Figure 6.

PIIO-1 attenuates canonical TGFβ signaling pathway in tumor-infiltrating immune cells and rejuvenates antitumor immunity in hLRRC32^KI mice. (A) 1×10⁵ MB-49 cells were injected s.c. on the right flank of hLRRC32^KI male mice. Humanized PIIO-1 (200 µg/mouse, i.p.) were administered on days 18 and 20. Tumors were collected on day 21. TILs were isolated and stained for intracellular pSMAD2/3 with indicated cell linage markers, followed by flow cytometry analysis. (B) Quantification of panel A. (C–E) 1×10⁵ MB-49 cells were injected s.c. on the right flank of hLRRC32^KI male mice. Humanized PIIO-1 (200 µg/mouse, i.p.) was delivered on days 6 and 9. Tumors were collected on day 10. Single cell suspension and RNA isolation were prepared, and then subjected to bulk RNA sequencing. (C) Volcano plot of transcript expression. Differential gene expression was shown in red (up) or blue (down). Representative transcripts such as Ccl3, Ccl9, Cxcl14, Cxcl15, Il6 and Tnfrsf25 were indicated. (D) GSEA of differential expression genes between tumors treated with PBS and PIIO-1. (E) Comparison of TILs between PBS and PIIO-1 treated tumors based on deconvolution of bulk RNA sequencing data. Data were performed using two-tailed Student’s t-test, data in (B) represented mean±SEM. *p<0.05, **p<0.01.