EOC tumors exhibit accelerated growth and attenuated sensitivity to cisplatin in antibiotic treated mice.
We tested the hypothesis that an antibiotic driven disruption of the microbiome impacts tumor development and chemotherapy sensitivity in EOC. To this end, the following study paradigm (Fig. 1A) was implemented wherein C57 BL/6J (BL/6) female mice at 6 weeks of age were provided water or antibiotic containing water (ABX; ampicillin, neomycin, metronidazole, and vancomycin), which was sustained for the duration of the study. After 2 weeks, mice were injected intraperitoneally (IP) with murine EOC lines ID8 or ID8-VEGF, syngeneic with BL/6 mice, and tumor growth was monitored by transabdominal ultrasound (TAUS) weekly for the course of the study (22). ID8 and ID8-VEGF cell lines were utilized as they are highly characterized and closely recapitulate human ovarian cancer progression. The ID8-VEGF cell line is slightly more severe causing an increase in angiogenesis and ascites development (23, 24). At 2 weeks post tumor cell injection, mice were injected IP with cisplatin or vehicle weekly for the remainder of the study. Mice met study endpoint at tumor burden of > 150mm3 or humane endpoint, including debilitating ascites development. Upon necropsy, EOC tumor phenotype was confirmed through histological assessment of H&E stained tissue sections as well as benign adjacent omentum (Supplemental Fig. 1A). Our findings indicate that EOC tumor growth is significantly increased by ABX treated mice compared to controls. In the presence of ABX, cisplatin therapy had attenuated efficacy compared to in the control treated mice indicating development of cisplatin resistance (Fig. 1B, D). As expected from the tumor burden, median survival was decreased in the ABX treatment groups compared to controls. The ID8 cohort exhibited a median survival of 66 and 64 days in the ABX placebo and cisplatin groups compared to 68.5 and 84 days in the H2O placebo and cisplatin groups, respectively (Fig. 1C). This result was paralleled in the ID8-VEGF cohort of mice with a median survival of 39 and 34 days in the ABX placebo and cisplatin groups compared to 42 and 53 days in the H2O placebo and cisplatin groups respectively (Fig. 1E). The ID8-VEGF mice reached the endpoint earlier than the ID8 cohort secondary to large volume ascites development.
Limited impact on immune populations in ascites of EOC by broad spectrum antibiotics.
The majority of EOC patients present with advanced disease (stage III or stage IV) that may include ascites (25, 26). Although the underlying mechanisms of ascites development are largely unknown, previous studies have reported that immune cells such as Th17 T cells, NK cells and macrophages found in peritoneal ascites may play a key role in tumor cell invasiveness and growth (27–30). To this end, we analyzed the immune cell populations within the peritoneal ascites fluid through flow cytometry. Our flow cytometry evaluation of immune cell populations within the peritoneal ascites was designed to focus on both myeloid and lymphoid populations (see Supplemental Fig. 2 for antibody panels and gating strategies). Our findings indicated no significant difference in myeloid (Supplemental Fig. 3A) or lymphoid (Supplemental Fig. 3B) cell populations following ABX therapy in the presence or absence of cisplatin therapy in the BL/6 ID8-VEGF cohort ascites collected at endpoint.
Disruption of the immune system does not significantly impact the accelerated EOC tumor growth or reduced cisplatin sensitivity in antibiotic treated mice.
To further assess whether the observed augmentation of tumor phenotype and cisplatin resistance following ABX treatment is dependent on an intact immune system, the same study paradigm was utilized in NOD.Cg-Prkdc < scid > Il2rg < tm1Wjl > SzJ (NSG) immuno-deficient mice. The NSG cohort displayed more significant impact of microbiome depletion through antibiotics on tumor growth as the BL/6 cohort, with no clinical benefit to the mice receiving cisplatin along with ABX (Fig. 2A, C). The time to tumor progression was accelerated and the effect on median survival was even more dramatic with the ID8 cohort exhibiting a median survival of 41 and 45 days in the ABX placebo and cisplatin groups compared to 55 and 69 days in the H2O placebo and cisplatin groups respectively (Fig. 2B). A similar phenotype was observed in the ID8-VEGF cohort of NSG mice (Fig. 2D). As before, EOC tumors were confirmed via H&E staining (Supplemental Fig. 1B). Additionally, the peritoneal ascites fluid at endpoint was analyzed by flow cytometry. NSG mice have immature T cells, DCs and macrophages, but functional neutrophils (31). NSG ID8-VEGF cohort ascites exhibited no significant alterations in myeloid (Supplemental Fig. 3C) populations in ABX treated mice, regardless of cisplatin therapy compared to H2O controls.
