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Defining the complex role of the microbiome in colorectal cancer and the discovery of novel, protumorigenic microbes are areas of active investigation. In the present study, culturing and reassociation experiments revealed that toxigenic strains of Clostridioides difficile drove the tumorigenic phenotype of a subset of colorectal cancer patient–derived mucosal slurries in germ-free ApcMin/+ mice. Tumorigenesis was dependent on the C. difficile toxin TcdB and was associated with induction of Wnt signaling, reactive oxygen species, and protumorigenic mucosal immune responses marked by the infiltration of activated myeloid cells and IL17-producing lymphoid and innate lymphoid cell subsets. These findings suggest that chronic colonization with toxigenic C. difficile is a potential driver of colorectal cancer in patients.Significance:Colorectal cancer is a leading cause of cancer and cancer-related deaths worldwide, with a multifactorial etiology that likely includes procarcinogenic bacteria. Using human colon cancer specimens, culturing, and murine models, we demonstrate that chronic infection with the enteric pathogen C. difficile is a previously unrecognized contributor to colonic tumorigenesis.See related commentary by Jain and Dudeja, p. 1838.This article is highlighted in the In This Issue feature, p. 1825

Supplementary Table from Human Colon Cancer–Derived Clostridioides difficile Strains Drive Colonic Tumorigenesis in Mice

Supplementary Figure from Human Colon Cancer–Derived Clostridioides difficile Strains Drive Colonic Tumorigenesis in Mice

Human gut microbial species found to associate with clinical responses to immune checkpoint inhibitors (ICIs) are often tested in mice using fecal microbiota transfer (FMT), wherein tumor responses in recipient mice may recapitulate human responses to ICI treatment. However, many FMT studies have reported only limited methodological description, details of murine cohorts, and statistical methods. To investigate the reproducibility and robustness of gut microbial species that impact ICI responses, we performed human to germ-free mouse FMT using fecal samples from patients with non-small cell lung cancer who had a pathological response or nonresponse after neoadjuvant ICI treatment. R-FMT mice yielded greater anti-tumor responses in combination with anti-PD-L1 treatment compared to NR-FMT, although the magnitude varied depending on mouse cell line, sex, and individual experiment. Detailed investigation of post-FMT mouse microbiota using 16S rRNA amplicon sequencing, with models to classify and correct for biological variables, revealed a shared presence of the most highly abundant taxa between the human inocula and mice, though low abundance human taxa colonized mice more variably after FMT. Multiple Clostridium species also correlated with tumor outcome in individual anti-PD-L1-treated R-FMT mice. RNAseq analysis revealed differential expression of T and NK cell-related pathways in responding tumors, irrespective of FMT source, with enrichment of these cell types confirmed by immunohistochemistry. This study identifies several human gut microbial species that may play a role in clinical responses to ICIs and suggests attention to biological variables is needed to improve reproducibility and limit variability across experimental murine cohorts.

Defining the complex role of the microbiome in colorectal cancer and the discovery of novel, protumorigenic microbes are areas of active investigation. In the present study, culturing and reassociation experiments revealed that toxigenic strains of Clostridioides difficile drove the tumorigenic phenotype of a subset of colorectal cancer patient–derived mucosal slurries in germ-free ApcMin/+ mice. Tumorigenesis was dependent on the C. difficile toxin TcdB and was associated with induction of Wnt signaling, reactive oxygen species, and protumorigenic mucosal immune responses marked by the infiltration of activated myeloid cells and IL17-producing lymphoid and innate lymphoid cell subsets. These findings suggest that chronic colonization with toxigenic C. difficile is a potential driver of colorectal cancer in patients. Significance: Colorectal cancer is a leading cause of cancer and cancer-related deaths worldwide, with a multifactorial etiology that likely includes procarcinogenic bacteria. Using human colon cancer specimens, culturing, and murine models, we demonstrate that chronic infection with the enteric pathogen C. difficile is a previously unrecognized contributor to colonic tumorigenesis. See related commentary by Jain and Dudeja, p. 1838. This article is highlighted in the In This Issue feature, p. 1825

