease with a median survival of 26 days. Surgical resection alone conferred a small but significant survival advantage (median survival, 28 days, P = 0.03). Primary CS-682 treatment at all doses also significantly prolonged survival compared with untreated animals (P < 0.05) and was more effective than surgery alone at doses of 50 and 60 mg/kg (median survival, 34 days, P = 0.045, and 38.5 days, P = 0.03, respectively). Maximal survival (median, 48 days, with 30% of animals surviving longer than 60 days) was achieved by adjuvant CS-682 (50 mg/kg), given after surgical resection of the primary pancreatic tumor (P = 0.004 compared with surgery alone). The results demonstrate that adjuvant oral administration of CS-682 for pancreatic cancer is highly effective with acceptable toxicity, suggesting its potential for cure of this disease in appropriate combinations.

Simultaneous serous cystadenoma of the pancreas and mucinous cystadenoma of the appendix.

CONTEXT: Serous cystadenoma of the pancreas and mucinous tumors of the vermiform appendix are rare. To our knowledge, the simultaneous occurrence of these two tumors has not been reported. CASE REPORT: Here, we report an adult female who presented with signs and symptoms of appendicitis. A preoperative CT scan confirmed the findings of appendicitis and also showed an incidental large mass in the head of the pancreas. The patient underwent uneventful appendectomy. Her pathology revealed an acutely inflamed appendix with a benign mucinous cystadenoma at the tip. Several months after her recovery, a Whipple procedure was performed. Pathologic examination showed a 5x5 cm serous cystadenoma of the head of the pancreas without evidence of malignancy. Two years later, the patient is alive and well without evidence of tumor recurrence. CONCLUSIONS: Cystadenomas of the pancreas and appendix are unusual and their simultaneous occurrence is a rare event.

Selective antimetastatic activity of cytosine analog CS-682 in a red fluorescent protein orthotopic model of pancreatic cancer.

In this study we demonstrate the ability of a novel, p.o.-administered cytosine analogue, CS-682, to effectively prolong survival and inhibit metastatic growth in an imageable orthotopic mouse model of pancreatic cancer. MIA-PaCa-2-RFP pancreatic cancer cells were transduced with the Discosoma red fluorescent protein (RFP) and orthotopically implanted onto the pancreas of nude mice. Tumor RFP fluorescence facilitated real-time, sequential imaging, and quantification of primary and metastatic growth and dissemination in vivo. Mice were treated with various p.o. doses of CS-682 on a five times per week schedule until death. At a dose of 40 mg/kg, CS-682 prolonged survival compared with untreated animals (median survival 35 days versus 17 days; P = 0.0008). At nontoxic doses, CS-682 effectively suppressed the rate of primary tumor growth. CS-682 also decreased the development of malignant ascites and the formation of metastases, which were reduced significantly in number in the diaphragm, lymph nodes, liver, and kidney. Selective RFP tumor fluorescence enabled noninvasive real-time comparison between groups during treatment and facilitated identification of micrometastases in solid organs at autopsy. Thus, we have demonstrated that CS-682 is an efficacious antimetastatic agent that significantly prolongs survival in an orthotopic model of pancreatic cancer. The antimetastatic efficacy of CS-682 and its p.o. availability confer significant advantages and clinical potential to this agent for pancreatic cancer.