BACKGROUND/AIM:Pancreatic stellate cells are involved in fibrosis of pancreatic cancer termed desmoplasia, which may contribute to both pancreatic cancer growth and metastasis, as well as to drug resistance. A better understanding of pancreatic cancer-cell interactions with stellate cells is therefore critical to our ability to develop effective anti-metastatic therapeutics for pancreatic cancer.MATERIALS AND METHODS:The human pancreatic cancer cell line XPA-1 was engineered to express green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm. Pancreatic stellate cells were engineered to express RFP. The pancreatic cancer cells and stellate cells were co-cultured and their interaction was imaged in vitro. The pancreatic cancer cells and stellate cells were then co-injected in the spleen of transgenic cyan fluorescent protein (CFP) nude mice and imaged in liver, lung and diaphragm metastasis.RESULTS:The interaction of the pancreatic cancer cells expressing GFP in the nucleus and RFP in the cytoplasm and stellate cells expressing RFP was first imaged in vitro. The intimate relationship between the two cell types could be seen. Three hours after splenic co-injection, dual-color pancreatic cancer cells and pancreatic stellate cells were found distributed in the host liver. By 28 days after splenic co-injection of the pancreatic cancer and stellate cells, liver metastases were observed in host CFP nude mice. Metastases were also observed in the lung and diaphragm. Stellate cells were observed along with the pancreatic cancer cells at all metastatic sites suggesting that stellate cells may be necessary for metastasis. With high-resolution intravital imaging afforded by the Olympus FV1000 confocal microscope, the interaction of the dual-colored pancreatic cancer cells and the RFP-expressing pancreatic stellate cells could be clearly imaged in the liver and other metastases, further suggesting that stellate cells participate in metastasis formation.CONCLUSION:Pancreatic cancer cells and stellate stem cells form a very close relationship and accompany each other to distant metastatic sties. Our hypothesis is that pancreatic stellate cells form a niche for metastasis of pancreatic cancer.

Objectives To develop a mouse model for multispectral fluorescence imaging of the pancreas and pancreatic microenvironment. Methods Cre/loxP technology was used to develop this model. We crossed mT/mG indicator mice, engineered to constitutively express a conditional tdTomato transgene that converts to green fluorescent protein (GFP) expression after exposure to Cre recombinase, with Pdx1-Cre transgenic mice. To characterize this model for studies of pancreas biology, we performed bright light and fluorescence imaging of body cavities and intact organs and confocal microscopy of pancreata from offspring of Pdx1-Cre and mT/mG crosses. Results Pdx1-Cre–mT/mG mice demonstrated bright GFP expression within the pancreas and duodenum and intense tdTomato expression in all other organs. Green fluorescent protein expression was mosaic in Pdx1-Cre–mT/mG pancreata, with most showing extensive conversion from tdTomato to GFP expression within the epithelial-derived elements of the pancreatic parenchyma. Because both GFP and tdTomato are membrane targeted, individual cell borders were clearly outlined in confocal images of mT/mG pancreata. Conclusions This mouse model enables multispectral fluorescence imaging of individual cells and cell processes at the microscopic level of the pancreatic microenvironment; it should prove valuable for a variety of fluorescence imaging studies, ranging from pancreatic development to pancreatic cancer biology.

BACKGROUND:We inquired if fluorescence-guided surgery (FGS) could improve surgical outcomes in fluorescent orthotopic nude mouse models of human colon cancer.METHODS:We established fluorescent orthotopic mouse models of human colon cancer expressing a fluorescent protein. Tumors were resected under bright light surgery (BLS) or FGS. Pre- and post-operative images with the OV-100 Small Animal Imaging System (Olympus Corp, Tokyo Japan) were obtained to assess the extent of surgical resection.RESULTS:All mice with primary tumor that had undergone FGS had complete resection compared with 58% of mice in the BLS group (P = 0.001). FGS resulted in decreased recurrence compared with BLS (33% versus 62%, P = 0.049) and lengthened disease-free median survival from 9 to >36 wk. The median overall survival increased from 16 wk in the BLS group to 31 weeks in the FGS group. FGS resulted in a cure in 67% of mice (alive without evidence of tumor at >6 mo after surgery) compared with only 37% of mice that underwent BLS (P = 0.049).CONCLUSIONS:Surgical outcomes in orthotopic nude mouse models of human colon cancer were significantly improved with FGS. The present study can be translated to the clinic by various effective methods of fluorescently labeling tumors.

