Subcutaneous inoculation position affects the immune environment in CT26 carcinomas

Abstract

Comprehensive knowledge on the murine CT26 colon carcinoma line is a classic model used in the pharmacodynamic experiments involving IDO-1 inhibitors, immune-related checkpoint antibodies and immune related mechanisms. In this study, we determined the impact of different subcutaneous inoculation locations on tumor growth and immune factor expression. CT26 cells were treated with the IDO-1 inhibitor, INCB024360, following INF-γ stimulation and analyzed for kynurenine concentration. Female Balb/c mice were inoculated with CT26 cells in either or both the right upper flank or the right lower flank. Isolated tumors were evaluated for changes in tumor volume following treatment with anti-PD-1, anti-CTLA-4, or no treatment. Isolated tumors were also evaluated for changes in immune cell subpopulations and expression of key immune factors using FACS. Treatment of two CT26 cell lines with INCB024360 produced similar results. IC50 values were 222.5 and 276.0, and the peak inhibitory rates were 97.99% and 91.85% respectively. Analysis of tumor growth revealed that tumor volumes were larger (1925 mm3 vs. 767 mm3), and the anti-tumor effects of both anti-PD-1 and anti-CTLA-4 were different in mice inoculated in the right lower flank than in those inoculated in the upper flank. FACS analysis revealed that the CD8+T subpopulation in the right upper flank was higher than that in the lower flank (*P < 0.05). By contrast, the myeloid cell populations was lower in the right upper flank than it was in the right lower flank (*P < 0.05). The INF-γ populations in CD8+T (*P < 0.05) and regulatory T (Treg) cell subpopulation (*P < 0.05) were also more abundant in the right upper flank than in the right lower flank.In contrast to the uniform of the results from the in vitro experiment, the anti-CTLA-4 and anti-PD-1 antibodies had different efficacies depending on the location of the subcutaneous inoculation of CT26 in mice. The differences in the percentages of CD8+T, myeloid cells, INF-γ in CD8+T, and Treg subpopulations indicated that the tumor microenvironment was affected by inoculation position. Taken together, these results suggest that tumors isolated from same cell line with different inoculation positions are different enough to be considered different models.