Integrated chromatin and transcriptomic profiling of patient-derived colon cancer organoids identifies personalized drug targets to overcome oxaliplatin resistance

Abstract

Colorectal cancer is a leading cause of cancer deaths. Most colorectal cancer patients eventually develop chemoresistance to the current standard-of-care therapies. Here, we used patient-derived colorectal cancer organoids to demonstrate that resistant tumor cells undergo significant chromatin changes in response to oxaliplatin treatment. Integrated transcriptomic and chromatin accessibility analyses using ATAC-Seq and RNA-Seq identified a group of genes associated with significantly increased chromatin accessibility and upregulated gene expression. CRISPR/Cas9 silencing of fibroblast growth factor receptor 1 (FGFR1) and oxytocin receptor (OXTR) helped overcome oxaliplatin resistance. Similarly, treatment with oxaliplatin in combination with an FGFR1 inhibitor (PD166866) or an antagonist of OXTR (L-368,899) suppressed chemoresistant organoids. However, oxaliplatin treatment did not activate either FGFR1 or OXTR expression in another resistant organoid, suggesting that chromatin accessibility changes are patient-specific. The use of patient-derived cancer organoids in combination with transcriptomic and chromatin profiling may lead to precision treatments to overcome chemoresistance in colorectal cancer.

Keywords: Chromatin accessibility; Drug screening; Patient-derived organoids; Personalized medicine; Target discovery.

Figure 1

Chemo-sensitivity of CRC 240, CRC344, and CRC159 PDOs. (A) Bright-field images of colorectal cancer organoids. From left to right: CRC240, CRC159, and CRC344. Scale bar = 400 μm. (B) Drug sensitivities to oxaliplatin, 5-FU, and SN-38 were assessed in CRC240, CRC159, and CRC344 organoids. Organoids were exposed to chemotherapy for 6 days, and cell viability was assessed by CellTiter-Glo 3D cell viability assay. The IC50 values were calculated by a nonlinear regression model in GraphPad Prism. Error bars represents the standard error of the mean.

Figure 2

Transcriptomic and chromatin accessibility profiling of oxaliplatin-treated organoids. (A) Integration of ATAC-Seq and RNA-Seq. The differential analyses of ATAC-Seq and RNA-Seq were performed by using DESeq2 and DiffBind respectively. The differential genes and peaks were filtered by P-values (p-value < 0.05). Red color represents increase changes (logFC >1) for expression (square) or chromatin accessibility (triangle). Blue color represents decreased changes (logFC < −1) for expression (square) or chromatin accessibility (triangle). The filled triangles represent both ATAC-Seq peaks and RNA-Seq expression significantly altered (P < 0.05). (B) Top ranked genes that display both increased chromatin accessibility nearby and increased gene expression in oxaliplatin-resistant CRC240.

Figure 3

Confirmation of drug-associated genes FGFR1 and OXTR in CRC organoids. (A) Gene set enrichment analysis of top genes identified by integrated analysis in Fig. 2B. Cancer modules curated by the Broad institute were applied for the enrichment analysis. Blue color in module-gene matrix indicates the significantly enriched cancer modules and the associate gene hits from the top gene list. (B) Bar diagram of drug-gene interactions. The known drugs targeting the identified cancer associated genes are classified into groups annotated by DGIdb based on the targeting mechanisms. Among these drugs, 33 drugs are inhibitors targeting FGFR1, 18 drugs are antagonists targeting OXTR, and one antagonist targets RARB. (C) Left: RT-qPCR showed mRNA expression of FGFR1 in CRC240 control (DMSO) organoids compared to oxaliplatin treatment. Right: RT-qPCR showed mRNA expression of OXTR in CRC240 control (DMSO) organoids compared to oxaliplatin treatment. Organoids were exposed to the IC50 concentration of oxaliplatin, and RNA was isolated after incubation. Expression levels are given relative to the housekeeping gene GAPDH. Data were mean ± SEM (n = 3) and the statistical significance was assessed by unpaired two-tailed student's t-test. *P < 0.05, **P < 0.01. (D) Protein expression was assessed by Western blot using antibodies to FGFR1, phospho-FGFR1, FGFR2, FGFR3, FGFR4, OXTR, and beta-tubulin in CRC240, CRC159, and CRC344 organoids with DMSO or oxaliplatin treatment. Immunoblots between 90KD to 120KD are different isoforms and glycosylated fibroblast growth factor receptors. (E) RT-qPCR showed mRNA expression of FGFR1, FGFR2, FGFR3, and FGFR4 in CRC240 control (DMSO) organoids compared to oxaliplatin treatment. Organoids were exposed to the IC50 concentration of oxaliplatin, and RNA was isolated after incubation. Expression levels are given relative to the housekeeping gene GAPDH. Data were mean ± SEM (n = 3) and the statistical significance was assessed by unpaired two-tailed student's t-test. *P < 0.05; **P < 0.01; ***P < 0.01; n.s., not significant.

