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Background Tumor-associated glycoprotein (TAG)–72 is a pancarcinoma antigen that is overexpressed in greater than 80% of colorectal adenocarcinomas. CC49 is a TAG-72–specific antibody. The aim of the present study was to demonstrate selective imaging of colon tumors and metastases with the humanized TAG-72 antibody (anti-huCC49) conjugated to a near-infrared fluorophore in orthotopic mouse models. Methods Anti-huCC49 was conjugated to near-infrared dye IR800CW. Mouse imaging was performed with the Pearl Trilogy Small Animal and FLARE Imaging Systems. Subcutaneous mouse models of colon cancer cell line LS174T were used to determine the optimal dose of administration and timing of imaging. Orthotopic mouse models of LS174T were established by surgical orthotopic implantation of LS174T tumors onto the serosa of the cecum. Peritoneal carcinomatosis models were established by injection of LS174T cells into the peritoneum of nude mice. Mice were administered anti-huCC49–IR800 via tail vein injection. Mice were euthanized 72 h later and imaged after laparotomy. Results Subcutaneous LS174T xenografts demonstrated optimal tumor detection 72 h after administration with 50 μg anti-huCC49-IR800CW. Tumors were visualized with fluorescence imaging with a mean tumor-to-liver ratio of 7.39 (standard deviation: 2.76). In the orthotopic model, metastases smaller than 1 mm were fluorescently visualized that were invisible with bright light. Conclusions Anti-huCC49–IR800CW provides sensitive and specific imaging of colon cancer and metastases at a submillimeter resolution in metastatic nude mice models. This provides a promising near-infrared probe for the imaging of colon cancer and metastases for preoperative diagnosis and fluorescence-guided surgery.

Background/Aim: Surgical orthotopic implantation (SOI) is used to establish patient-derived orthotopic xenograft (PDOX) and other orthotopic mouse models. Orthotopic liver models can be challenging, as the liver parenchyma is prone to bleeding. The present report describes a sutureless method to implant tumors in the liver that reduces bleeding and procedural time. Materials and Methods: Human HCC cell-line (Huh-7-GFP) and CM2, a patient-derived colon-cancer liver metastasis, were used for sutureless SOI of tumor fragments in the liver of nude mice. A small cavity was formed on the liver surface. A solitary tumor fragment was implanted in the cavity without suturing to create hemostasis. Results: Six weeks after sutureless SOI, the tumor volume of Huh-7-GFP (n=5) was 584.41±147.64 mm(3) and the tumor volume of CM2 (n=5) was 1336.54±1038.20 mm(3). The engraftment rate was 100%. Conclusion: This novel method for establishing orthotopic liver-implantation mouse models is suitable for studies of liver cancer and liver metastases due to its simple procedure and potential high engraftment rate.

Background/aim Fluorescence imaging has been shown to improve intra-operative detection of liver metastasis. The present study aimed to determine whether humanized anti-TAG-72 antibody (huCC49) conjugated to a near-infrared dye provides selective labeling of colorectal-cancer liver metastasis in orthotopic mouse models. Materials and methods Humanized anti-TAG-72 (huCC49) was conjugated to IRDye800CW (huCC49-IR800). Orthotopic liver-metastasis nude-mouse models (n=5) were established with the human colon-cancer LS174T cell-line. Three weeks later, mice were administered huCC49-IR800 and intra-vital imaging was performed 48 h later. The mean tumor-to-liver ratio (TLR) was calculated. Results Intra-vital imaging demonstrated clear tumor margins with minimal liver fluorescence 48 h after administration of 50 μg huCC49-IR800 with mean TLR=7.53 (SD±2.76). Conclusion Anti-TAG-72 monoclonal antibody conjugated to IRDye800 provides distinct and bright labeling of colorectal tumors in orthotopic nude-mouse models of liver metastasis. TAG-72 may be a useful target for intra-operative imaging of colorectal cancer liver metastasis in the clinic.

