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.

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.

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. J. Cell. Biochem. 114: 1738–1743, 2013. © 2013 Wiley Periodicals, Inc.

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.

Abstract In this study, CD44+ HCT-116 cells were considered as colon cancer stem-like cells (CSCs) and CD44− HCT-116 cells were considered colon non-cancer stem cells (NCSCs). CD44+ cells were isolated by magnetic bead sorting. CD44+ cells outgrew CD44− cells in vitro. GFP-CD44+ cells preferentially migrated from the center of the spheroids after co-culture with RFP-CD44− cells. GFP-CD44+ cells grew to a greater extent subcutaneously in nude mice, when compared to the CD44− cells. Equal numbers of GFP CSCs and the RFP NCSCs were mixed and co-injected in the spleen of nude mice where the cells metastasized to the liver. GFP CSCs were found in greater numbers compared to RFP NCSCs in the liver of nude mouse. GFP CSCs and RFP NCSCs were co-injected in the wall of the cecum. GFP CSCs were observed in metastases formed in the small intestine and colon wall, to a much greater extent than RFP-NCSC. Simultaneous color-coded imaging of CSCs and NCSCs in the present study has demonstrated two very different subsets of cells within the HCT-116 cell line with respect to tumor growth and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5208. doi:10.1158/1538-7445.AM2011-5208

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.

Background. The lymphatic system is a major route for cancer cell dissemination, and a potential target for antitumor therapy. Despite ongoing interest in this area of research, the real-time behavior of cancer cells trafficking in the lymphatic system is poorly understood due to lack of appropriate tools to image this process.Materials and methods. We have used monoclonalantibody and fluorescence technology to color-code lymphatic vessels and the cancer cells inside them in a living animal. Monoclonal anti-mouse LYVE-1 antibody was conjugated to a green fluorophore and delivered to the lymphatic system of a nude mouse, allowing imaging of mouse lymphatics. Tumor cells engineered to express red fluorescent protein were then imaged traveling within the labeled lymphatics in real time.Results. AlexaFluor-labeled monoclonal anti-mouse LYVE-1 created a durable signal with clear delineation of lymphatic architecture. The duration of fluorescent signal after conjugated LYVE-1 delivery was far superior to that of fluorescein isothiocyanatedextran or control fluorophore-conjugated IgG. Tumor cells engineered to express red fluorescent protein delivered to the inguinal lymph node enabled real-time tracking of tumor cell movement within the green fluorescent-labeled lymphatic vessels.Conclusions. This technology offers a powerful tool for the in vivo study of real-time trafficking of tumor cells within lymphatic vessels, for the deposition of the tumor cells in lymph nodes, as well as for screening of potential antitumor lymphatic therapies. (C) 2009 Elsevier Inc. All rights reserved.

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.

CONTEXTIn 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.OBJECTIVESince 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.INTERVENTIONIn 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 MEASUREPancreatic 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.RESULTSFive 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.CONCLUSIONCommon bile duct injection is a novel technique for the development of fluorescent mouse models of metastatic pancreatic cancer.

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.

1006 Cancer cells are antigenically different from normal cells. Due to the immune system, most cancer cells should be eliminated due to the recognition of cancer-specific antigens on cancer cells. However, in clinical cancer, this is usually not the case. We report here development of a tri-color imageable mouse model to visualize the interaction between host immune cells and cancer cells during the rejection process. To observe subcellular cancer cell dynamics during the rejection process HT-1080 human fibrosarcoma cells were labeled in the nucleus with histone H2B-GFP and with retroviral RFP in the cytoplasm. HT-1080-GFP-RFP cells were sprinkled over a skin-flap in transgenic GFP immunocompetent mice. After 24 hr, the mice were imaged with the Olympus IV100 laser scanning microscope. HT-1080-GFP-RFP cells were visualized surrounded by host-derived lymphocytes and macrophages both expressing GFP. The tumor cell cytoplasm appeared red, with the GFP nucleus visualized as yellow. The host cells appeared green due to GFP. We could observe host GFP macrophages contacting, engulfing and digesting dual-color HT-1080-GFP-RFP cells in real-time. The dual-color cancer cells were readily visible after being engulfed in the GFP macrophages. In addition, other cancer cells were being eliminated without phagocytosis. The cancer cells, tagged with GFP in the nucleus and RFP in the cytoplasm in transgenic GFP mice, visualized with the IV100 laser scanning microscope enable visualization of GFP lymphocyte and GFP macrophage reaction with cancer cells at the subcellular level in live mice. This multicolor imageable model of cancer cell rejection should be useful for understanding and pharmacological intervention in this process.

1063 Xeroderma pigmentosum (XP) complementation group E gene product, damaged DNA binding protein 2 (DDB2), is a subunit of DDB heterodimeric protein complex with high specificity for binding to a variety of DNA lesions. DDB is believed to play a role in the initial step of damage recognition in mammalian nucleotide excision repair (NER) of ultraviolet light (UV)-induced photolesions. It has been shown that DDB2 is rapidly degraded after cellular UV irradiation. However, the relevance of DDB2 degradation to its functionality in NER is still unknown. Here, we provide evidence that Cullin 4A (CUL-4A), a key component of CUL-4A-based ubiquitin ligase, mediates DDB2 degradation at the damage sites and regulates the recruitment of XPC and the repair of cyclobutane pyrimidine dimers (CPD). We show that CUL-4A can be identified in an UV-responsive protein complex containing both DDB subunits. CUL-4A was visualized in localized UV-irradiated sites together with DDB2 and XPC. Degradation of DDB2 could be blocked by silencing CUL-4A using siRNA or by treating cells with proteasome inhibitor MG132. This blockage resulted in prolonged retention of DDB2 at the subnuclear DNA damage foci within micropore irradiated cells. Knockdown of CUL-4A also decreased recruitment of the damage recognition factor, XPC, to the damaged foci and concomitantly reduced the removal of CPD from the entire genome. These results suggest that CUL-4A mediates the proteolytic degradation of DDB2 and that this degradation event, initiated at the lesion sites, regulates damage recognition by XPC during the early steps of NER. (Supported by NIH grants, ES2388, ES12991 and CA93413)

The stem cell marker nestin has recently been shown to be expressed in nascent blood vessels in nestin-driven green fluorescent protein (ND-GFP) nude mice. In addition, ND-GFP is expressed in the acinar cells of the pancreas in the ND-GFP transgenic mice. In the present study, we visualized by dual-color fluorescence imaging tumor angiogenesis in the ND-GFP transgenic nude mice after orthotopic transplantation of the MIA PaCa-2 human pancreas cancer line expressing red fluorescent protein (RFP). Nestin was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumor. Results of immunohistochemical staining showed that CD31 co-localized in ND-GFP-expressing nascent blood vessels. The density of nascent blood vessels in the tumor was readily quantitated. Gemcitabine significantly decreased the tumor volume and mean nascent blood vessel density in the tumor. These results suggest that the dual-color model of the ND-GFP nude mouse and RFP tumor cells is useful for the visualization and quantitation of tumor angiogenesis and evaluation of angiogenesis inhibitors.

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.