{"paper_id":"16b8da40-6748-44b4-9a96-76c48c4aa68f","body_text":"Discovery and characterization of Christensenella hongkongensis as a novel bacterium in the adenoma-carcinoma progression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Discovery and characterization of Christensenella hongkongensis as a novel bacterium in the adenoma-carcinoma progression Wenqing Zhang, Qi Su, Haiyun Shi, Yang Sun, Xiaobo Li, Mengbin Li, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7436037/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Journal of Translational Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Background Colorectal cancer (CRC) is one of the most prevalent malignancies worldwide and commonly starts from a pre-cancerous stage. This study aimed to identify potential fecal bacterial candidates associated with progression of CRC from the adenoma-carcinoma sequence and to explore underlying mechanisms of carcinogenesis. Methods Publicly metagenomic datasets were analyzed using MaAsLin2 to identify bacterial species enriched in CRC patients compared to healthy controls. Additionally, we established a large cohort in mainland China, consisting of 686 subjects (285 CRC patients, 73 advanced adenoma patients, 134 non-advanced adenoma patients, and 194 healthy controls). Fecal samples from this cohort were analyzed by duplex quantitative polymerase chain reaction (qPCR) to validate the abundance of key bacterial candidate and its association with tumor node metastasis (TNM) stages. In vitro experiments and transcriptome sequencing were performed to explore the effects of Christensenella hongkongensis ( C. hongkongensis ) and its mechanisms in CRC progression. Results MaAsLin2 analysis identified seven bacterial species were significantly more abundant in fecal samples of CRC patients than in healthy controls ( P < 0.05). Among them, Christensenella hongkongensis , an obligately anaerobic, catalase-positive, motile, non-sporulating, gram-positive coccobacillus was distinguished by its lowest abundance in healthy controls and significant enrichment in CRC patients. Validation in our recruited cohort showed that the abundance of C. hongkongensis progressively increased from non-advanced adenomas to advanced adenomas and CRC. Linear regression analysis revealed a significant positive association between C. hongkongensis and TNM stages in CRC. In vitro experiments showed that C. hongkongensis promoted CRC cell proliferation, inhibited apoptosis, and enhanced the growth of patient-derived CRC organoids. RNA-seq analysis identified activation of the Wnt/β-catenin signaling pathway, which was further validated by the upregulation of downstream targets such as c-Jun and Cyclin-D1. Conclusions Our findings suggest that C. hongkongensis promotes colorectal tumorigenesis via Wnt/β-catenin activation, and highlights its potential as a novel non-invasive bacteria marker for early detection and monitoring of CRC progression. Colorectal adenoma biomarker gut microbiota Christensenella hongkongensis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Colorectal cancer (CRC) is the third most common malignancy and the second leading cause of cancer-related mortality worldwide, with its incidence continuing to rise and imposing substantial medical and economic burdens [ 1 , 2 ]. Most cases of CRC originate from precursor lesions, including non-advanced adenomas, advanced adenomas, and serrated polyps, each carrying distinct risks of progression to cancer [ 3 ]. Among these, advanced adenomas present a significantly higher risk of developing into CRC compared to non-advanced adenomas [ 4 ]. Early detection and endoscopic removal of precursor lesions are effective strategies for markedly reducing CRC risk [ 5 ]. Although the fecal immunochemical test (FIT) is the most widely used non-invasive method for early CRC detection, its sensitivity for identifying advanced adenomas remains suboptimal, ranging from 25 to 42% [ 3 ]. Consequently, there is an urgent need for novel biomarkers with greater accuracy to improve the detection of precancerous lesions and further reduce the incidence and mortality of CRC [ 6 , 7 ]. Accumulating evidence, including findings from animal studies, has linked dysregulation of the gut microbiota to the progression of CRC, which is characterized by reduced microbial diversity and expansion of pathobiont [ 8 ]. Microbial depletion, achieved either through germ-free conditions [ 9 ] or antibiotic intervention [ 10 ], significantly reduces colorectal tumor formation compared to conventional microbiota. Pathogenic bacteria such as Fusobacterium nucleatum ( F. nucleatum ) [ 11 ], Peptostreptococcus stomatis [ 12 ], pks + Escherichia coli [ 13 ], and Enterotoxigenic Bacteroides