Elizabeth M. Hechenbleikner

UNLABELLED: We examined the immune microenvironment of primary colorectal cancer using immunohistochemistry, laser capture microdissection/qRT-PCR, flow cytometry, and functional analysis of tumor-infiltrating lymphocytes. A subset of colorectal cancer displayed high infiltration with activated CD8(+) cytotoxic T lymphocyte (CTL) as well as activated Th1 cells characterized by IFNγ production and the Th1 transcription factor TBET. Parallel analysis of tumor genotypes revealed that virtually all of the tumors with this active Th1/CTL microenvironment had defects in mismatch repair, as evidenced by microsatellite instability (MSI). Counterbalancing this active Th1/CTL microenvironment, MSI tumors selectively demonstrated highly upregulated expression of multiple immune checkpoints, including five-PD-1, PD-L1, CTLA-4, LAG-3, and IDO-currently being targeted clinically with inhibitors. These findings link tumor genotype with the immune microenvironment, and explain why MSI tumors are not naturally eliminated despite a hostile Th1/CTL microenvironment. They further suggest that blockade of specific checkpoints may be selectively efficacious in the MSI subset of colorectal cancer. SIGNIFICANCE: The findings reported in this article are the first to demonstrate a link between a genetically defined subtype of cancer and its corresponding expression of immune checkpoints in the tumor microenvironment. The mismatch repair-defective subset of colorectal cancer selectively upregulates at least five checkpoint molecules that are targets of inhibitors currently being clinically tested.

Biofilms provide refuge for cancerous bacteria Familial adenomatous polyposis (FAP) causes benign polyps along the colon. If left untreated, FAP leads to a high incidence of colon cancer. To understand how polyps influence tumor formation, Dejea et al. examined the colonic mucosa of FAP patients. They discovered biofilms containing the carcinogenic versions of the bacterial species Escherichia coli and Bacteroides fragilis . Colon tissue from FAP patients exhibited greater expression of two bacterial genes that produce secreted oncotoxins. Studies in mice showed that specific bacteria could work together to induce colon inflammation and tumor formation. Science , this issue p. 592

Environmental factors clearly affect colorectal cancer (CRC) incidence, but the mechanisms through which these factors function are unknown. One prime candidate is an altered colonic microbiota. Here we show that the mucosal microbiota organization is a critical factor associated with a subset of CRC. We identified invasive polymicrobial bacterial biofilms (bacterial aggregates), structures previously associated with nonmalignant intestinal pathology, nearly universally (89%) on right-sided tumors (13 of 15 CRCs, 4 of 4 adenomas) but on only 12% of left-sided tumors (2 of 15 CRCs, 0 of 2 adenomas). Surprisingly, patients with biofilm-positive tumors, whether cancers or adenomas, all had biofilms on their tumor-free mucosa far distant from their tumors. Bacterial biofilms were associated with diminished colonic epithelial cell E-cadherin and enhanced epithelial cell IL-6 and Stat3 activation, as well as increased crypt epithelial cell proliferation in normal colon mucosa. High-throughput sequencing revealed no consistent bacterial genus associated with tumors, regardless of biofilm status. However, principal coordinates analysis revealed that biofilm communities on paired normal mucosa, distant from the tumor itself, cluster with tumor microbiomes as opposed to biofilm-negative normal mucosa bacterial communities also from the tumor host. Colon mucosal biofilm detection may predict increased risk for development of sporadic CRC.

BACKGROUND: Enterotoxigenic Bacteroides fragilis (ETBF) produces the Bacteroides fragilis toxin, which has been associated with acute diarrheal disease, inflammatory bowel disease, and colorectal cancer (CRC). ETBF induces colon carcinogenesis in experimental models. Previous human studies have demonstrated frequent asymptomatic fecal colonization with ETBF, but no study has investigated mucosal colonization that is expected to impact colon carcinogenesis. METHODS: We compared the presence of the bft gene in mucosal samples from colorectal neoplasia patients (cases, n = 49) to a control group undergoing outpatient colonoscopy for CRC screening or diagnostic workup (controls, n = 49). Single bacterial colonies isolated anaerobically from mucosal colon tissue were tested for the bft gene with touch-down polymerase chain reaction. RESULTS: The mucosa of cases was significantly more often bft-positive on left (85.7%) and right (91.7%) tumor and/or paired normal tissues compared with left and right control biopsies (53.1%; P = .033 and 55.5%; P = .04, respectively). Detection of bft was concordant in most paired mucosal samples from individual cases or controls (75% cases; 67% controls). There was a trend toward increased bft positivity in mucosa from late- vs early-stage CRC patients (100% vs 72.7%, respectively; P = .093). In contrast to ETBF diarrheal disease where bft-1 detection dominates, bft-2 was the most frequent toxin isotype identified in both cases and controls, whereas multiple bft isotypes were detected more frequently in cases (P ≤ .02). CONCLUSIONS: The bft gene is associated with colorectal neoplasia, especially in late-stage CRC. Our results suggest that mucosal bft exposure is common and may be a risk factor for developing CRC.