Background/aim The visualization of hepatic segments with indocyanine green (ICG) fluorescence can aid in anatomic liver resection. The present study aimed to develop a method to specifically label an hepatic segment in a nude mouse model with liver metastasis. Materials and methods An orthotopic mouse model was established by surgical orthotopic implantation (SOI) of a patient-derived colon-cancer liver metastasis in the left lobe of the liver. Three weeks after SOI, the left Glissonean pedicle was ligated and 10 μg ICG was administrated intravenously. Images were obtained with the Pearl Trilogy Imaging System. Results All mice expressed an 800 nm signal from ICG on the right lobe of the liver. The left lobe of the liver, in which the tumor was located, showed no fluorescence and had ischemia due to successful ligation of the Glissonean pedicle. Conclusion The ligation of the Glissonean pedicle enables specific liver-segment labeling with ICG, which has potential clinical application for liver metastasectomy.

Monocytes are important regulators for the maintenance of homeostasis in innate and adaptive immune system and have been reported to play important role in cancer progression. CD47-SIRPα recognition is a coinhibitory immune signal to inhibit phagocytosis in monocytes and macrophages and has been well-known as the "Don't eat me" signal. By using an approach of integrated sensing and activating proteins (iSNAPs), we have rewired the CD47-SIRPα axis to create iSNAP-M which activates pathways in engineered human monocytes (iSNAP-MC). The mRNA expression levels of the monocyte/macrophage markers CD11b, CD14, and CD31 are upregulated in iSNAP-monocytes (iSNAP-MC). With PMA induction, the iSNAP-MC-derived macrophages (iSNAP-MΦ) showed upregelation in CD86 and CD80, but not CD206. TNFα expression and secretion were also increased in iSNAP-MΦ. Furthermore, the injection of iSNAP-MC into mice bearing human B-lymphoma tumors led to the suppression of tumor progression. Therefore, the engineered monocytes, via blockage of coinhibitory immune signals by rewiring CD47-SIRPα axis, can be applied to suppress target tumors for cancer immunotherapy.

Introduction Claudins are tight-junction proteins, which maintain an epithelial barrier in normal colon cells. Overexpression of Claudin-1 has been implicated for development of colon cancer. We postulated that Claudin-1 may be a useful target in near-infrared imaging and fluorescence-guided surgery. Methods We conjugated Claudin-1 antibody to LI-COR IR800DyeCW (Claudin-1-IRDye800CW). Western blotting of 9 human colon cancer cell lysates was performed. Animal imaging was performed with the LI-COR Pearl Trilogy Fluorescence Imaging System. A dose-response study was carried out with subcutaneous LS174T colon cancer cell line models. Increasing doses of Claudin-1-IRDye800CW via tail vein injection were administered to three groups of mice. Two groups of mice were used as controls (antibody alone, and dye alone). In vivo imaging was performed at 24, 48, and 72 h after administration of the conjugated dye. Orthotopic implantation of patient-derived tumors and cell lines was performed and peritoneal carcinomatosis models were created. After tumor growth, mice were administered Claudin-1-IRDye800CW and imaged in vivo 48 h later. The mice were euthanized and laparotomy was performed to assess internal organs and toxicity. Results Western blotting revealed that all colon cancer cell lysates expressed varying amounts of Claudin-1. All tumors demonstrated strong and specific fluorescence labeling at 800 nm, even with the lowest dose of 12.5 μg of Claudin-1-IRDye800CW. Conclusions Claudin-1 is a useful target for near-infrared antibody-based imaging for visualization of colorectal tumors for future use in fluorescence-guided surgery.