ABX treatment results in comparable disruptions of the gut microbiome in BL/6 and NSG EOC tumor bearing mice.
The ABX treatment regimen we applied was previously shown to be sufficient to deplete detectable commensal bacteria (32, 33). However, to define the response of the gut microbiome to broad spectrum antibiotic therapy, stool was collected at baseline, at 2 weeks, at tumor engraftment, at 5 weeks and at endpoint necropsy and then processed for 16S rRNA gene sequencing. The microbiome was analyzed from the following cohorts: BL/6 ID8 BL/6 ID8-VEGF, NSG ID8, and NSG ID8-VEGF.
C57 BL/6 cohort. In total, 2,801,471 and 2,411,554 high-quality and usable reads were obtained from fecal samples of 5 mice per treatment group in the ID8 and ID8-VEGF BL/6 cohorts from sequencing the 16S rRNA gene, with an average length of 210 base pairs (bps). There were 3,184 amplified sequence variants (ASVs) in all ID8 and ID8-VEGF BL/6 samples. Pairwise comparisons within both the ID8-VEGF and ID8 cohorts revealed statistically significant differences in alpha diversity between temporal collections, as analyzed by the Shannon diversity index (ANOVA p < 0.05) (Supplemental Fig. 4A, B). Additionally, the Bray-Curtis dissimilarity based beta diversity comparisons between collection time points in the ID8-VEGF and ID8 cohorts were significant at p = 0.003 and p = 0.001 respectively (Supplemental Fig. 4C, D).
Antibiotic treated groups displayed significantly less diverse fecal microbial communities compared to control water treated groups regardless of cisplatin therapy. Specifically, the relative abundance of some Proteobacteria including Enterobacteriaceae and Parasutterella was increased in the antibiotic treated ID8 groups while the abundance of Enterobacteriaceae was increased in the antibiotic treated ID8-VEGF groups (Fig. 3A, B). These were significantly increased by 2 weeks post antibiotic therapy in the mice being co treated with cisplatin, but took up to 5 weeks for the mice treated with placebo to obtain the same increase. Being Gram-negative, and mostly facultative anaerobes, the Proteobacteria are generally resistant to vancomycin which affects mainly Gram-positive bacteria and are also less susceptible to metronidazole which affects more the anaerobes (34). Furthermore, Enterobacteriaceae have multiple antibiotic resistance genes allowing for their survival post ABX therapy (35, 36). Increased abundance of Parasutterella has been associated with dysbiosis of the gut microbiome, but the mechanism has not been fully interrogated (37, 38).
NSG cohort. In total, 4,784,498 and 2,822,423 high-quality and usable reads were obtained from fecal samples of 5 mice per treatment group in the ID8 and ID8-VEGF NSG cohorts from sequencing the 16S rRNA gene, with an average length 210 bps. There were 6,484 ASVs in all ID8 and ID8-VEGF NSG samples. As with the BL/6 cohort, the pairwise comparisons revealed statistically significant (ANOVA, p < 0.05) patterns for alpha diversity (i.e. Shannon Index) between the collection time points in both the ID8-VEGF and ID8 cohorts (Supplemental Fig. 5A, B). Additionally, the Bray-Curtis dissimilarity based beta diversity between collection time points in the ID8-VEGF and ID8 cohorts were both significant at p = 0.001 (Supplemental Fig. 5C, D).