Background: Mucus-invasive colonic bacterial biofilms are dense, polymicrobial communities that are in direct contact with the epithelium. Prior data from our group has demonstrated that these invasive biofilms are prevalent in colorectal cancer, are directly tumorigenic in germ-free and specific pathogen-free ApcMin/+ mouse models, and are associated with enrichment of the polyamine N1,N12-diactylspermine (DAS) in patient samples. However, whether the composition of these biofilms impacts their pro-tumorigenic behavior and/or metabolism is presently unknown. Methods: Carnoy’s-fixed CRC resections from 115 patients from the University of Malaya in Malaysia were stained with the all-bacterial EUB338 probe and screened for mucus-invasive biofilms (BF). All BF+ samples were subsequently stained with oligonucleotide probes targeting Bacteroidetes, Fusobacteria, Lachnospiraceae, and γ/β-Proteobacteria. Imaging was performed on a Zeiss780 confocal microscope with linear unmixing. Untargeted metabolomics was performed on 5-10 samples of each biofilm type using a Waters UPLC Xevo GS-XS quadrupole time-of-flight (QTOF) mass spectrometer. Five Fusobacterium nucleatum (Fn) strains derived from CRC cohorts were singly gavaged into germ-free ApcMin/+ mice and colons were harvested for tumors 11 wk later. Results: Seventy-nine of 115 Malaysian tumors (69%) were BF+ using EUB338 staining. As with our prior US CRC cohort, BFs were more prevalent on the tumors proximal to the hepatic flexure (35/39, 90%) vs. those that were distal (44/76, 58%) (Chi-square p < 0.001). Of the BF+ tumors (BF+T), 55% were polymicrobial (predominantly Bacteroidetes/Lachnospiraceae), 40% were polymicrobial with fusobacterial blooms, and 5% were Proteobacteria dominant. Untargeted metabolomics of BF- healthy biopsies, BF-T, and BF+T with various BF composition subtypes revealed a gradient in levels of the polyamine DAS, with BF+T with fusobacterial blooms having the highest levels (p = 0.055 vs. Proteo-dominant BF+T, p = 0.043 vs. polymicrobial BF+T, p = 0.015 vs. BF-T, p < 0.001 vs. BF- healthy biopsies), suggesting that fusobacteria may drive proliferation and polyamine production in CRC tissues. However, inoculation of 5 different CRC-derived Fn strains including a strain isolated from a Malaysia CRC patient did not promote tumorigenesis in germ-free ApcMin/+ mice. Conclusions: Fusobacterial-dominant biofilms are prevalent in a Malaysian CRC cohort and are associated with high levels of the polyamine N1,N12-diacetylspermine. However, F. nucleatum strains were not tumorigenic in germ-free ApcMin/+ mice, suggesting that the association between Fn, DAS, and tumorigenesis may require other bacteria in order to confer a procarcinogenic state. Citation Format: Julia L. Drewes, Jessica Queen, Jada C. Domingue, Caroline R. Wensel, Yuping Cai, Jane Wanyiri, April C. Roslani, Jamuna Vadivelu, Caroline H. Johnson, Cynthia L. Sears. Fusobacterial-dominant colorectal cancer biofilms are associated with high levels of the polyamine N1,N12-diacetylspermine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3052.

The human microbiota is a complex ecosystem of diverse microorganisms consisting of bacteria, viruses, and fungi residing predominantly in epidermal and mucosal habitats across the body, such as skin, oral cavity, lung, intestine and vagina. These symbiotic communities in health, or dysbiotic communities in disease, display tremendous interaction with the local environment and systemic responses, playing a critical role in the host’s nutrition, immunity, metabolism and diseases including cancers. While the profiling of normal microbiota in healthy populations is useful and necessary, more recent studies have focused on the microbiota associated with disease, particularly cancers. In this paper, we review current evidence on the role of the human microbiota in four cancer types (colorectal cancer, head and neck cancer, pancreatic cancer, and lung cancer) proposed as affected by both the oral and gut microbiota, and provide a perspective on current gaps in the knowledge of the microbiota and cancer.

There is mounting evidence of the role of the immune response to microbiota in orchestrating homeostatic mucosal immunity, cancer pathogenesis, and, furthermore, the response to immunotherapy. It is only recently that the causative contribution of bacteria in colorectal cancer and the mechanisms used to trigger oncogenesis and/or tumor growth are being unraveled. Coopting mucosal immunity by “driver” pathogens is the main common theme of colon carcinogenesis. The cross talk between the microbiota, the epithelium, and the immune system is the center stage to the homeostatic mucosal microbiome:host mutualism. If the balance is altered, bacteria and/or immune response to these bacteria will contribute to pathogenesis of colon carcinogenesis. In this chapter, we will discuss the roles of the microbiome-associated inflammation (consortia of bacteria paradigm) versus immune response to specific bacteria (single-species paradigm) as microbial triggers of unbalanced mucosal immunity that can initiate colon carcinogenesis and/or promote tumor progression.

