Fig. 4: Dual HSF1-HSP90 pathway inhibition reduces colonic tumor progression in a mouse model by remodeling the immune system.

A Treatment scheme for the p53-proficient AOM/DSS mouse model. After tumor visualization by repeat colonoscopy and tumor scoring for at least one S2-sized tumor and three S1-sized tumors per mouse, mice were treated with single drugs or combination treatment for 19 days. 4 h after the final dose, mice were dissected and analyzed. B, C Representative images of entire resected colons (B) and H&E-stained colon sections (C) from single or combination-treated C57BL6/J mice at endpoint as described in (A). Mice received 50 mg/kg RG-7388 orally 5× per week, or 50 mg/kg Ganetespib intravenously 2× per week, or both. B scale bars, 5 mm. C ×20 magnification, scale bars, 1 cm. D Tumor surface area from AOM/DSS mice that had received single or combination treatment for 19 days as in (A). Cross-sections of Swiss roles were H&E stained (Supplementary Fig. 4A). Tumor areas of all tumors per H&E-stained Swiss role were measured using ImageJ. Bar, mean. vehicle n = 35 tumors from 5 mice; RG-7388 and Ganetespib n = 33 tumors from 6 mice each; combination n = 45 tumors from 9 mice. E, F GSEA hallmark analysis of RNAseq data from HCT116 cells treated with drug combination or single drugs for 24 h. Enriched pathways are plotted according to NES. Gray bars: Enrichment in (F) RG-7388-only or (G) Ganetespib-only versus DMSO controls. Red bars: further enrichment with combination treatment relative to single treatment. NES: normalized enrichment score. G–I Multiplex immunohistochemistry of p53-proficient tumors from AOM/DSS mice receiving drug treatments for 19 days as in (A). G Representative images of indicated groups. Ly6G staining represents neutrophils, and panCytokeratin represents tumor-epithelial cells. DAPI counter staining. Scale bars, 50 µm. H Quantification of Ly6G + CD11b + F4/80− neutrophils using the “Vectra Polaris” platform and the inForm Advanced Image Analysis software. n = 5 mice per group. All tumors from stained Swiss roles were analyzed for the indicated treatment groups. I Plot of the neutrophil-lymphocyte-ratio (NLR) as a marker of an inflammatory response. A low NLR indicates an inflammatory response. J, K Multiplex immunohistochemistry of tumor-bearing AOM/DSS mice who received a short drug treatment of 6 days. Quantification of Ly6G + CD11b + F4/80− neutrophils of the indicated treatment groups as in (H). DMSO, Ganetespib, and RG-7388 mice n = 3 each, combination-treated mice n = 4. C, H–K Mean ± SEM. Student’s t-test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; ns not significant. Ganet: Ganetespib, RG: RG-7388.