BACKGROUND: Nanobodies, derived from camelid antibodies made of only heavy chains, are the smallest, biologic, antigen-binding fragments (~15kDa) with faster pharmacokinetics and better tumor penetration efficiency than standard antibodies. The present study evaluates the efficacy of a fluorescent, anti-carcinoembryonic antigen nanobody for rapid tumor labeling in an orthotopic mouse model of pancreatic cancer. METHODS: Anti-carcinoembryonic antigen or control nanobodies were conjugated with the near-infrared fluorophore IRDye 800CW. Fragments of BxPC3 or MiaPACA2 human pancreatic cancer cell lines were orthotopically implanted into the pancreatic tail of nude mice. After tumors reached 7 to 10 mm in size, 2 nmol anti-carcinoembryonic antigen or control nanobody-IRDye800CW was injected intravenously. Mice were imaged at various time points hours postinjection. RESULTS: Carcinoembryonic antigen-expressing, BxPC3 pancreatic orthotopic tumors were fluorescently labeled with aCEA-nb-800 3 hours after injection. The signal was present as early as 15 minutes after injection and was robust at 1 to 3 hours after injection with a tumor-to-background ratio of 2.66. In contrast, there was very low accumulation in the low carcinoembryonic antigen-expressing, MiaPACA2 pancreatic orthotopic tumors. The fluorophore-conjugated nanobody was specific for carcinoembryonic antigen-expressing tumors, while the control nanobody did not show any tumor-specific signal. Both nanobodies had strong kidney uptake as expected for small-molecule probes. The fluorescence signal was detectable using 2 clinical, Food and Drug Administration-approved, 800 nm imaging devices as well as small animal imaging systems. CONCLUSION: This anti-carcinoembryonic antigen, nanobody-based, fluorescent probe labeled pancreatic orthotopic tumors within 15 minutes of intravenous injection. Fluorescent anti-carcinoembryonic antigen nanobodies have labeling kinetics that approach the speed of nonspecific dyes such as indocyanine green but with the specificity of antibodies. The use of fluorescently-labeled, intact antibodies leads to a labeling delay of 48 to 96 hours between probe administration and the necessarily delayed time of operation, which can be avoided with nanobodies. The kinetics of a nanobody-based probe makes it a practical agent for same-day, patient administration and fluorescence-guided surgery.

BackgroundPhotoimmunotherapy (PIT) employs the use of a near-infrared (NIR) laser to activate an antibody conjugated to a NIR-activatable dye to induce cancer cell death. PIT has shown to be effective in a number of studies, however, there are no data on its use in colorectal cancer in an orthotopic model.MethodsHumanized anti-CEA antibody (M5A) was conjugated to NIR-activatable IRDye700DX (M5A-700). PIT was validated in vitro with a colon cancer cell-line, using a laser intensity of either 4 J/cm2, 8 J/cm2, or 16 J/cm2. Orthotopic colon cancer mouse models were established by surgical implantation of LS174T tumor fragments onto the cecum. M5A-700 was administered and PIT was performed 24 hours later using a 690 nm laser. Repeat PIT was performed after 7 days in one group. Control mice received laser treatment only.ResultsIn vitro PIT demonstrated tumor cell death in a laser intensity dose-dependent fashion. In orthotopic models, control mice demonstrated persistent tumor growth. Mice that underwent PIT one time had tumor growth arrested for one week, after which re-growth occurred. The group that received repeated PIT exposure had persistent inhibition of tumor growth.ConclusionPIT arrests tumor growth in colon cancer orthotopic nude-mouse models. Repeated PIT arrests colon cancer growth for a longer period of time. PIT may be a useful therapy in the future as an adjunct to surgical resection or as primary therapy to suppress tumor progression.

