A novel murine colorectal cancer cell line mirroring key clinically relevant mutations

Colorectal cancer (CRC) is a highly prevalent and lethal disease, with tumor heterogeneity contributing to varied responses to standard therapies. Preclinical models are essential for developing new strategies, yet existing models are limited in the specific tumor subtypes that they represent. To address this, we established a novel murine CRC cell line that represents tumors with mutations in Kras , Apc and Trp53 , the three most common mutations in human CRC. These novel cells, which we termed KAP, give rise to highly aggressive, invasive rectal tumors when implanted orthotopically. KAP cells were developed from colorectal organoids derived from mice harboring the oncogenic Kras G12D mutations as well as deletion of Trp53 . Organoids were further modified to knockout Apc using CRISPR-Cas9. KAP cells from organoids were grown on tissue culture treated flasks and used in a murine orthotopic rectal model using syngeneic B6 mice. KAP tumors were analyzed by immunohistochemistry to assess structural and cellular features of the tumor microenvironment. Additionally, to assess whether KAP tumors also mimic treatment response, we administered FOLFOX and radiation, standard therapies for CRC. We determined that KAP tumors recapitulate key features of human CRC, including poorly differentiated gland-like structures, dense areas of collagen deposition, immature vasculature, and a low abundance of intratumoral immune cells. This tumor microenvironment closely resembles immune-excluded CRC, a subtype known for its resistance to therapy. Importantly, following administration of standard therapy to mice bearing KAP rectal tumors, we observed similar outcomes to those seen in patients. The establishment of this novel cell line enhances the translational relevance of murine studies for CRC and provides a valuable tool for studying tumor biology and the therapeutic response in the context of low immune infiltration.