Gene expression profiles of melanoma tumor samples from patients treated with checkpoint inhibitors (mouse)

Checkpoint inhibitors have improved outcomes for patients with metastatic melanoma, but primary or acquired resistance often occurs. We used the mouse melanoma cell line B16, originally derived from C57BL6 mice, to model changes in tumor cell gene expression that occur with acquired resistance to checkpoint inhibitors. B16 cells used were engineered to express the fluorescent marker tdTomato, to assist monitoring and cell sorting. Previously-untreated B16 cells (F0) were inoculated into C57BL6 mice, followed by treatment with triple-checkpoint inhibitor therapy (3I) with combined antibodies to CTLA4, PD-1, and PD-L1. Tumors that emerged despite immune selection pressure were harvested, grown again in vitro as a cell line, and then re-inoculated into a new generation of mice. This was repeated for 4 generations (F4) until inoculations were 100% successful at generating immune-resistant tumors. F0 or F4 B16 cells, obtained from in vitro culture, were inoculated into C57BL6 mice and tumors allowed to form. Mice received 3 doses of 3I. Tumors were harvested when they reached 100-400 mm3 in size, pooled from 3 mice per replicate, disaggregated into single-cell suspensions, and sorted into tumor (tdTomato-positive) and microenvironment (tdTomato-negative) fractions.