Abstract In this study, we evaluated multiple wavelengths of different fluorophores of the AlexaFlour (Invitrogen) and Dylight (Thermofisher scientific) series of fluorescent probes for labeling of mouse models of pancreatic cancer. BxPC-3 human pancreatic cancer cells were cultured in-vitro and subsequently injected subcutaneously in nude mice. After the development of subcutaneous tumors, the tumors were harvested and orthotopically implanted into the pancreatic tails of nude mice (1x1 mm fragments). The following fluorescent dyes were evaluated: 488 group (Alexa 488 and Dylight 488); 550 group (Alexa 555 and Dylight 550); 650 group (Alexa 660 and Dylight 650). The dyes were conjugated to chimeric anti-CEA antibody at a ratio of 3-4moles of dye per mole of protein. 50-100 μg of the fluorophore conjugated antibody was injected per mouse intravenously. Mice were then imaged with the OV100 Small Animal Imager (Olympus Corp, Tokyo, Japan). Whole body and intravital imaging were done at various time points (post implantation day 7-14). Mice that had tumor visible on whole body imaging were sacrificed, after which tumor depth, size, weight and fluorescence intensity were recorded. The smallest tumor size was first discernible on whole body imaging with the 650 group of dyes, followed by the 550 group and finally the 488 group with significantly larger tumors. The fluorescence intensity of the Dylight panel of dyes overall was brighter than the AlexaFluor dyes and therefore able to detect smaller and deeper tumors. The 550 and 650 group of dyes had the least amount background fluorescence with the highest amount of tumor to normal tissue contrast. In evaluation of tumor depth the 650 group of dyes were able to detect the deepest tumors; of the group the 488 group had the least tissue penetrance. In this study, the Dylight group of dyes had brighter conjugates with better tissue penetration and tumor delineation when compared to the AlexaFluor dyes. The 650 group of dyes had the most tissue penetration. The 550 group of dyes had similar background autofluorescence to the 650 group that was signficantly less than the 488 group. Both the 550 and 650 group had the highest tumor to background ratio of fluoresence that allows for very high fidelity in the delineation of neoplastic tissue from normal tissue. Citation Format: Ali A. Maawy, Sharmeela Kaushal, Cynthia S. Snyder, George A. Luiken, Mark A. Talamini, Robert M. Hoffman, Michael Bouvet. Evaluation of a chimeric anti CEA-conjugated antibody with AlexaFlour or Dylight fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 722. doi:10.1158/1538-7445.AM2013-722

Abstract The aim of this study was to improve disease-free survival and overall survival in orthotopic nude mouse models of human colon cancer with fluorescence-guided surgery (FGS). Fluorescent orthotopic or carcinomatosis models were established in nude mice using human colon cancer cell lines HCT-116 and HT-29 expressing either green fluorescent protein (GFP) or red fluorescent protein (RFP). The tumors were later resected by bright light surgery (BLS) or FGS. In orthotopic and carcinomatosis models, pre- and post-operative images were obtained with the OV-100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan) to assess the extent of surgical resection. In the orthotopic model, whole body imaging of the mice was performed in the postoperative period to assess cancer recurrence and to follow subsequent tumor progression. The mice were sacrificed when they became premorbid and their abdomens were exposed for intravital and ex vivo imaging. Tumor burden was measured in mm2 using ImageJ v1.440. A greater extent of tumor resection in mice with carcinomatosis was achieved using FGS compared to BLS (99.9% vs. 76.9%, p = 0.006). Furthermore, all mice with localized disease had a complete surgical resection with FGS. In contrast, complete resection was achieved in only 56% of the mice undergoing BLS. (p=0.001) Fewer mice in the FGS group had evidence of tumor recurrence (33%) compared to mice in the BLS group (58%), lengthening disease-free survival from 9 weeks in the BLS group to 27 weeks in the FGS group. Overall survival of the mice also increased from 17 weeks in the BLS group to 29 weeks in the FGS group. The results of the present study demonstrate that improved surgical outcomes in the treatment of colon cancer can be achieved with FGS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 372. doi:1538-7445.AM2012-372