The impact of the human microbiome on health and disease is of utmost importance and has been studied intensively in recent years. Microbes promote immune system development and are essential to the production and absorption of nutrients for the host but are also implicated in disease pathogenesis. Particularly, bacterial biofilms have long been recognized as contributors to chronic infections and diseases in humans. However, our understanding of how the host responds to the presence of biofilms, specifically the immune response to biofilms, and how this contributes to disease pathogenesis is limited. This review aims to highlight what is known about biofilm formation and in vivo models available for the biofilm study. We critique the contribution of biofilms to human diseases, focusing on the lung diseases, cystic fibrosis and chronic obstructive pulmonary disease, and the gut diseases, inflammatory bowel disease and colorectal cancer.

Purpose: Approximately 10% of patients with mismatch repair–proficient (MMRp) colorectal cancer showed clinical benefit to anti-PD-1 monotherapy (NCT01876511). We sought to identify biomarkers that delineate patients with immunoreactive colorectal cancer and to explore new combinatorial immunotherapy strategies that can impact MMRp colorectal cancer. Experimental Design: We compared the expression of 44 selected immune-related genes in the primary colon tumor of 19 patients with metastatic colorectal cancer (mCRC) who responded (n = 13) versus those who did not (n = 6) to anti-PD-1 therapy (NCT01876511). We define a 10 gene–based immune signature that could distinguish responder from nonresponder. Resected colon specimens (n = 14) were used to validate the association of the predicted status (responder and nonresponder) with the immune-related gene expression, the phenotype, and the function of tumor-infiltrating lymphocytes freshly isolated from the same tumors. Results: Although both IL17Low and IL17High immunoreactive MMRp colorectal cancers are associated with intratumor correlates of adaptive immunosuppression (CD8/IFNγ and PD-L1/IDO1 colocalization), only IL17Low MMRp tumors (3/14) have a tumor immune microenvironment (TiME) that resembles the TiME in primary colon tumors of patients with mCRC responsive to anti-PD-1 treatment. Conclusions: The detection of a preexisting antitumor immune response in MMRp colorectal cancer (immunoreactive MMRp colorectal cancer) is not sufficient to predict a clinical benefit to T-cell checkpoint inhibitors. Intratumoral IL17-mediated signaling may preclude responses to immunotherapy. Drugs targeting the IL17 signaling pathway are available in clinic, and their combination with T-cell checkpoint inhibitors could improve colorectal cancer immunotherapy. See related commentary by Willis et al., p. 5185

Bile acids (BAs) play a complex role in colonic fluid secretion. We showed that dihydroxy BAs, but not the monohydroxy BA lithocholic acid (LCA), stimulate Cl− secretion in human colonic T84 cells (Ao M, Sarathy J, Domingue J, Alrefai WA, Rao MC. Am J Physiol Cell Physiol 305: C447–C456, 2013). In this study, we explored the effect of LCA on the action of other secretagogues in T84 cells. While LCA (50 μM, 15 min) drastically (>90%) inhibited FSK-stimulated short-circuit current ( Isc), it did not alter carbachol-stimulated Isc. LCA did not alter basal Isc, transepithelial resistance, cell viability, or cytotoxicity. LCA's inhibitory effect was dose dependent, acted faster from the apical membrane, rapid, and not immediately reversible. LCA also prevented the Isc stimulated by the cAMP-dependent secretagogues 8-bromo-cAMP, lubiprostone, or chenodeoxycholic acid (CDCA). The LCA inhibitory effect was BA specific, since CDCA, cholic acid, or taurodeoxycholic acid did not alter FSK or carbachol action. While LCA alone had no effect on intracellular cAMP concentration ([cAMP]i), it decreased FSK-stimulated [cAMP]i by 90%. Although LCA caused a small increase in intracellular Ca2+ concentration ([Ca2+]i), chelation by BAPTA-AM did not reverse LCA's effect on Isc. LCA action does not appear to involve known BA receptors, farnesoid X receptor, vitamin D receptor, muscarinic acetylcholine receptor M3, or bile acid-specific transmembrane G protein-coupled receptor 5. LCA significantly increased ERK1/2 phosphorylation, which was completely abolished by the MEK inhibitor PD-98059. Surprisingly PD-98059 did not reverse LCA's effect on Isc. Finally, although LCA had no effect on basal Isc, nystatin permeabilization studies showed that LCA both stimulates an apical cystic fibrosis transmembrane conductance regulator Cl− current and inhibits a basolateral K+ current. In summary, 50 μM LCA greatly inhibits cAMP-stimulated Cl− secretion, making low doses of LCA of potential therapeutic interest for diarrheal diseases.

