Novel Syngeneic Cell Lines for Studying High-Risk BRAFV600E-Driven Colorectal Cancer In Vivo

Combining anti-EGFR antibodies with BRAF/MEK/ERK pathway blockade opened new treatment options for BRAFV600E-driven colorectal cancer. Nevertheless, responses of these poor prognostic cancers are short-lived and heterogeneous, thereby highlighting the unmet need for novel concepts integrating targeted therapy and immuno-oncology. To this end, an immunocompetent mouse model with reliable disease onset and responding to clinically relevant drugs is essential but unavailable. In this study, we generated three cell lines from peritoneal metastases originating from orthotopically transplanted organoids carrying BrafV600E, Trp53R172H, and Apc mutations and characterized their ground state and responses to targeted therapy compounds in detail by RNA sequencing, whole-exome sequencing, and various functional assays. Interestingly, these so-called NaJa lines displayed distinct differentiation states and responses to the clinically relevant RAF inhibitors (RAFi) encorafenib and exarafenib, thereby resembling the clinical heterogeneity of BRAFV600E-driven colorectal cancer. RAFi resistance was overcome by the EGFR family inhibitor afatinib. RAFis also influenced the expression of the antigen presentation machinery, cytokines, and other immunomodulatory factors. Upon retransplantation into syngeneic mice, all NaJa lines established aggressive tumors with distinct tumor microenvironments matching their differentiation states. Thus, the NaJa lines provide a unique tool to study tumor heterogeneity, drug resistance, and the interplay between tumor, stroma, and immune cells in BRAFV600E-driven colorectal cancer.

We present three newly isolated syngeneic BRAFV600E-driven colorectal cancer cell lines with distinct in vitro and in vivo phenotypes. They differ in their response to drug treatments, resemble patient tumor heterogeneity, and enable efficient tumor growth in immunocompetent mice for tumor-immune microenvironment studies.