Abstract The aim of this study was to select a more aggressive variant of a human pancreatic cancer cell line by serially passaging primary pancreatic tumor in nude mice and to evaluate for changes in tumor morphology as well as expression of stem cell markers and PEAK. Mouse models of human pancreatic cancer were established by orthotopically injecting 1x106 brightly-fluorescent BxPC-3 RFP pancreatic cancer cells into nude mice. Mice were subsequently followed with weekly whole body imaging using the Olympus OV-100 Small Animal Imaging System to follow tumor progression and time to metastasis. When deemed premorbid, the mice were sacrificed and their abdomens exposed for intravital imaging. Primary pancreatic tumor, a periportal metastatic deposit and ascites were removed from the premorbid mouse to establish cell lines. One million cells of the passaged primary pancreatic cell line were then orthotopically injected into another set of nude mice. Serially passaging continued until the life-span of the mouse with tumor stabilized. We subsequently analyzed samples of primary and metastatic lesions from the serially passaged tumors for analysis of stem cell markers and PEAK expression. With serial in vivo passaging of tumor, we were able to generate a more aggressive variant of human pancreatic cancer cell line BxPC-3 that displayed a more rapid primary tumor growth and a shortened overall survival in mice harboring tumor. Overall survival decreased from a mean of 20 weeks in mice with the parental cell line (P0) tumor to 7 weeks in mice with third-passage primary (P3) tumor. Additionally, tumor take was more readily established with the more aggressive cell line and time to metastasis within the abdominal cavity was shortened despite implanting a consistent number of cells at each passage. While mice harboring the parental line did not demonstrate evidence of metastasis until approximately three months post implantation, mice with P3 tumor developed metastatic disease by the second month after implantation. Real-time PCR evaluation demonstrated enrichment of the stem cell population in P3 compared to the parental cell line (P0). While there was an ∼2.5-fold increase in CD24 and EpCAM markers in the P3 primary tumor, CD44 had a 4-fold increase. Furthermore, P3 primary tumor had a 4-fold increase in PEAK expression while the periportal metastatic lesion from a P3 primary tumor had a 5.5-fold increase in the expression of PEAK. Further understanding of these in vivo effects on tumor initiation, progression and metastasis can aide in discovery of effective therapies to treat this dismal disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3375. doi:1538-7445.AM2012-3375

The aim of this study was to determine if fluorescence-guided surgery (FGS) could improve surgical outcomes, reduce recurrence rates and improve overall survival in orthotopic mouse models of human pancreatic cancer. Orthotopic mouse models of human pancreatic cancer were established using the BxPC-3 RFP-expressing pancreatic cancer cell line in nude mice. Two weeks after implantation, the mice were randomized to undergo bright light surgery (BLS) or fluorescence-guided surgery (FGS). Pre- and postoperative images were obtained with the Olympus OV-100 Small Animal Imaging System to assess completeness of surgical resection. Whole body imaging of the mice was performed weekly in the postoperative period to assess for recurrence and follow tumor progression. Additionally, half of the mice were randomly selected to undergo 4 weeks of postoperative gemcitabine treatment. At six weeks postoperatively, or when premorbid, the mice were sacrificed and primary pancreatic tumor burden was measured using ImageJ v1.440. A more complete resection of pancreatic cancer was achieved using FGS compared to BLS: 98.9% vs 77.1%, p=0.005. Sixty-three percent of the mice undergoing BLS had evidence of gross residual disease, whereas 20% of mice undergoing FGS received a complete resection and an additional 75% of the mice were left with minimal residual disease (p=0.0001). At six weeks, the primary pancreatic tumor burden was significantly less with FGS compared to BLS: 19.341 ± 5.26 mm2 vs. 6.194 ± 3.61 mm2, p=0.048. In a pilot survival study, FGS alone was associated with a decreased risk of tumor recurrence (HR=0.093, 95% CI 0.013-0.645), and mean overall survival was lengthened from 18.5 weeks to 28.2 weeks. The addition of adjuvant gemcitabine (GEM) to FGS further lengthened mean overall survival to 42.75 weeks (p=0.019). Three of the four mice in the FGS with adjuvant gemcitabine group were alive at 12 months postoperatively and all of the mice (n=4) were free of tumor at time of sacrifice. In this study, we achieved improved surgical outcomes in primary pancreatic cancer under fluorescence-guidance. FGS afforded a more complete resection of primary tumor and improved disease-free survival and overall survival. The combination of FGS and GEM lengthened disease-free survival and improved overall survival. This novel approach has potential to improve outcomes in the surgical treatment of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 375. doi:1538-7445.AM2012-375

