Preclinical mouse models suggest that the gut microbiome modulates tumor response to checkpoint blockade immunotherapy; however, this has not been well-characterized in human cancer patients. Here we examined the oral and gut microbiome of melanoma patients undergoing anti–programmed cell death 1 protein (PD-1) immunotherapy (n = 112). Significant differences were observed in the diversity and composition of the patient gut microbiome of responders versus nonresponders. Analysis of patient fecal microbiome samples (n = 43, 30 responders, 13 nonresponders) showed significantly higher alpha diversity (P < 0.01) and relative abundance of bacteria of the Ruminococcaceae family (P < 0.01) in responding patients. Metagenomic studies revealed functional differences in gut bacteria in responders, including enrichment of anabolic pathways. Immune profiling suggested enhanced systemic and antitumor immunity in responding patients with a favorable gut microbiome as well as in germ-free mice receiving fecal transplants from responding patients. Together, these data have important implications for the treatment of melanoma patients with immune checkpoint inhibitors.

SUMMARY Treatment of cancer has been revolutionized by immune checkpoint blockade therapies. Despite the high rate of response in advanced melanoma, the majority of patients succumb to disease. To identify factors associated with success or failure of checkpoint therapy, we profiled transcriptomes of 16,291 individual immune cells from 48 tumor samples of melanoma patients treated with checkpoint inhibitors. Two distinct states of CD8+ T cells were defined by clustering, and associated with patient tumor regression or progression. A single transcription factor, TCF7, was visualized within CD8+ T cells in fixed tumor samples and predicted positive clinical outcome in an independent cohort of checkpoint-treated patients. We delineated the epigenetic landscape and clonality of these T cell states, and demonstrated enhanced anti-tumor immunity by targeting novel combinations of factors in exhausted cells. Our study of immune cell transcriptomes from tumors demonstrates a strategy for identifying predictors, mechanisms and targets for enhancing checkpoint immunotherapy.

Drug resistance remains a vexing problem in the treatment of cancer patients. While many studies have focused on cell autonomous mechanisms of drug resistance, we hypothesized that the tumor microenvironment may confer innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anti-cancer drugs. We found that stroma-mediated resistance is surprisingly common – particularly to targeted agents. We further characterized the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibition because most of these patients exhibit some degree of innate resistance1-4. Proteomic analysis showed that stromal secretion of the growth factor hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the MAPK and PI3K/AKT pathways, and immediate resistance to RAF inhibition. Immunohistochemistry confirmed stromal HGF expression in patients with BRAF-mutant melanoma and a statistically significant correlation between stromal HGF expression and innate resistance to treatment. Dual inhibition of RAF and MET resulted in reversal of drug resistance, suggesting RAF/MET combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, these studies indicate that the systematic dissection of tumor-microenvironment interactions may reveal important mechanisms underlying drug resistance.

Bacteria were first detected in human tumors more than 100 years ago, but the characterization of the tumor microbiome has remained challenging because of its low biomass. We undertook a comprehensive analysis of the tumor microbiome, studying 1526 tumors and their adjacent normal tissues across seven cancer types, including breast, lung, ovary, pancreas, melanoma, bone, and brain tumors. We found that each tumor type has a distinct microbiome composition and that breast cancer has a particularly rich and diverse microbiome. The intratumor bacteria are mostly intracellular and are present in both cancer and immune cells. We also noted correlations between intratumor bacteria or their predicted functions with tumor types and subtypes, patients’ smoking status, and the response to immunotherapy.

Growing evidence suggests that microbes can influence the efficacy of cancer therapies. By studying colon cancer models, we found that bacteria can metabolize the chemotherapeutic drug gemcitabine (2′,2′-difluorodeoxycytidine) into its inactive form, 2′,2′-difluorodeoxyuridine. Metabolism was dependent on the expression of a long isoform of the bacterial enzyme cytidine deaminase (CDDL), seen primarily in Gammaproteobacteria. In a colon cancer mouse model, gemcitabine resistance was induced by intra-tumor Gammaproteobacteria, dependent on bacterial CDDL expression, and abrogated by co-treatment with the antibiotic ciprofloxacin. Gemcitabine is commonly used to treat pancreatic ductal adenocarcinoma (PDAC), and we hypothesized that intra-tumor bacteria might contribute to drug resistance of these tumors. Consistent with this possibility, we found that of the 113 human PDACs that were tested, 86 (76%) were positive for bacteria, mainly Gammaproteobacteria.