Bile acids are known to initiate intricate signaling events in a variety of tissues, primarily in the liver and gastrointestinal tract. Of the known bile acids, only the 7α-dihydroxy species, deoxycholic acid and chenodeoxycholic acid (CDCA), and their conjugates, activate processes that stimulate epithelial Cl−secretion. We have previously published that CDCA acts in a rapid manner to stimulate colonic ion secretion via protein kinase A (PKA)-mediated activation of the dominant Cl−channel, the cystic fibrosis transmembrane conductance regulator (CFTR) (Ao M, Sarathy J, Domingue J, Alrefai WA, and Rao MC. Am J Physiol Cell Physiol 305: C447–C456, 2013); however, PKA signaling did not account for the entire CDCA response. Here we show that in human colonic T84 cells, CDCA's induction of CFTR activity, measured as changes in short-circuit current ( Isc), is dependent on epidermal growth factor receptor (EGFR) activation and does not involve the bile acid receptors TGR5 or farnesoid X receptor. CDCA activation of Cl−secretion does not require Src, mitogen-activated protein kinases, or phosphoinositide 3-kinase downstream of EGFR but does require an increase in cytosolic Ca2+. In addition to PKA signaling, we found that the CDCA response requires the novel involvement of the exchange protein directly activated by cAMP (EPAC). EPAC is a known hub for cAMP and Ca2+cross talk. Downstream of EPAC, CDCA activates Rap2, and changes in free cytosolic Ca2+were dependent on both EPAC and EGFR activation. This study establishes the complexity of CDCA signaling in the colonic epithelium and shows the contribution of EGFR, EPAC, and Ca2+in CDCA-induced activation of CFTR-dependent Cl−secretion.

ever since its discovery in 1989, the cystic fibrosis transmembrane conductance regulator has proven time and again that its name as a regulator and moniker, CFTR, are fully justified. The pleiotropic effects of CFTR continue to ingratiate themselves into the intricacies of cell regulation. In a

Epithelial apoptosis and tight junction (TJ) changes contribute to intestinal barrier dysfunction in Crohn's disease and colitis. We have reported that in T84 monolayers, CDCA decreased transepithelial resistance (TER) and caused dose- and time-related cytotoxicity. CDCA increased cascade blue 10kDa dextran (CB10D) flux from apical to basal surfaces and this was enhanced by pro-inflammatory cytokines (PiC: TNFα+IL-1s+IFNγ; FASEB J:28, 1113.3, '14). In this study we examined if LCA, a dehydroxylated product of CDCA, had similar effects. Confluent T84 cells (TER >1000 Ω.cm2) were treated with 5-500µM LCA for 1 and 24 hr ± PiCs. Cell viability and apoptosis (Accuri flow cytometry), toxicity (lactate dehydrogenase), and paracellular permeability (TER and CB10D fluxes) were assessed. One hour or overnight (O/N) LCA (5 – 500μM) exposure neither affected cell viability nor apoptosis. LCA (1 Hr or O/N) did not increase LDH release and therefore was not cytotoxic. Time-dependent studies (0.5 – 18 Hr) show that LCA...

We recently showed that the BA chenodeoxycholate (CDCA) stimulates CFTR-mediated Cl- secretion in T84 cells via protein kinase A (PKA; AJP 305:C447-56, '13). We now demonstrate that CDCA signaling ...

