Colorectal cancers comprise a complex mixture of malignant cells, non-transformed cells, and microorganisms. Fusobacterium nucleatum is among the most prevalent bacterial species in colorectal cancer tissues. Here we show that colonization of human colorectal cancers with Fusobacterium and its associated microbiome, —including Bacteroides, Selenomonas, and Prevotella species, —is maintained in distal metastases, demonstrating microbiome stability between paired primary and -metastatic tumors. In situ hybridization analysis revealed that Fusobacterium is predominantly associated with cancer cells in the metastatic lesions. Mouse xenografts of human primary colorectal adenocarcinomas were found to retain viable Fusobacterium and its associated microbiome through successive passages. Treatment of mice bearing a colon cancer xenograft with the antibiotic metronidazole reduced Fusobacterium load, cancer cell proliferation, and overall tumor growth. These observations argue for further investigation of antimicrobial interventions as a potential treatment for patients with Fusobacterium-associated colorectal cancer.

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Background Accumulating evidence has identified Fusobacterium as an important pathogenic gut bacterium associated with colorectal cancer. Nevertheless, only limited data exist about the role of this bacterium in locally advanced rectal cancer (LARC). In this study, we quantified Fusobacterium nucleatum in untreated and post-neoadjuvant chemoradiotherapy (nCRT) samples from LARC patients and investigated its association with therapy response and survival. Patients and methods A total of 254 samples from 143 patients with rectal adenocarcinomas were analyzed for the presence and abundance of F. nucleatum using RNA in situ hybridization and digital image analysis. Assay accuracy was determined using infected cell lines and tumor samples with available quantitative PCR data. We studied the impact of F. nucleatum load on pathologic complete response and relapse-free survival. Treatment-induced changes were evaluated in paired pre- and post-nCRT samples ( n = 71). Finally, tumor microenvironment changes during nCRT were assessed in paired samples ( n = 45) by immune contexture analysis. Results F. nucleatum tissue levels by RNA in situ hybridization strongly correlated with quantitative PCR ( r = 0.804, P < 0.001). F. nucleatum abundance was higher in untreated [median, 7.4; 95% confidence interval (3.7–16.2)] compared with treated [median, 1.6; 95% confidence interval (1.3–2.4)] tumors ( P <0.001) with 58% (73/126) and 26% (22/85) positive tumors, respectively ( P < 0.001). Baseline F. nucleatum levels were not associated with pathologic complete response. F. nucleatum positivity after nCRT, but not baseline status, significantly increased risk of relapse [hazard ratio = 7.5, 95% confidence interval (3.0–19.0); P < 0.001]. Tumors that turned F. nucleatum- negative after nCRT had a strong increase in CD8+ T cells post-nCRT ( P < 0.001), while those that persisted F. nucleatum -positive after nCRT lacked CD8+ T cells induction in post-nCRT samples compared with baseline ( P = 0.69). Conclusion F. nucleatum persistence post-nCRT is associated with high relapse rates in LARC, potentially linked to suppression of immune cytotoxicity.

Brain metastases are the most common tumor of the brain with a dismal prognosis. A fraction of patients with brain metastasis benefit from treatment with immune checkpoint inhibitors (ICI) and the degree and phenotype of the immune cell infiltration has been used to predict response to ICI. However, the anatomical location of brain lesions limits access to tumor material to characterize the immune phenotype. Here, we characterize immune cells present in brain lesions and matched cerebrospinal fluid (CSF) using single-cell RNA sequencing combined with T cell receptor genotyping. Tumor immune infiltration and specifically CD8+ T cell infiltration can be discerned through the analysis of the CSF. Consistently, identical T cell receptor clonotypes are detected in brain lesions and CSF, confirming cell exchange between these compartments. The analysis of immune cells of the CSF can provide a non-invasive alternative to predict the response to ICI, as well as identify the T cell receptor clonotypes present in brain metastasis.