Abstract 2926: CT26.WT-luc orthotopic syngeneic tumor model of colon cancer: development and immunotherapy response

Colorectal cancer (CRC) is a major contributor to the whole of cancer as a disease, representing 10% of all cases of cancer. Recent clinical studies and FDA approvals demonstrate strong possibilities for the future of immunotherapy in the treatment of CRC. Highly translational preclinical models of the disease are required to assess the effectiveness of novel immuno-oncology agents. The CT26.WT murine colon carcinoma model is a widely used syngeneic tumor model to evaluate the preclinical efficacy of immuno-oncology agents. Subcutaneous tumor models are limited in translatability compared to the orthotopic implant site including differential stromal interactions, immune cell infiltrate, and response to immunotherapy. We present the development of a robust murine orthotopic colon tumor model (CT26.WT-luc) assessed by bioluminescence imaging (BLI) and assessed for response to common checkpoint targets. All animal work was performed in an AAALAC accredited facility, in alignment with applicable animal welfare regulations and with predetermined humane euthanasia criteria on all studies. Subcutaneous CT26.WT-luc tumors were dissected into fragments and grafted onto the cecum of naive Balb/c mice. Mice were subjected to BLI twice weekly for four weeks for longitudinal monitoring of tumor growth. The tumor engraftment rate was 95% based on BLI and necropsy, no spontaneous regressions were observed. The BLI derived tumor volume doubling time was 1.5 days and the median time on study was 21 days. Common clinical observations associated with disease progression were abdominal distension due to tumor growth and bodyweight gain. Necropsy revealed large primary masses attached to the cecum with large vascular involvement, and small nodules on the liver and abdominal wall. To assess the model response to immunotherapy, Balb/c mice bearing orthotopic CT26.WT-luc tumors were treated by intraperitoneal administration of 10mg/kg isotype control, anti-PD-L1, anti-CTLA-4 or anti-PD-1 twice weekly for two weeks (Bio X Cell). Administration of anti-PD-L1 resulted in one complete regression. Anti-CTLA-4 treatment resulted in a day 23 ΔT/ΔC of 0.1% and five partial regressions with no visibly detectable disease at necropsy. Administration of anti-PD-1 did not result in regressions or tumor free survivors. Administration of either anti-PD-L1 or anti-CTLA-4 provided an increase of life span over isotype control of 13 and 24 days, respectively. Terminal necropsy revealed no evidence of tumors or other lesions in 1 out of 8 animals administered anti-PD-L1, and 5 out of 8 animals administered anti-CTLA-4. This model represents a powerful tool with translational potential for assessing the efficacy of novel immune modulating agents with the possibility of combination therapy. Assessment of tumor response, cell and molecular differences between subcutaneous and orthotopic CT26.WT-luc tumors is ongoing. Citation Format: Patrick Allison, Derrik Germain, Meredith Baugher, Maryland Franklin. CT26.WT-luc orthotopic syngeneic tumor model of colon cancer: development and immunotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2926.