Fig. 3: Activated p53 represses the HSF1-mediated heat-shock response in HSP90-based therapies in murine and human colorectal tumor-derived organoids.

A Cell viability matrix of murine tumor organoids treated with Ganetespib plus RG-7388 combination for 48 h. Organoids were generated from AOM/DSS-treated C57BL6/J mice. Four independent biological replicates with 3 in-plate technical replicates each. B mRNA expression levels of HSF1 target genes in murine tumor organoids generated from AOM/DSS mice treated with 10 nM (left) and 15 nM (right) Ganetespib in combination with 0.5 µM RG-7388 for 24 h. qRT-PCRs for the indicated mRNAs normalized to RPLP0 mRNA. Mean ± SEM from min. 2 independent biological replicates. C Quantification of dead tumor-derived organoids treated with indicated drug combination for 48 h. Organoids were generated from AOM/DSS-treated C57BL6/J mice. Mean ± SEM from 2 independent biological replicates. D Quantification of normal murine small intestinal mucosa-derived organoids treated with the indicated combinations for 48 h. Mean ± SEM from 3 independent biological replicates. E Top, Cell viability matrices of p53-proficient (wild-type p53, wtp53) patient-derived organoids (PDOs) treated with Ganetespib, RG-7388, or in combination for 48 or 72 h. A PDO case represents one PDO culture (PDO #). For each PDO culture, four replicates (different passages) with 3 in-plate technical replicates each were measured. Organoids were cultivated and treated as large organoids. Bottom, Representative brightfield images of large CRC tumor organoids. Scale bars, 200 μm. F Cell viability matrix of PDO #2 culture, cultivated and treated as small organoids with Ganetespib, RG-7388, or in combination for 3 or 5 days. Three replicates (different passages) with 3 in-plate technical replicates each were measured. Bottom, Representative brightfield images of small PDOs #2 culture. Scale bars, 200 μm. G Quantification of dead organoids via their morphology. Normal human colon mucosa-derived organoids were treated with the indicated combinations for 48 h. Mean ± SEM from 3 independent biological replicates. A, E, F Color scheme represents changes in cell viability. Numbers in the matrix are HSA synergy scores. Synergy scores: <−10 antagonistic; −10 to 10 additive; >10 synergistic. Ganet: Ganetespib, RG: RG-7388. B, C, D, G Ganet: Ganetespib, RG: RG-7388. Student’s t-test (B) or one-way ANOVA (C, D, G), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; when ns not significant or p-value indicated.