Fig. 2 (abstract P385).

Induction of CXCL5 in Melanomas Undergoing Treatment with Anti-PD-1 Ab Therapy is Dependent Upon a Paracrine HSP-TLR-Wnt5a Signaling Pathway. A. Qrt-PCR analysis shows that Wnt5a stimulates the expression of several CXCR2 ligands in a BRAF(V600E)-PTEN-/- melanoma cell line. B. Western blot analysis of nuclear YAP1 levels in response to Wnt5a stimulation in a BRAF(V600E)-PTEN-/- melanoma cell line. C. Immunohistochemistry studies show that CXCL5 expression is significantly suppressed in Wnt5a-silenced BRAF(V600E)-PTEN-/- melanomas. D. SILAC-AHA metabolic labeling mass spectrometry-based differential secretome analysis and RNAseq differential gene expression analysis of BRAF(V600E)-PTEN-/- melanomas following treatment with anti-PD-1 ab vs IgG isotype control ab. Several cellular stress pathways identified as being upregulated in those melanomas undergoing anti-PD-1 ab therapy. Several heat shock proteins including HSP70 noted to be induced in anti-PD-1 ab-treated melanomas. E. BRAF(V600E)-PTEN-/- melanomas express elevated levels of TLR2 and TLR4 based on qrt-PCR. Western blot demonstrates that HSP70 and agonistic anti-TLR2 ab stimulates Wnt5a expression in the BRAF(V600E)-PTEN-/- melanoma cell line. F. top, CD8+ T cell levels and Gr-MDSC levels correlate significantly in BRAF(V600E)-PTEN-/- melanomas only when treated with anti-PD-1 ab therapy (p = 0.044). bottom, Ablation of CD8+ T cells diminishes the recruitment of Gr-MDSCs in response to anti-PD-1 ab therapy. G. top, Whole tissue Western blot analysis shows that anti-PD-1 ab therapy induces the upregulation of Wnt5a and YAP1 in BRAF(V600E)-PTEN-/- melanomas. bottom, Dot blot analysis shows that CXCL5 expression is upregulated in the serum of transgenic BRAF(V600E)-PTEN-/- mice undergoing treatment with anti-PD-1 ab therapy (red, blot density analysis). Representative blots shown. All data is mean +/- SEM. *P<0.05