Disruption of the Gut Microbiota Confers Cisplatin Resistance in Epithelial Ovarian Cancer

Abstract

Epithelial ovarian cancer (EOC) is the leading cause of gynecologic cancer death. Despite initial responses to intervention, up to 80% of patient tumors recur and require additional treatment. Retrospective clinical analysis of patients with ovarian cancer indicates antibiotic use during chemotherapy treatment is associated with poor overall survival. Here, we assessed whether antibiotic (ABX) treatment would impact growth of EOC and sensitivity to cisplatin. Immunocompetent or immunocompromised mice were given untreated control or ABX-containing (metronidazole, ampicillin, vancomycin, and neomycin) water prior to intraperitoneal injection with EOC cells, and cisplatin therapy was administered biweekly until endpoint. Tumor-bearing ABX-treated mice exhibited accelerated tumor growth and resistance to cisplatin therapy compared with control treatment. ABX treatment led to reduced apoptosis, increased DNA damage repair, and enhanced angiogenesis in cisplatin-treated tumors, and tumors from ABX-treated mice contained a higher frequency of cisplatin-augmented cancer stem cells than control mice. Stool analysis indicated nonresistant gut microbial species were disrupted by ABX treatment. Cecal transplants of microbiota derived from control-treated mice was sufficient to ameliorate chemoresistance and prolong survival of ABX-treated mice, indicative of a gut-derived tumor suppressor. Metabolomics analyses identified circulating gut-derived metabolites that were altered by ABX treatment and restored by recolonization, providing candidate metabolites that mediate the cross-talk between the gut microbiome and ovarian cancer. Collectively, these findings indicate that an intact microbiome functions as a tumor suppressor in EOC, and perturbation of the gut microbiota with ABX treatment promotes tumor growth and suppresses cisplatin sensitivity.

Significance: Restoration of the gut microbiome, which is disrupted following antibiotic treatment, may help overcome platinum resistance in patients with epithelial ovarian cancer. See related commentary by Hawkins and Nephew, p. 4511.

Figure 1.. Tumor growth is increased and median survival is decreased in ABX treated Bl/6 mice, regardless of cisplatin therapy.

A. Schematic of study design highlighting treatment paradigm. B. and C. ID8 and ID8-VEGF tumor growth kinetics in H₂O and ABX C57Bl/6J mice treated without or with cisplatin. D. and E. ID8 and ID8-VEGF survival analysis kinetics in H₂O and ABX C57Bl/6J mice treated without or with cisplatin. Cisplatin therapy (dashed lines) increased H₂O treated murine survival (black, ID8 p<0.01), but did not alter ABX treated murine survival (red), in either cohort. (n=6 or otherwise denoted in graphs, 2-way ANOVA *p<0.05, **p<0.01, ****p<0.005 survival curve analysis: Gehan-Breslow-Wilcoxon test placebo vs cisplatin). F. Tumors from ID8 cell injected mice from H₂O + Cisplatin or ABX + Cisplatin treated mice were stained for 53BP1 to assess DNA damage and H&E. Representative sections shown. G. Tumors from H₂O +/− Cisplatin or ABX +/− Cisplatin mice were processed for TUNEL staining to detect apoptosis. H. Tumors from H₂O + Cisplatin or ABX + Cisplatin mice were harvested and qRT-PCR analysis performed for 53BP1, BRCA1, and CD31. (n = 4, * p<0.01). (Fig. 1A, Reprinted with permission, Cleveland Clinic Foundation ©2022. All Rights Reserved.)

Figure 2.. Tumor growth is increased and median survival is decreased in ABX treated NSG mice, regardless of cisplatin therapy.

Mice treated with antibiotics exhibited increased tumor growth regardless of cisplatin therapy following injection of both ID8 (A) and ID8 VEGF (C) cells in C57 BL/6 mice. Antibiotic treated ID8 (B) and ID8 VEGF (D) injected NSG mice (red) had significantly decreased median survival compared to H₂O treated mice (black). Further, cisplatin therapy (dashed lines) increased H₂O treated murine survival (black, ID8 p<0.01), but did not alter ABX treated murine survival (red), in either cohort. (n=6 or otherwise denoted in graphs, 2-way ANOVA, *p<0.05, **p<0.01, p<0.005 survival curve analysis: Gehan-Breslow-Wilcoxon test placebo vs cisplatin).

Figure 3.. ABX therapy results in microbial alterations over time in the gut microbiome of C57Bl/6 mice.

