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) (26, 27), which was sustained for the duration of the study. This poorly absorbed antibiotic cocktail was used because it has been previously shown to broadly suppress major phyla in the mouse gut microbiome over an extended duration (23). After 2 weeks of ABX treatment, 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 (28). 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 (29, 30). 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 > 150 mm3 or humane endpoint, including debilitating ascites development. Upon necropsy, EOC tumor phenotype was confirmed through histological assessment of hematoxylin and eosin (H&E) stained tissue sections as well as benign adjacent omentum (data not shown). 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 (31, 32). Although the underlying mechanisms of ascites development are largely unknown, previous studies have reported that immune cells such as natural killer (NK) cells and macrophages found in peritoneal ascites may play a key role in tumor cell invasiveness and growth (33–36). 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 Table 1 and Supplemental Fig. 1 for antibody panels and gating strategies). Our findings indicated no statistically significant difference in myeloid (Supplemental Fig. 2A) or lymphoid (Supplemental Fig. 2B) cell populations following ABX therapy in the presence or absence of cisplatin therapy in the BL/6 ID8-VEGF cohort ascites collected at endpoint. Additionally, no significant differences were observed between myeloid or lymphoid cell populations within collected splenocytes (Supplemental Fig. 3A, B) or bone marrow (Supplemental Fig. 3C, D) 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 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. NSG mice have immature T cells, DCs and macrophages, but functional neutrophils (37). The NSG cohort treated with ABX displayed accelerated tumor growth with more rapid onset compared to the BL/6 cohort, with no benefit of cisplatin (Fig. 2A, C). The time to tumor progression was accelerated and the effect on median survival was even more significant 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). Additionally, the peritoneal ascites fluid at endpoint was analyzed by flow cytometry. NSG ID8-VEGF cohort ascites exhibited no significant alterations in myeloid (Supplemental Fig. 2C) populations in ABX treated mice, regardless of cisplatin therapy compared to H2O controls. Overall, these findings provide evidence for a previously unappreciated role for the microbiome in the absence of an intact immune system in the modulation of cancer progression and sensitivity to chemotherapy.
ABX treatment results in comparable disruptions of the gut microbiome in BL/6 and NSG EOC tumor bearing mice.
To confirm that the ABX cocktail used was able to chronically suppress gut microbial communities, 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, > 2.4 million 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 and ID8-VEGF ABX placebo or cisplatin treated cohorts revealed statistically significant differences in alpha diversity between temporal collections, as analyzed by the Shannon diversity index (Supplemental Fig. 4A, B). These differences were not observed as expected in the H2O treated groups. Additionally, the Bray-Curtis dissimilarity based beta diversity comparisons between collection time points in the ID8 and ID8-VEGF cohorts were significant in the ABX treated groups demonstrating a strong change in microbial diversity in these groups from baseline over time, in contrast to the H2O treated groups (Supplemental Fig. 4C).
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 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. Proteobacteria are Gram-negative, mostly facultative anaerobes, which are generally resistant to vancomycin, an antibiotic that affects mainly Gram-positive bacteria. Proteobacteria are also less susceptible to metronidazole compared to strictly anaerobic gut bacteria (39). Furthermore, Enterobacteriaceae have multiple antibiotic resistance genes, which might explain their occurrence in in the ABX treated groups (40, 41). In addition, we also saw a relative increase in Enterococcacae. Interestingly, The alpha diversity is done at the ASV level, the most abundant taxa in Post 2 weeks is Enterobactereace, that means there is increase in ASV with in Enterobactereacee and that’s why the alpha diversity is higher because it is showing the diversity at ASV level, however overall, there is only one group that’s increasing its abundance and that’s Enterobactereace.
NSG cohort. In total, > 2.8 million 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 patterns for alpha diversity between the collection time points in both the ID8 and ID8-VEGF ABX placebo and ABX cisplatin treated cohorts, whereas the control H2O treated groups had similar alpha diversity over time (Supplemental Fig. 5A, B). Additionally, the Bray-Curtis dissimilarity based beta diversity between collection time points in the ID8 and ID8-VEGF ABX treated cohorts were significantly different from baseline over time demonstrating the effect of ABX on microbial diversity (Supplemental Fig. 5C). This alteration in beta diversity was not observed in the H2O treated groups regardless of placebo or cisplatin therapies.
Like the BL/6 cohort, the NSG cohort exhibited a marked 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 (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 in vitro assessment of the effects of ABX on growth of these tumor cell lines. ID8 and ID8-VEGF cell lines were co-cultured with various concentrations of ABX in the same respective ratios utilized in the murine studies Following 7 days culture, there were no appreciable growth differences in cell number between ABX treated cell lines compared to cell lines cultured in non-supplemented media (Supplemental Fig. 6A, B). Additionally, there were no changes in the determined half maximal inhibitory concentration (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 5x106 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 cancer 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). When comparing ABX Placebo over H2O Placebo, multiple genes were significantly enriched including PAX2, PAX8, WNT7a, CALB1, A2ML, IHH and PLEKHS1, among others that play key roles in cancer stem cell development and tumor progression and invasiveness (Fig. 5A) (42–49). Interestingly, the most enriched Gene Ontology (GO) terms included cell differentiation, proliferation, and locomotor activity (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) (50–57). Upon gene set enrichment analysis (GSEA), epithelial mesenchymal transition, NOS targets, NFkB signaling via TNFα, and hypoxia were all enriched in ABX treated tumors compared to H2O treated controls (Fig. 5D) (58–60).
As we observed increased stem cell markers based on RNAseq of tumors, we performed self-renewal assays to assess stem cell frequency in tumors from H2O and ABX treated mice with and without cisplatin therapy. Following endpoint necropsy, tumor tissue from each group was dissociated to single cells and plated in a limiting dilution assay. Following 14 days of incubation, sphere initiation frequency was determined indicating 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). The observed effect on cancer stem cells (CSCs) was replicated in the NSG cohorts demonstrating an apparent increase in the stem cell population following ABX therapy that is further increased in the presence of cisplatin even in the absence of an intact immune system (Fig. 6B).
Cecal Microbial Transplant (CMT) from control treated mice is sufficient to inhibit tumor growth of ABX treatment on EOC tumor progression and cisplatin resistance.
We tested the hypothesis that the gut microbiome of control mice (i.e. mice not receiving ABX treatment) can inhibit the accelerated EOC tumor growth by performing cecal microbial transplant (CMT) studies. Cecal content was harvested from either H2O or ABX treated mice following the study paradigm outlined in Fig. 7A. BL/6 mice treated with ABX were orally gavaged with ABX or H2O cecal content, followed by IP injection with ID8 EOC cells and monitored for tumor progression with bi weekly IVIS imaging. When ABX CMT treated mice met endpoint, all groups were compared for total tumor progression from baseline (7 days post EOC cell injection) (Fig. 7B). H2O-CMT treated mice responded to cisplatin therapy and had survival similar to H2O treated mice throughout. Interestingly, ABX-CMT vehicle treated mice had survival similar to ABX vehicle treated mice, but ABX-CMT cisplatin treated mice had significantly worse overall survival compared to ABX cisplatin treated mice with a median survival of 57 and 72 days respectively (Supplemental Fig. 8). Survival analysis showed that ABX-CMT treated mice were not responsive to cisplatin therapy with the median survival of vehicle and cisplatin treated mice at 61 and 57 days post cell injection respectively. The H2O-CMT treated mice however, were sensitized to cisplatin therapy showing median survivals of 83 and 91 days in vehicle and cisplatin treated groups respectively (Fig. 7C).