Similar to the BL/6 cohort, the NSG cohort exhibited a marked relative increase in Enterobacteriaceae in the antibiotic treated ID8 and ID8-VEGF groups’ fecal samples, increasing more quickly in the Cisplatin treated groups compared to the placebo as well. In addition, this cohort also saw a marked relative increase in Paenibacillus and Enterococcus (Fig. 4A, B).
ABX treatment does not significantly alter ID8 or ID8-VEGF EOC tumor cell proliferation in vitro
To determine if tumor growth was a direct effect of ABX interaction with the EOC ID8 and ID8-VEGF cell lines, we performed tissue culture analyses. ID8 and ID8-VEGF cell lines were co-cultured with various concentrations of ABX (metronidazole, vancomycin, ampicillin and neomycin) in the same respective ratios utilized in the murine studies Following 7 days culture, there were no appreciable differences in ABX treated cell lines compared to cell lines in normal media (Supplemental Fig. 6A, B). Additionally, there were no changes in the determined IC50 of cisplatin on ID8 of ID8-VEGF cells following ABX treatment when compared to controls (Supplemental Fig. 6C, D).
Antibiotic treatment leads to accelerated tumor growth and attenuated sensitivity to cisplatin in patient derived EOC.
To ensure our observed phenotype was not unique to syngeneic EOC cell lines (ID8 and ID8-VEGF). We repeated our study paradigm in NSG mice with a human derived OV81 cell line. Following 2 weeks of ABX or control water, mice were IP injected with 5 × 106 OV81 cells. To monitor tumor growth is this cohort of mice, cells were transduced with a luciferase reporter prior to IP injection. Following 2 weeks IP cell injection, mice were randomized into 4 groups: H2O Vehicle, H2O Cisplatin, ABX Vehicle, and ABX Cisplatin and treated as previously outlined. At endpoint, mice underwent Perkin Elmer in Vivo Imaging System (IVIS) imaging and total flux of photons/second was calculated to determine total tumor burden. Overall ABX therapy resulted in a drastic increase in total tumor burden when normalized to initial tumor burden compared to H2O controls in both the vehicle and cisplatin treated groups (Supplemental Fig. 7).
ABX therapy induces a stem cell phenotype in ID8 EOC tumors in NSG mice
To better understand the effect of antibiotic therapy on tumor cells and their growth in the presence and absence of cisplatin, we performed RNAseq on tumors collected from H2O Placebo, H2O Cisplatin, ABX Placebo, and ABX Cisplatin treated NSG mice at endpoint (8 weeks post ID8 cell injection). The top 100 most significantly affected genes by p-value following a pairwise comparison between the H2O Placebo and ABX Placebo groups (Fig. 5A) were analyzed utilizing DAVID software for the enrichment of GO terms. Interestingly, the most enriched GO terms included cell differentiation, proliferation, and locomotor activity in line with the increased self-renewal ability observed in the previous tumor sphere initiation assays (Fig. 5B). Genes commonly associated with cancer stem cell signatures were also increased in the ABX treatment group compared to the H2O control group such as SOX2, WNT7a, HOXb5, HOXb6, DLX5, MSX1, EFNA4, SALL1, and PAX2 genes. SHOX2 and GATA5 genes that promote cell differentiation were also significantly decreased (Fig. 5C) (39–46). Upon KEGG pathway analysis of genes with a p-value < 0.01 in the ABX Placebo group vs. the H2O Placebo group, multiple signaling pathways were implicated including PI3-Akt, MAPK, WNT, and Hedgehog (Fig. 5D) (47–49).
As we observed increased stem cell markers based on RNAseq of tumors, we performed tumorsphere assays to assess stem cell frequency in tumors from H2O and ABX treated mice without and with cisplatin. Following endpoint necropsy, tumor tissue from each group was dissociated to single cells and plated in a tumor sphere formation assay. Following 14 days of incubation, sphere initiation frequency was determined demonstrating a significant increase in sphere initiation frequency of ID8 cells from ABX treated BL/6 mice compared to control H2O in both the presence and absence of cisplatin therapy (Fig. 6A). This observed effect was increasingly evident in the NSG cohorts demonstrating an apparent increase in the stem cell population following ABX therapy, further increased in the presence of cisplatin (Fig. 6B).