Abstract The aim of this study was to evaluate the utility of fluorescence laparoscopy for resection of primary pancreatic cancer. Orthotopic mouse models of human pancreatic cancer were established with the brightly-fluorescent BxPC-3 RFP human pancreatic cancer cell line. Two weeks after implantation, mice were randomized to undergo either bright light laparoscopic resection (BLR) or fluorescence-guided laparoscopic resection (FLR). Fluorescence laparoscopy (FL) was performed with a Stryker L9000 LED light source 24 hours after tail vein injection of CEA antibodies conjugated to Alexa 488. Bright light laparoscopy (BL) was performed with a Stryker X8000 xenon light source. Pre- and postoperative images were taken with the Olympus OV-100 Small Animal Imaging System to assess completeness of resection. Postoperatively, whole body images were obtained to assess for recurrence and follow tumor progression. All resected tumors were collected for histologic review. Fluorescence laparoscopy with a 495-nm emission filter and an LED light source enabled real-time identification and localization of the brightly-fluorescent BxPC-3 RFP tumor additionally labeled with anti-CEA-Alexa 488. The combination of red and green fluorophores optimized the fluorescence signal of the tumor thereby enabling accurate distinction of tumor margins from normal surrounding tissue without compromising background illumination. Furthermore, adequate visualization of surrounding structures enabled surgical navigation for resection of primary pancreatic tumor. Tumor was more readily detected and resected under fluorescence guidance than under standard bright light. In addition, sub-millimeter lesions within the pancreas that were undetected by bright light laparoscopy were easily identified and resected by fluorescence laparoscopy. This study demonstrates the capabilities of laparoscopic fluorescence-guided surgery of primary pancreatic cancer. The optimal combination of fluorophores with the use of an LED light source enhanced the fluorescence tumor-to-background ratio while permitting enough light leakage for background illumination for surgical navigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 366. doi:1538-7445.AM2012-366

Abstract Conditional fluorescent (mTmG) indicator mice constitutively express a conditional tdTomato transgene (mTmG) that converts to the expression of EGFP following exposure to Cre recombinase. PdxCre+, mTmG+ mice express bright, membrane-targeted, green fluorescent protein (mG) specifically in the pancreatic epithelial parenchyma. Non-epithelial pancreatic tissues and non-pancreatic tissues ubiquitously express membrane-targeted tdTomato (mT), providing a red fluorescent backlight in imaging studies. In the present report, we have applied the strengths of the PdxCre+, mTmG+ dual color system to the study of spontaneous tumor development in a triple transgenic PdxCre, LSL-KrasG12D/+, LSL-Trp53R172H/+ mouse model of human pancreatic ductal adenocarcinoma (PDAC). We generated mice that follow the well-described progression of pre-neoplastic changes in the pancreas, and ultimately develop fluorescent pancreatic tumors, recapitulating the sequence of events that occur in human PDAC in an imagable mouse model system. To establish this model, we intercrossed four lines of transgenic mice [PdxCre (C), LSL-KrasG12D/+(K), LSLTrp53R172H/+(P), and mTmG (mTmG)] and generated small cohorts of quadruple transgenics (CKPmTmG) and littermate controls. Like PdxCre+, mTmG+ dual transgenic mice, CKPmTmG mice demonstrated green fluorescent pancreata and red fluorescent non-pancreatic tissues. As in the non-fluorescent PdxCre, LSL-KrasG12D/+, LSL-Trp53R172H/+ (CKP) parental line, CKPmTmG mice developed pancreatic dysmorphia and dysplasia within the first several weeks of life. These changes were accompanied by development of pancreatic tumors and were associated with early mortality not seen in littermate controls. Ex vivo imaging of CKPmTmG mice at necropsy demonstrated a spectrum of pathologic findings, including cystic and solid pancreatic tumor masses surfaced by distorted and distended green fluorescent pancreatic ducts and duct-like structures. The non-epithelial pancreatic tissues provided a red fluorescent background. Histologic findings included ductal dilatation, ductal atypia with features of pancreatic intraepithelial neoplasia (PanIN) as previously described in CKP mice, atrophy of pancreatic acini, and duct-centered desmoplasia entrapping the occasional isolated pancreatic islet. This dual color, quadruple transgenic mouse model enables us to apply fluorescence imaging technology to the study of tumor microenvironment in mice genetically engineered to develop pancreatic cancer spontaneously. Based on preliminary studies, this mouse model promises to be a valuable tool with which to address many intriguing questions and timely controversies regarding the pathogenesis of human pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-507. doi:1538-7445.AM2012-LB-507