Specific strains of bacteria are altered in the ID8 A. and ID8 VEGF B. cohorts of C57 Bl/6 mice following antibiotic treatment over time. n= 8 mice per group (HC: H₂O Cisplatin, HP: H₂O Placebo, AC: ABX Cisplatin, AP: ABX Placebo) n=8 mice per group per time point.

Figure 4.. ABX therapy results in microbial alterations over time in the gut microbiome of NSG mice.

Specific strains of bacteria are altered in the ID8 A. and ID8 VEGF B. cohorts of NSG mice following antibiotic treatment over time. n= 8 mice per group (HC: H₂O Cisplatin, HP: H₂O Placebo, AC: ABX Cisplatin, AP: ABX Placebo) n= 8 mice per group per time point.

Figure 5.. RNAseq of NSG ID8 tumors from metastasis to the omentum at endpoint demonstrates differentially expressed genes between treatment groups.

A. Top 100 differentially expressed genes (DEGs) ordered by p-value in the ABX Placebo group over the H₂O Placebo group. B. Top significant GO terms enriched in the ABX Placebo group over H₂O Placebo group. C. Stem cell genes enriched in the ABX Placebo group over the H₂O Placebo group ordered by p-value. D. qRT-PCR analysis of DEG from RNAseq analysis using tumors from an independent cohort of mice. Unpaired t-test *p<0.05, **p<0.01. E. Representative GSEA plots demonstrating enriched gene sets in ABX over H₂O treated tumors including EMT, HYPOXIA, NOS, and NFKβ pathways.

Figure 6.. Tumor sphere initiation frequency is increased in ABX treated mice and further increased upon cisplatin therapy.

A. ID8 tumor cells isolated from tumors of C57Bl/6J H₂O and ABX treated mice without and with cisplatin were dissociated to single cells followed by spheroid analysis for CSC frequency. n= 4 mice per group in 96 well format. B-D. Tumors from C57Bl/6J H₂O and ABX treated mice without and with cisplatin were processed for RNA and validated by qRT-PCR of the stem cell gene NANOG, OCT-4, and SOX-2, respectively. E. ID8 tumor cells isolated from tumors of NSG H₂O and ABX treated mice without and with cisplatin were dissociated to single cells followed by spheroid analysis for CSC frequency. n= 4 mice per group in 96 well format. 2-way ANOVA *p<0.05, **p<0.01, ****p<0.005 n= 4 mice per group in 96 wells per condition.

Figure 7.. H₂O CMT ameliorates the effects of ABX on cisplatin sensitivity and tumor progression.

A. Schematic depicting the study design for cecal microbial transplant following ABX treatment. B. Overall fold change in tumor burden based on luminescence (photons/sec) and corrected to baseline detection of tumor implantation. (n=10/group). C. Survival analysis of ABX treated CMT mice regardless of cisplatin therapy compared to H₂O treated CMT mice. n=10 mice per group, 2-way ANOVA used to test for significance and p values indicated in figure. (Fig. 7A, Reprinted with permission, Cleveland Clinic Foundation ©2022. All Rights Reserved.)

Figure 8.. Gut derived microbial metabolites in mouse plasma are suppressed by ABX treatment highlighting the impact of ABX on EOC.

Plasma from C57Bl/6J mice that were treated with H₂O or ABX without or with cisplatin were processed for mass spec analysis of 29 metabolites previously shown to be gut microbe derived. A-C. Shown is analysis of indole-3-propionic acid, indoxyl sulfate, and hippuric acid. (n = 14/group). CMT study plasma was processed as above with focus on indole-3-propionic acid, indoxyl sulfate, and hippuric acid. (n = 10/group). One way ANOVA used to test for significance (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). D. Summary of our findings indicating the gut microbiome confers a protective cue in ovarian cancer. Use of broad-spectrum antibiotics results in disruption of the microbial communities and reduced microbial metabolites with subsequent unchecked ovarian cancer growth and cisplatin resistance. Microbial metabolites (shown as dots) found around the bacteria in the gut as well as circulating in the blood of control of no antibiotic treated mice treated and suppressed in antibiotic treated mice. Schematic highlights tumors from mice treated with broad spectrum antibiotic exhibit increased cell proliferation, angiogenesis, and cancer stem cells. (Fig. 8D, Reprinted with permission, Cleveland Clinic Foundation ©2022. All Rights Reserved.)