Fig 2: Combination effect of HSP90 peptides/STING agonist/anti-CTLA-4 Ab induces immune environment and epitope spreading by effective antitumor immunity. (A) Experimental design (n=4 per groups) and schedule of the combination therapy of HSP90 peptide in the mouse model. (B) Tumor growth is shown for individual mice in control (PBS+hamster IgG+DMSO), HSP90 peptides, HSP90 peptides+anti-CTLA-4 Ab, STING agonist+anti-CTLA-4 Ab and HSP90 peptides+STING agonist+anti-CTLA-4 Ab groups. (C) HSP90-specific T-cell responses and (D) potential epitope-spreading responses of HER2, c-MET and HIF-1α peptide were evaluated by enzyme-linked immunospot using splenocytes from experimental groups. Bars represent CSPW and error bars represent SD. N.S, not significant. *P<0.05, **P<0.01, ***P<0.001 in Tukey’s multiple comparison test of one-way ANOVA and in Bonferroni post-tests of two-way ANOVA. Ab, antibody; ANOVA, analysis of variance; CTLA-4, cytotoxic T lymphocyte-associated antigen-4; HER2, human epidermal growth factor receptor 2; HSP, heat shock protein; HSP90, heat shock protein 90; MMC, mouse mammary carcinoma; STING, stimulator of interferon genes; TCR, T-cell receptor; TT, tetanus toxoid.

Fig 3: Increased stem marker expression in tumor cells with elevated surface-bound Hsp90A. Representative flow cytometry scatter plots for surface-bound Hsp90-alpha in ARCaPE and DU145. Samples treated with secondary Rabbit IgG-PE were used as a negative control (denoted PE Control). Surface-bound Hsp90-PE was defined as the area of events with higher PE staining not found in the negative control population, which ranged from 2-5% for each cell line. B. RNA was harvested from each of these respective cell populations and qRT-PCR was performed for each of the indicated stem-like gene targets. All statistics were performed using the Student's t-test for the treated versus the untreated control. * = p<0.05, ** = p<0.01, *** = p<0.001.

Fig 4: Surface-bound Hsp90 co-segregates with a subset of PSA(lo)-expressing cellsA. Androgen receptor (AR) responsive LNCaP and Myc-CaP prostate cancer cells were transiently transduced with lentiviral particles encoding PSA-GFP and GFP expression was assessed by flow cytometry. Approximately 70-80% of LNCaP are GFP+, while approximately 40% of MycCaP demonstrate similar positivity. PSA-Con refers to nontransduced control cells, while PSA-GFP refers to non-sorted bulk cells. B. Quantitative PCR analysis was performed on the indicated cell populations to determine the relative DNA expression of GFP, indicative of transduction efficiency. The populations are identified as follows: untransduced (PSA-con), transduced unsorted (PSA-GFP), and flow sorted PSA-GFP positive and negative (PSA-GFP(Hi)) and PSA-GFP(Lo)). C. Representative flow cytometry scatter plots demonstrating the relation between surface-bound Hsp90-alpha and PSA-GFP expression in LNCaP and MycCaP cells. D. Tabular depiction of replicate flow cytometry analyses for surface-bound Hsp90 in LNCaP and Myc-CaP cells. E. Model depicting the role of eHsp90 in supporting stem-like cellular heterogeneity. Collectively, our data support a model whereby secreted Hsp90 increases stem-like properties in prostate tumor cells. This was demonstrated by increased prostasphere growth, and expansion of both the side population (indicative of dye efflux and drug transporter activity) and ALDH activity. As indicated, the effectors EZH2 and Snail appear to differently impact seveal of these metrics. In tandem, tumor cells with elevated surface Hsp90 appear to correlate with lower levels of PSA-regulated GFP, indicating a potential inverse correlation between surface Hsp90 and androgen receptor (AR) regulation.

Fig 5: Human T-cell responses specific to HSP90 peptides can be recognized. (A) HSP90 peptides with the highest binding affinities across multiple MHC class II alleles. Colors represent final scores from five algorithms for each peptide sequences from dark red to light blue in the order of rank scores. Color strata are as follows: dark red: >9000, red: 8000–9000, orange: 7000–8000, light orange: 6000–7000, gold: 5000–6000, yellow: 4000–5000, light yellow: 3000–4000, light green: 2000–3000, and light blue: 1000–2000. (B, C) HSP90 peptides were profiled antigen-specific responses in human PBMCs from 10 healthy donors using IFN and IL-10 enzyme-linked immunospot assay. Per cent responding donors to HSP90 epitopes (B) and CSPW (C). White columns, IFN-γ; black columns, IL-10; horizontal bars indicate mean CSPW. CSPW, corrected spots per well; HSP90, heat shock protein 90; IFN, interferon; IFN-γ, interferon gamma; IL, interleukin; PBMC, peripheral blood mononuclear cell.