Abstract The aim of the present study was to improve detection and treatment of colon cancer with the use of fluorescence laparoscopy in an orthotopic mouse model of human colon cancer. The orthotopic model was established by intra-cecal injection of GFP-expressing HCT-116 human colon cancer cells into 12 six-week-old female athymic mice. Two models were then established: one of early disease and the second of widely metastatic disease. Two weeks after implantation, 6 mice underwent diagnostic laparoscopy. Laparoscopy was performed first under standard brightfield lighting, followed by fluorescent lighting. The number of metastatic foci identified within each of the four quadrants of the peritoneal cavity was recorded in three mice in each light mode. This process was repeated in the second group 4 weeks after implantation. Following laparoscopy, all animals were sacrificed. Fluorescence imaging was used as a positive control to identify metastasis at necropsy. Tumors and metastases were collected and processed for histologic review. Fluorescence laparoscopy enabled visualization of colon cancer metastatic foci which could not be visualized with standard brightfield laparoscopy in both the early (p= 0.028) and late (p= 0.001) models of colon cancer. When compared to the positive control of open fluorescence imaging, standard brightfield laparoscopy was significantly inferior in both the early (p= 0.03) and late (p= 0.0005) groups. Conversely, fluorescence laparoscopy was not significantly different from open fluorescence imaging in either the early (p= 0.94) or late (p= 0.56) groups. Fluorescence laparoscopy thus allowed identification and localization of sub-millimeter micrometastases that could not be distinguished from surrounding tissue under brightfield laparoscopy. Metastatic lesions were histologically confirmed. This report illustrates the important clinical potential of fluorescence laparoscopy in the surgical treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5530. doi:1538-7445.AM2012-5530

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pancreatic stellate cells are involved in fibrosis of pancreatic cancer. An understanding of pancreatic cancer-cell interactions with stellate cells is critical to our ability to develop effective anti-tumor therapeutics for pancreatic cancer. We report here imaging of the interaction of pancreatic cancer cells and pancreatic stellate cells in liver metastasis. Human pancreatic cancer cell lines (XPA1 and MiaPaCa-2) were engineered to express green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm. Pancreatic stellate cells, engineered to express RFP, were co-injected with the cancer cells into the spleen of transgenic cyan fluorescent protein (CFP) nude mice. Three hours after splenic injection dual-color pancreatic cancer cells and pancreatic stellate cells were found distributed in the host liver. Seven days after cancer cell-stellate cell co-injection, most pancreatic cancer cells and stellate cells were dead. However, by 28 days after injection, liver metastases were observed in the host CFP nude mice. With the high-resolution intravital imaging afforded by the Olympus FV1000 confocal microscope, the interaction of the dual-colored pancreatic cancer cells and the RFP-expressing pancreatic stellate cells could be clearly imaged in the liver metastasis, suggesting that stellate cells participate in metastasis formation. Our hypothesis is that pancreatic stellate cells form a niche for liver metastasis of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1695. doi:1538-7445.AM2012-1695

