Figure 4

Silencing PORCN expression in the B16 melanoma model enhances anti-tumor immunity and suppresses tumor progression in vivo. A. Silencing PORCN expression by B16/F10 cells suppresses paracrine β-catenin activation in a 293T-TCF/LEF1-luciferase reporter cell line. NTC, non-targeted control. KD, knock-down. Performed in triplicate. Representative of 3 independent experiments. P value based on two-tailed student's t test. B. PORCN silencing suppresses Wnt5a secretion from B16/F10 melanoma cells. Top, CM was harvested from HA-Wnt5a transfected B16-NTC cells treated with increasing concentrations of C59 (left) and HA-sRIII or HA-Wnt5a transfected B16-PORCN^KD cells (right) and immunoprecipitated with an anti-HA antibody. Bottom, Total cell lysates from HA-Wnt5a transfected B16-NTC were treated with increasing concentrations of C59 (left) and HA-sRIII or HA-Wnt5a transfected B16-PORCN^KD cells (right) were analyzed by anti-HA Western blot. HA-sRIII, HA-tagged soluble ectodomain of type III TGF-β receptor used as a positive control for protein secretion. Representative of two independent experiments. C. Wnt5a detection in the CM of B16-NTC and B16-PORCN^KD cells by ELISA. Cumulative data from 3 independent experiments. P value based on two-tailed student's t test. D. The B16-NTC and B16-PORCN^KD cell lines were implanted in C57BL/6 mice and tumor growth was monitored by orthogonal caliper measurements. 10 mice per group. Statistical significance based on Mann-Whitney U-test. *P < 0.05. E. Tumor-infiltrating CD3⁺CD8⁺ T lymphocyte populations are increased in PORCN silenced B16/F10 tumors based on flow cytometry. Performed in triplicate. Representative of 2 independent experiments. P value based on two-tailed student's t test. F. Tumor-infiltrating CD8⁺ T lymphocytes in PORCN silenced B16/F10 tumors exhibit diminished levels of surface PD-1 expression based on flow cytometry. Performed in triplicate. Representative of 2 independent experiments. P value based on two-tailed student's t test.