The Human Embryonic Kidney 293 cell line (HEK‐293) readily lends itself to genetic manipulation and is a common tool for biologists to overexpress proteins of interest and study their function and molecular regulation. Although these cells have some limitations, such as an inability to form resistive monolayers necessary for studying transepithelial ion transport, they are nevertheless valuable in studying individual epithelial ion transporters. We report the use of HEK‐293 cells to study the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. While HEK‐293 cells endogenously express mRNA for the Cl− channels, ClC‐2 and TMEM16A, they neither express CFTR mRNA nor protein. Therefore, we stably transfected HEK‐293 cells with EGFP‐CFTR (HEK‐CFTR) and demonstrated CFTR function by measuring forskolin‐stimulated iodide efflux. This efflux was inhibited by CFTRinh172, and the protein kinase A inhibitor H89, but not by Ca2+ chelation. In contrast to intestinal epithelia, forskolin stimulation does not increase surface CFTR expression and does not require intact microtubules in HEK‐CFTR. To investigate the role of an endogenous GαS‐coupled receptor, we examined the bile acid receptor, TGR5. Although HEK‐CFTR cells express TGR5, the potent TGR5 agonist lithocholic acid (LCA; 5–500 μmol/L) did not activate CFTR. Furthermore, forskolin, but not LCA, increased [cAMP]i in HEK‐CFTR suggesting that endogenous TGR5 may not be functionally linked to GαS. However, LCA did increase [Ca2+]i and interestingly, abolished forskolin‐stimulated iodide efflux. Thus, we propose that the stable HEK‐CFTR cell line is a useful model to study the multiple signaling pathways that regulate CFTR., In this study, we characterize a HEK‐293 cell line, stably transfected with EGFP‐CFTR (HEK‐CFTR). We examined its regulation by endogenously expressed signaling pathways, in particular the cAMP and the GαS‐coupled bile acid receptor, TGR5, signaling pathways.

A role for EGFR signaling in BA action has been well described in colon cancer, but not as well in BA induced Cl- secretion. Our preliminary report (EB LB 10695, ‘13) showed that the EGFR inhibitor, AG1478, reduced CDCA (500 µM) mediated Cl- secretion by 69%, measured as short circuit current (Isc, µA.cm-2), in T84 cells. Here we aim to further elucidate this mechanism. To determine if BA action involves matrix metalloproteinases (MMP), CDCA (500 µM) was added to T84 cells ± batimastat (BAT, 20 µM, 30’). CDCA itself increases Isc in 2-3’ and the effect is sustained for > 20’. BAT inhibited the sustained phase (∆Isc: CDCA: 11.6±1.4; +BAT: 3.9±2.6, n=3). Interestingly the PKA inhibitor H89 has a similar effect (∆Isc: CDCA: 21±4.6; +H89 (30 µM): 1±0.3, n=5), suggesting that EGF and cAMP are involved in sustaining CDCA-stimulated secretion. The distal steps of EGFR action do not involve Src or MEK as their respective inhibitors PP2 (10 µM, n=3), and PD98059 (20 µM, n=3) did not alter CDCA-induced Isc. Immunob...

High levels of chenodeoxycholic acid (CDCA) and deoxycholic acid stimulate Cl−secretion in mammalian colonic epithelia. While different second messengers have been implicated in this action, the specific signaling pathway has not been fully delineated. Using human colon carcinoma T84 cells, we elucidated this cascade assessing Cl−transport by measuring I−efflux and short-circuit current ( Isc). CDCA (500 μM) rapidly increases I−efflux, and we confirmed by Iscthat it elicits a larger response when added to the basolateral vs. apical surface. However, preincubation with cytokines increases the monolayer responsiveness to apical addition by 55%. Nystatin permeabilization studies demonstrate that CDCA stimulates an eletrogenic apical Cl−but not a basolateral K+current. Furthermore, CDCA-induced Iscwas inhibited (≥67%) by bumetanide, BaCl2, and the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh-172. CDCA-stimulated Iscwas decreased 43% by the adenylate cyclase inhibitor MDL12330Aand CDCA increases intracellular cAMP concentration. The protein kinase A inhibitor H89 and the microtubule disrupting agent nocodazole, respectively, cause 94 and 47% reductions in CDCA-stimulated Isc. Immunoprecipitation with CFTR antibodies, followed by sequential immunoblotting with Pan-phospho and CFTR antibodies, shows that CDCA increases CFTR phosphorylation by approximately twofold. The rapidity and side specificity of the response to CDCA imply a membrane-mediated process. While CDCA effects are not blocked by the muscarinic receptor antagonist atropine, T84 cells possess transcript and protein for the bile acid G protein-coupled receptor TGR5. These results demonstrate for the first time that CDCA activates CFTR via a cAMP-PKA pathway involving microtubules and imply that this occurs via a basolateral membrane receptor.