BACKGROUND:The aim of this study was to improve fluorescence laparoscopy of pancreatic cancer in an orthotopic mouse model with the use of a light-emitting diode (LED) light source and optimal fluorophore combinations.STUDY DESIGN:Human pancreatic cancer models were established with fluorescent FG-RFP, MiaPaca2-GFP, BxPC-3-RFP, and BxPC-3 cancer cells implanted in 6-week-old female athymic mice. Two weeks postimplantation, diagnostic laparoscopy was performed with a Stryker L9000 LED light source or a Stryker X8000 xenon light source 24 hours after tail-vein injection of CEA antibodies conjugated with Alexa 488 or Alexa 555. Cancer lesions were detected and localized under each light mode. Intravital images were also obtained with the OV-100 Olympus and Maestro CRI Small Animal Imaging Systems, serving as a positive control. Tumors were collected for histologic analysis.RESULTS:Fluorescence laparoscopy with a 495-nm emission filter and an LED light source enabled real-time visualization of the fluorescence-labeled tumor deposits in the peritoneal cavity. The simultaneous use of different fluorophores (Alexa 488 and Alexa 555), conjugated to antibodies, brightened the fluorescence signal, enhancing detection of submillimeter lesions without compromising background illumination. Adjustments to the LED light source permitted simultaneous detection of tumor lesions of different fluorescent colors and surrounding structures with minimal autofluorescence.CONCLUSIONS:Using an LED light source with adjustments to the red, blue, and green wavelengths, it is possible to simultaneously identify tumor metastases expressing fluorescent proteins of different wavelengths, which greatly enhanced the signal without compromising background illumination. Development of this fluorescence laparoscopy technology for clinical use can improve staging and resection of pancreatic cancer. (JAmColl Surg 2012;214:997-1007. (C) 2012 by the American College of Surgeons)

212 Background: Standard laparoscopy for pancreatic cancer often leads to false negative results, causing understaging of the disease. Improved sensitivity and resolution are necessary. Methods: Orthotopic and carcinomatosis mouse models of human pancreatic cancer were established with red fluorescent protein (RFP)-expressing or non-fluorescent BxPC-3 human pancreatic cancer cells. The mice with orthotopic unlabeled pancreatic cancer were administered Alexa 488- or 555-conjugated anti-CEA by tail-vein injection 2-4 weeks after tumor implantation. Diagnostic laparoscopy was performed with a Stryker L9000 LED light source or X8000 xenon light source 24 hours later. Pancreatic tumors were detected and localized under each light mode. After laparoscopy, intravital images were obtained with the OV-100 and Maestro CRI Small Animal Imaging Systems as positive controls. Tumors were collected for histologic analysis. Results: Fluorescence laparoscopy (FL) with the use of 495-nm excitation filter and an LED light source enabled more rapid and accurate identification and localization of primary tumors and metastases than bright light laparoscopy (BL). The use of fluorescent conjugates antibody-labeled tumors improved the accuracy of staging laparoscopy, increasing the sensitivity from 40% in BL to 96% in FL (p<0.001). FL was sufficiently sensitive to detect sub-millimeter tumor deposits that went undetected under BL. With adjustments to the LED light source, we could simultaneously detect tumor lesions of different fluorescent colors and surrounding structures with minimal autofluorescence. Conclusions: The use of FL and fluorophore-labeled anti-CEA antibodies permits rapid detection and accurate localization of primary and metastatic CEA-expressing human pancreatic cancer, including tumors that were undetectable with BL. The introduction of an LED light source allows simultaneous identification of fluorescent tumor of different wavelengths without compromising background illumination. Further development of this technology for clinical use can improve the staging and treatment of pancreatic cancer.