Figure 4

Inhibition of fibroblast growth factor 1 (FGFR1) and oxytocin receptor (OXTR) reduce tumor growth. (A) RT-qPCR measurement validated the efficiency of FGFR1 and OXTR knockout by CRISPR-Cas9 editing in CRC240 organoids. Left: RT-qPCR measurement of FGFR1 mRNA levels in either wild-type or FGFR1 knockout CRC240 organoids. Right: RT-qPCR measurement of OXTR mRNA levels in either wild-type or OXTR knockout CRC240 organoids. Expression was normalized to GAPDH. Data represent mean ± SEM (n = 3), and the statistical significance was assessed by unpaired two-tailed student's t-test. **P < 0.01, ***P < 0.001. (B) Western blot analysis of knockout efficiency of either FGFR1 or OXTR in CRC240 organoids. Beta-tubulin was used as an internal control. (C) Cell viability of either FGFR1 (top) or OXTR (bottom) knockout organoids after oxaliplatin treatment. FGFR1 knockout or OXTR knockout CRC240 organoids were treated with IC50 of oxaliplatin, and wild-type CRC240 organoids were treated with same IC50 for comparison. Cell viability of wild-type CRC240 organoids without oxaliplatin treatment were measured as control. Data represent mean ± SEM (n = 3). One-way ANOVA with Tukey's post hoc test was performed. ***P < 0.001, ****P < 0.0001. (D) Dose-response curves of CRC240 organoids treated with monotherapy or a combination therapy. Top: Combination treatment with oxaliplatin (OXA) and PD166866 (PD). Bottom: Combination treatment with oxaliplatin (OXA) and L-368,899 (L368). Organoids were treated with a series of six different drug doses of oxaliplatin (OXA), PD166866 (PD), and L-368,899 (L368) or a combination of both agents for 6 days. Then, cell viability was measured via CellTiter-Glo 3D cell viability assay (Promega). Purple (PD or L368) line, orange (OXA) line, and black (PD/OXA or L368/OXA combination therapy) line represent the dose-response curves. (E) Combination index of combination treatments. Top: Combination treatment of oxaliplatin (OXA) and PD166866 (PD). Bottom: Combination treatment with oxaliplatin (OXA) and L-368,899 (L368). Left: 5 × 5 dose matrix of combination index. Right: 5 × 5 dose matrix of dose effect. CRC240 organoids were treated with increasing concentrations of oxaliplatin, and PD166866, or L-368,899 or co for 6 days in conditional medium. Combination index (CI) was calculated using CompuSyn software. Additive area was selected by CI between 0.9 and 1.1. CI ≥ 1.1 indicates antagonism; <0.9 indicates synergism. Red and green color indicate synergism and antagonism in CI matrix. Dose effect represents fraction of cells killed by drug treatment. Dark red indicates 100% killing, while light blue indicates 0% killing.