HopQ is an outer-membrane protein of Helicobacter pylori that binds to human carcinoembryonic antigen-related cell-adhesion molecules (CEACAMs) with high specificity. We aimed to investigate fluorescence targeting of CEACAM-expressing colorectal tumors in patient-derived orthotopic xenograft (PDOX) models with fluorescently labeled recombinant HopQ (rHopQ). Western blotting, flow cytometry and ELISA were performed to determine the efficiency of rHopQ binding to CEACAMs. rHopQ was conjugated to IR800DyeCW (rHopQ-IR800). Nude mice received orthotopic implantation of colon cancer tumors. Three weeks later, mice were administered 25 μg or 50 μg HopQ-IR800 and imaged 24 or 48 h later. Intravital images were analyzed for tumor-to-background ratio (TBR). Flow cytometry and ELISA demonstrated binding of HopQ to CEACAM1, 3 and 5. Dose-response intravital imaging in PDOX models demonstrated optimal results 48 h after administration of 50 μg rHopQ-IR800 (TBR = 3.576) in our protocol. Orthotopic models demonstrated clear tumor margins of primary tumors and small regional metastases with a mean TBR = 3.678 (SD ± 1.027). rHopQ showed specific binding to various CEACAMs in PDOX models. rHopQ may be useful for CEACAM-positive tumor and metastasis detection for pre-surgical diagnosis, intra-operative imaging and fluorescence-guided surgery. CA extern

Background: Liver surgery is the mainstay curative treatment of liver cancer and metastasis. There are two main procedures for liver surgery. One is anatomic resection which is based on the portal flow and the other is partial resection for securing the surgical margin. Indocyanine green (ICG) fluorescence-guided surgery (FGS) has recently been developed in the field of liver surgery. Preoperative ICG administration provides tumor location for partial resection. Intraoperative ICG administration shows the liver segmentation for anatomic resection. However, it is difficult to distinguish between them, especially in the case that tumor lies close to a fluorescent liver segment in a single color image. Our laboratory previously developed color-coded FGS in an orthotopic mouse model of cancer that improved outcome over single color FGS (1). Our aim is to investigate a color-coded FGS to enhance the boundary between the tumor and the transection plane using ICG in combination with a fluorescent-labeled tumor-specific antibody. Methods: Anti-CEACAM monoclonal antibody (mAb 6G5j) was conjugated to near-infrared dye LI-COR IR700DX (6G5j-IR700DW). A fresh sample of colon-cancer liver metastasis was initially subcutaneously implanted in nude mice. Grown tumors were harvested and cut into small fragments and implanted into the left lobe of the liver of nude mice under anesthesia. After three weeks, mice were administered 50 μg of 6G5j-IR700DW via tail vein injection 48 hours before surgery. 10 μg of ICG was intravenously injected after isolation and ligation of the left Glissonean pedicle during surgery resulting in labeling of the right lobe of the liver. For intra-vital imaging and the FGS procedure, the Pearl Trilogy Small Animal Fluorescence Imaging system (LI-COR) and FLARE Imaging System (Curadel) were used. Results: On the 700 nm channel, the tumor had a clear fluorescence signal of 6G5j-IR700DW. On the 800 nm channel, the right lobe in the liver had the clear fluorescence signal of ICG and the left lobe had no fluorescence signal due to ligation of the left Glissonean pedicle. Overlay mode on the FLARE Imaging System (bright light, 700 nm and 800 nm) showed clear differentiation between tumor and segmental boundary on the same image. On the overlay mode, FGS for partial liver resection was performed using the 700 nm fluorescence signal of 6G5j-IR700DW. FGS for anatomic left hepatectomy as non-colored area was also well performed using the 800 nm fluorescence signal of ICG. Conclusions: This preliminary result shows that the combination of ICG (800 nm) and anti-CEACAM antibody conjugated to 700 nm dye has potential for visualization of transection lines and detection of colon-cancer liver metastases for FGS in a single image. (1) Yano S, et al. Cancer Gene Ther. 2015;22:344-50. Citation Format: Hiroto Nishino, Hannah M. Hollandsworth, Siamak Amirfakhri, Filemoni Filemoni, Jun Yamamoto, Yoshihiko Tashiro, Robert M. Hoffman, Michael Bouvet. Color-coded fluorescence-guided surgery in a patient-derived orthotopic xenograft (PDOX) nude mouse model of colon-cancer liver metastasis with ICG and a fluorescent antibody [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2786.