OBJECTIVETo improve detection of colon cancer metastases using fluorescence laparoscopy (FL).DESIGNAn orthotopic mouse model of human colon cancer was established by intracecal injection of HCT-116 human colon cancer cells expressing green fluorescent protein into 12 mice. One group modeled early disease and the second modeled late metastatic disease. For the early-disease model, 2 weeks after implantation, 6 mice underwent 2 modalities of laparoscopy: bright field laparoscopy (BL) and FL. The number of metastases identified within each of the 4 abdominal quadrants was recorded with both laparoscopy modalities. This process was repeated in the late-metastatic disease group 4 weeks after implantation. All animals were then humanely sacrificed and imaged using open fluorescence laparoscopy (OL) as a positive control to identify metastases.SETTINGBasic science laboratory.PARTICIPANTSTwelve female, 6-week-old nude mice.INTERVENTIONSDetection of tumor foci by FL compared with BL.MAIN OUTCOME MEASURESNumber of tumors identified in each quadrant. RESULTS Fluorescence laparoscopy enabled superior visualization of colon cancer metastases compared with BL in the early (P = .03) and late (P = .002) models of colon cancer. Compared with OL, BL was significantly inferior in the early (P = .04) and late (P < .001) groups. Fluorescence laparoscopy was not significantly different from OL in the early (P = .85) or late (P = .46) group. Thus, FL allowed identification of micrometastases that could not be distinguished from surrounding tissue using BL.CONCLUSIONSThe use of FL enables identification of metastases that could not be visualized using standard laparoscopy. This report illustrates the important clinical potential for FL in the surgical treatment of cancer.

Abstract The aim of this study was to improve detection of primary and metastatic lesions of human pancreatic cancer in mouse models with fluorescence laparoscopy by enhancing the fluorescence signal of tumor with different combinations of fluorophores without compromising background illumination. Human pancreatic cancer models were established with combinations of FG-RFP, MiaPaca2-GFP, BxPC-3-RFP, or BxPC-3 cancer cells implanted in 6-week-old female athymic mice. Two weeks post-implantation, diagnostic laparoscopy was performed with a Stryker L9000 LED light source 24 hours after tail vein injection of CEA antibodies conjugated with Alexa 488- and/or Alexa 555. Cancer lesions were detected and localized under the fluorescence light mode. Intravital images were obtained with the Maestro CRI Small Animal Imaging System, serving as a positive control. Tumors were collected for histologic review. Fluorescence laparoscopy with a 495-nm emission filter and an LED light source enabled real-time visualization of differently fluorescence-labeled tumors in the peritoneal cavity simultaneously. The combination of BxPC-3 RFP labeled with anti-CEA-Alexa 488 afforded the brightest signal of all established mouse models. We were able to reproduce this enhanced fluorescence signaling by doubly labeling non-color BxPC-3 tumor with anti-CEA-Alexa 488 and anti-CEA-Alexa 555. The doubly labeled tumor provided a greater signal intensity profile compared to tumors labeled with either anti-CEA-Alexa 488 or -Alexa 555 alone. This enhanced fluorescence signal permitted improved detection of sub-millimeter lesions without compromising background illumination. The ability to visualize tumor deposits expressing fluorescent proteins of different wavelengths simultaneously with an LED light source enabled us to establish the optimal combination of fluorophores that enhanced the signal intensity of fluorescence. With this optimal fluorophore combination, we improved the detection of sub-millimeter tumor lesions without impairing the visualization of surrounding structures necessary for surgical navigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 369. doi:1538-7445.AM2012-369

BACKGROUND/AIMS:Laparoscopy is important in staging pancreatic cancer, but false negatives remain problematic. Making tumors fluorescent has the potential to improve the accuracy of staging laparoscopy.METHODOLOGY:Orthotopic and carcinomatosis models of pancreatic cancer were established with BxPC-3 human pancreatic cancer cells in nude mice. Alexa488-antiCEA conjugates were injected via tail vein 24 hours prior to laparoscopy. Mice were examined under bright field laparoscopic (BL) and fluorescence laparoscopic (FL) modes. Outcomes measured included time to identification of primary tumor for the orthotopic model and number of metastases identified within 2 minutes for the carcinomatosis model.RESULTS:FL enabled more rapid and accurate identification and localization of primary tumors and metastases than BL. Using BL took statistically significantly longer time than FL (p<0.0001, fold change and 95% CI for BL vs. FL: 8.12 (4.54,14.52)). More metastatic lesions were detected and localized under FL compared to BL and with greater accuracy, with sensitivities of 96% vs. 40%, respectively, when compared to control. FL was sensitive enough to detect metastatic lesions <1mm.CONCLUSIONS:The use of fluorescence laparoscopy with tumors labeled with fluorophore-conjugated anti-CEA antibody permits rapid detection and accurate localization of primary and metastatic pancreatic cancer in an orthotopic model. The results of the present report demonstrate the future clinical potential of fluorescence laparoscopy.

