Over the past decade, our view of human-associated microbes has expanded beyond that of a few species along the gastrointestinal tract and towards an appreciation of the diverse and niche-specialized microbial communities that develop into complex relationships with the host. Not surprisingly, perturbation of these established relationships between humans and the bacteria within them (dysbiosis) has been associated with the development of numerous malignancies including gastric, colorectal, and cervical cancer.¹

Expanded culture techniques or culture-independent techniques, primarily through the use of metagenomics, have enabled assessment of the urinary microbiome, discrediting the historical notion that the bladder is a “sterile” environment.^2–6 While local microbial-driven immunomodulation in the gut is well described,⁷ more recent studies have demonstrated a role of microbes in the bladder, including their ability to influence cellular activity, contribute to pathogenesis, and/or predict response to treatment.⁸ However, there are limited data characterizing the urine and gut microbiome and its potential relationship with treatment efficacy in patients with muscle-invasive bladder cancer.

Studies have characterized bacterial communities within the bladders of patients with non–muscle-invasive bladder cancer.⁹ Our study sought to determine a unique consortium of bacterial species that were associated with response status to neoadjuvant chemotherapy in muscle-invasive bladder cancer.

To accomplish this, fecal and urine samples were collected from 29 patients with muscle-invasive bladder cancer before undergoing cystectomy, in addition to 31 healthy controls. Taxonomic profiling via 16S rRNA gene sequencing was performed on all available urine and stool samples. Eligible patients were classified as responders versus nonresponders based on final cystectomy staging.

Since compositional differences in the microbiome may also influence cancer development and response to therapy, we first characterized the overall differences of the gut microbiome using high dimensional class comparisons via linear discriminant analysis of effect size. Within gut microbiome, presence of Escherichia coli/Shigella was associated with lack of response to gemcitabine/cisplatin neoadjuvant chemotherapy (Figure 1, E and F). Biologically, this makes sense, as E. coli and Shigella are able to metabolize gemcitabine into its inactive form. We suspect that compositional differences in the microbiome of the gut alter the efficacy of chemotherapy through bacteria degradation and formation of inactive byproducts.

Furthermore, when further classified based on overall microbiome signature called enterotype, zero patients with enterotype II (Bacteroidales predominant) experienced a complete response (CR; Figure 1, C). Historically, patients on a high animal fat diet (Western diet) aggregate within Bacteroidales enterotype with decreased short chain fatty acid producing bacteria, and an exaggerated inflammatory cascade activation. We hypothesize that production of secondary inflammatory markers promotes a pro-oncogenic environment, limiting therapeutic efficacy of chemotherapy.

To explore how specific urinary bacterial taxa impact neoadjuvant chemoresponse, we compared urine microbiome of complete responders (n = 3) and nonresponders (n = 4), focusing on characterization of these differences as well as prediction of response status. Overall, patients with history of bladder cancer showed an increased abundance of Bacteroidales and Burkholderiales, while healthy controls’ urine was enriched with Enterobacteriales and Lactobacillales. Among the patients who received NAC, we further evaluated compositional differences within context of therapy. Pairwise comparisons were then performed for operational taxonomy unit at all levels by treatment response, highlighting that increased abundance of Lactobacillales at order, genus, and family were predictive of complete response status within urine (Figure 2).

Our study highlights that patients with “favorable” gut and urine composition (high diversity, presence of Lactobacillus within urine, and enterotype II within gut) are associated with improved NAC response. Although the mechanism of this association is yet to be determined, we hypothesize that it revolves around improved immune cell function and tumor microenvironment. We hope that functional microbiome studies in the next phase of our research will enable identification of microbial-derived products that facilitate interspecies interactions in the human host and improve treatment of bladder cancer.