Background The Helicobacter outer protein (HopQ) is a novel surface-located Helicobacter pylori adhesion protein that binds to various human carcinoembryonic antigen-related cell adhesion molecules (CEACAM1, 3, 5, 6). Our aim was to validate fluorescence targeting of colorectal tumors in patient derived orthotopic xenograft models with fluorescently labeled HopQ. Materials/Methods CEACAM binding protein HopQ was conjugated to near-infrared dye IR800CW (LI-COR). Western blotting was performed with various colon cancer cell lysates to determine CEACAM expression. Nude mice (n=4) received orthotopic implantation of patient-derived primary colon tumors and patient-derived colon cancer metastases. Mice were administered either 25 ug or 50 ug of HopQ-IR800CW via tail vein injection and imaged 48 hours later. Intravital images were analyzed using the Image Studio Software Small Animal Imaging Analysis to determine tumor-to-background ratio (TBR). Results Western blotting demonstrated varied expression of CEACAM binding in all 15 patient derived colon cancer lysates. Dose response intravital imaging demonstrated TBR of 1.41 when administered 25 ug of HopQ-IR800CW and TBR 3.78 when administered 50 ug of HopQ-IR800CW. Conclusion CEACAM binding protein HopQ shows promising use for fluorescence imaging of patient derived orthotopic colon cancer. HopQ may be useful for tumor detection for pre-surgical diagnosis and fluorescence-guided surgery. Citation Format: Hannah M. Hollandsworth, Siamak Amirfakhri, Filemoni Filemoni, Verena Schmitt, Gunther Wennemuth, Alexej Schmidt, Robert M. Hoffman, Bernhard B. Singer, Michael Bouvet. Anti-CEACAM binding protein for fluorescence visualization of primary colon cancer in patient-derived orthotopic xenograph mouse models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1658.

Pancreatic cancer is a highly lethal disease in part due to incomplete tumor resection. Targeting by tumor-specific antibodies conjugated with a fluorescent label can result in selective labeling of cancer in vivo for surgical navigation. In the present study, we describe a patient-derived orthotopic xenograft model of pancreatic cancer that recapitulated the disease on a gross and microscopic level, along with physiologic clinical manifestations. We additionally show that the use of an anti-CEA antibody conjugated to the near-infrared (NIR) fluorescent dye, IRDye800CW, can selectively highlight the pancreatic cancer and its metastases in this model with a tumor-to-background ratio of 3.5 (SEM 0.9). The present results demonstrate the clinical potential of this labeling technique for fluorescence-guided surgery of pancreatic cancer.

Introduction: Photoimmunotherapy (PIT) uses tumor specific monoclonal antibodies conjugated to photosensitizer phthalocyanine dye IR700 to produce cytotoxicity. To date, there is no published data on the use of PIT in colorectal cancers, which could be useful as an adjunct to surgery to prevent local recurrence, or as a primary therapy. Methods: Humanized anti-CEA antibody (m5A) was conjugated to IR700CW dye (LI-COR). In vitro PIT was performed using a human colon cancer cell line LS174T. Cancer cells (2x103) were seeded on a 96-well plate and incubated for 24 hours. m5A-IR700CW containing media was added to the cells. Cells received either 2 minutes of PIT or no treatment. PIT was administered at 4 J/cm2, 8 J/cm2 or 16 J/cm2. Cell viability was measured on a microplate reader with CellTiter 96 Aqueous One Solution (Promega, Madison, WI). Subcutaneous implantation of LS174T was performed on the bilateral flanks of four male nude mice. Tail vein injection of 25 micrograms of m5A-IR700CW reconstituted in 100 microliters PBS was performed for each mouse. Animal imaging was performed with a LI-COR Pearl Trilogy imaging system. Right flank tumors of three mice were treated with a 690 nm laser at 150 mW/cm2 for 30 minutes, delivering a total of 270 J/cm2. A non-treated mouse served as the control. Images were obtained prior to PIT, immediately after PIT and 10 minutes, 20 minutes, 30 minutes and 24 hours after treatment. Maximum fluorescence intensity was measured at each time point. Tumor size was measured prior to treatment, 24 hours, 48 hours, 5 days and 10 days after treatment. Results: In vitro PIT was effective to kill the cancer cells in a dose response manner. In vivo PIT decreased tumor fluorescence intensity in a time-dependent manner. PIT arrested tumor growth in all mice, compared to the control, which demonstrated continuous tumor growth. Conclusion: PIT affects cancer cell death in vitro and arrests tumor growth in colon cancer models. Citation Format: Hannah Hollandsworth, Siamak Amirfakhri, Filemoni Filemoni, Sahar Razemjooie, Paul Yazaki, Robert Hoffman, Michael Bouvet. Development of photoimmunotherapy for colon cancer in nude mice models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3733.