Background The use of fluorescent proteins to label tumors is revolutionizing cancer research, enabling imaging of both primary and metastatic lesions, which is important for diagnosis, staging, and therapy. This report describes the use of fluorescence laparoscopy to image green fluorescent protein (GFP)-expressing tumors in an orthotopic mouse model of human pancreatic cancer. Methods The orthotopic mouse model of human pancreatic cancer was established by injecting GFP-expressing MiaPaCa-2 human pancreatic cancer cells into the pancreas of 6-week-old female athymic mice. On postoperative day 14, diagnostic laparoscopy using both white and fluorescent light was performed. A standard laparoscopic system was modified by placing a 480-nm short-pass excitation filter between the light cable and the laparoscope in addition to using a 2-mm-thick emission filter. A camera was used that allowed variable exposure time and gain setting. For mouse laparoscopy, a 3-mm 0° laparoscope was used. The mouse’s abdomen was gently insufflated to 2 mm Hg via a 22-gauge angiocatheter. After laparoscopy, the animals were sacrificed, and the tumors were collected and processed for histologic review. The experiments were performed in triplicate. Results Fluorescence laparoscopy enabled rapid imaging of the brightly fluorescent tumor in the pancreatic body. Use of the proper filters enabled simultaneous visualization of the tumor and the surrounding structures with minimal autofluorescence. Fluorescence laparoscopy thus allowed exact localization of the tumor, eliminating the need to switch back and forth between white and fluorescence lighting, under which the background usually is so darkened that it is difficult to maintain spatial orientation. Conclusion The use of fluorescence laparoscopy permits the facile, real-time imaging and localization of tumors labeled with fluorescent proteins. The results described in this report should have important clinical potential.

Introduction: Metastatic pancreatic cancer has a very poor prognosis. Aggressive surgical resection provides no additional survival benefit. Staging laparoscopy has improved the diagnostic yield of peritoneal and liver metastases, otherwise undetectable by preoperative imaging. However, false-negative rates may be as high as 28%. The use of fluorescent proteins to label tumors enables imaging of primary and metastatic lesions, thus improving the diagnostic yield of staging laparoscopy. This report describes the use of fluorescence laparoscopy to image green fluorescent protein (GFP)-expressing tumors in a carcinomatosis mouse model of human pancreatic cancer. Methods: A carcinomatosis mouse model of human pancreatic cancer was established by intraperitoneal injection of 2×106 GFP-expressing MiaPaca-2 human pancreatic cancer cells into 6-week-old female athymic mice. On postoperative day 21, diagnostic laparoscopy, using white and fluorescent light, was performed. To standardize the procedure, all four quadrants of the peritoneal cavity were examined under both light modes in the same time frame, using staging laparoscopic methods. An attempt was made to detect and localize all cancer lesions under each light mode. After laparoscopy, the animals were sacrificed and abdominal cavities exposed. Images of the lesions were obtained with the OV-100 Small Animal Imaging System under GFP filter, serving as a positive control. Tumors were collected and processed for histologic review. Results: Fluorescence laparoscopy enabled visualization of the brightly fluorescent tumor metastases in the peritoneal cavity. With the use of a 480-nm filter, we could simultaneously detect tumor lesions and surrounding structures with minimal autofluorescence. Fluorescence laparoscopy afforded accurate detection of more lesions compared to standard bright field laparoscopy. In one quadrant of a representative mouse, over 28 small (<1 mm) metastatic lesions were identified under fluorescence, while bright field revealed only the one large lesion and yielded three false positive lesions (Figure). Fluorescence compared well to the positive control OV-100 images. Fluorescence laparoscopy thus allowed identification and exact localization of numerous tumor metastases less than 1 mm. Lesions of this size were not detected under bright field laparoscopy. All identified lesions, when possible, were verified histologically. Conclusion: The use of fluorescence laparoscopy improves the diagnostic yield of staging laparoscopy in identifying areas of tumor metastases labeled with fluorescent proteins. The results described in this report should have important clinical potential and would be instrumental in future clinical applications of fluorescence laparoscopy.