Figure 5.

Inhibition of PD1 modulated the expression of pleiotropic cytokines and cell cycle-related genes in the TME. Single-cell suspensions were obtained from tumors harvested at day 3 or 7 after treatment with IgG isotype control or α-PD1. RT-qPCR showing changes in (A) in cytokine expression in the CD45⁺ tumor-infiltrating cells or (B) in VEGF-A and G1/S cyclins in the CD45⁻ tumor cell enriched fraction. Results were normalized to RPL13a housekeeping gene and expressed as a fold difference relative to the IgG isotype control expression levels. Each bar represents the combined result of three independent experiments, containing pooled tumor samples from 2–3 mice in each experiment. Prior to normalizing the data, results were analyzed using the comparative ΔΔCT method and significance determined by using ANOVA followed with Bonferroni correction. (C) VEGF-A levels were determined by ELISA assay using fresh tumor tissue lysates obtained from mice treated with α-PD1 (n = 6) or IgG isotype control (n = 5) for 7 d. Each symbol represents an individual mouse. Comparison of VEGF-A protein levels between α-PD1 versus IgG isotype control treated mice was calculated using non-parametric Mann–Whitney test. N.S. = not significant. *p < 0.05, ***p < 0.001.