// Hannah M. Hollandsworth 1 , 2 , 5 , Siamak Amirfakhri 1 , 2 , 5 , Filemoni Filemoni 1 , 2 , 5 , Verena Schmitt 3 , Gunther Wennemuth 3 , Alexej Schmidt 6 , Robert M. Hoffman 1 , 2 , 4 , 5 , Bernhard B. Singer 3 , * and Michael Bouvet 1 , 2 , 5 , * 1 Department of Surgery, University of California, La Jolla, CA, USA 2 Moores Cancer Center, University of California San Diego, La Jolla, CA, USA 3 Institute of Anatomy, University Hospital, University Duisburg-Essen, Essen, Germany 4 AntiCancer, Inc., San Diego, CA, USA 5 VA San Diego Healthcare System, San Diego, CA, USA 6 Department of Medical Biosciences, Pathology, Umea University, Umea, Sweden * These authors contributed equally to this work Correspondence to: Michael Bouvet, email: mbouvet@ucsd.edu Keywords: carcinoembryonic antigen; CEACAM; colon cancer; fluorescence; near-infrared Received: September 25, 2019 Accepted: January 04, 2020 Published: January 28, 2020 ABSTRACT Background: Monoclonal antibody (mAb) 6G5j is a novel anti-CEACAM monoclonal antibody. Our aim was to investigate mAb 6G5j binding characteristics and to validate fluorescence targeting of colorectal tumors and metastases in patient derived orthotopic xenograft (PDOX) models with fluorescently labeled 6G5j. Materials/Methods: The MAb 6G5j binding profile was analyzed with ELISA, Western blot and immunohistochemistry. MAb 6G5j was conjugated to near-infrared dye IR800CW (LI-COR). Western blotting was performed with various colon cancer cell lysates to determine CEACAM expression. Nude mice received orthotopic implantation of patient-derived primary colon cancer and patient-derived colon cancer metastases. Mice were administered varying doses of 6G5j-IR800CW via tail vein injection and imaged 24 and 48 hours later. Results: MAb 6G5j bound to human CEACAM1, 3, 5, 6 and 8. Western blotting demonstrated varied expression of CEACAMs in 15 of 16 colon cancer lysates. Dose and time-response imaging demonstrated optimal imaging 48 hours after administration of 50 μg 6G5j-IR800CW (Tumor-to-liver ratio (TLR) 3.17, SEM ± 0.45). Primary cancers and multiple metastases were fluorescently visualized. Conclusions: Anti-CEACAM antibody 6G5j binds multiple CEACAMs which may lead to improved detection of tumor margins for tumors and metastases that have variable expression of CEA and other CEACAMs. 6G5j mAb may be useful for colon cancer detection for pre-surgical diagnosis and fluorescence-guided surgery.