fragilis [ 14 ] are enriched in CRC patients relative to healthy controls. These pathobionts contribute to CRC initiation and progression through multiple mechanisms, including the production of carcinogenic genotoxins and tumorigenic metabolites, bacterial adhesin-host cell receptor interactions, genetic and epigenetic modifications, and the induction of inflammation [ 8 ]. These findings underscore the crucial role of the gut microbiota in CRC pathogenesis and highlights its potential as a potential biomarker for non-invasive CRC diagnosis. Stool-based bacterial markers have been shown to serve as non-invasive diagnostic tools for early CRC detection via targeted quantification using quantitative polymerase chain reaction (PCR) [ 15 ]. For example, F. nucleatum and the novel gene marker “m3” from Lachnoclostrium sp . are significantly enriched during the progression from colorectal adenoma to CRC, showing good diagnostic performance for CRC [ 5 ]. However, their accuracy for adenoma diagnosis decreases significantly to 48%, and “m3” does not significantly distinguish between non-advanced and advanced adenomas [ 5 ].These findings highlight the current limitations on biomarkers discovery for effective colorectal adenoma detection. Hence, this study aims to identify potential bacterial marker for early CRC detection through metagenomic sequencing analysis and qPCR validation, and to explore its molecular mechanisms in promoting CRC development via in vitro experiments. The findings of this study are expected to contribute to the development of new diagnostic tool for tracking the adenoma-to-carcinoma progression in colorectal cancer and provide valuable insights for its prevention and treatment. Materials and methods Data download and metagenomic analysis A total of 1 656 fecal metagenomic sequencing samples from colorectal cancer patients and healthy controls, were collected from three datasets conducted in Hong Kong and Japan. The Hong Kong cohort (174 CRC, 893 controls) derived from our previous study was used as the discovery cohort. Two public cohorts from Japan were designated as validation cohort 1 (40 CRC, 40 controls) and validation cohort 2 (251 CRC, 258 controls). Bacterial taxonomic profiles were generated using MetaPhlAn 3 [ 42 ] and accessed by the curated Metagenomic Data package [ 43 ] in R. Differential species between CRC patients and controls were identified by MaAslin2 across all cohorts. The abundance of C. hongkongensis was specifically compared using Mann-Whitney test. The characteristics of all datasets are provided in Supplementary Table 1 . Human subject recruitment Participants were included if they were between 40 and 75 years old and had undergone a standard colonoscopy. Participants were excluded from the study if they had active gastrointestinal or had used antibiotics within the past month. Healthy controls are individuals with normal colorectal mucosa. Subjects with colorectal adenoma and CRC were diagnosed through colonoscopy and confirmed by histological examination. The TNM stage for CRC patients was assessed by the AJCC (American Joint Committee on Cancer) TNM system. Finally, we recruited 686 consecutive subjects, including 285 CRC, 73 advanced adenomas, 133 non-advanced adenomas and 194 healthy controls) from five different sites (Guangzhou, Beijing, Shanghai, Kunming and Xi’an) of mainland China. Advanced adenomas (AA) were defined as adenomas with any of the following criteria: size > 10 mm, more than three adenomas, the presence of a tubulovillous or villous component, or high-grade intraepithelial dysplasia. Non-advanced adenomas were defined as 1–2 adenomas, each < 10 mm in size [ 44 ]. Tumor anatomic locations were typically divided into three categories: rectum, proximal colon, and distal colon. The rectum refers to the terminal segment of the larger intestine. The proximal colon includes the ileocecal junction, ascending colon, hepatic flexure and transverse colon. The distal colon includes the splenic flexure, descending colon and sigmoid colon. Clinical characteristics of healthy subjects and colorectal adenoma/cancer patients are shown in Supplementary Table 2 . Fecal sample collection Fecal samples were collected within one month prior to the scheduled colonoscopy and before any bowel preparation. The fresh fecal sample was collected from each participant using a sealed, sterile collection tube containing preservative media (Norgen Biotek Corp, Thorold, Ontario Canada). All samples were kept capped when not in use to prevent cross-contamination. Following collection, samples were sent to the lab within 24 h and stored at -80°C until analysis. Fecal DNA extraction For mainland China cohort, fecal DNA was extracted following manufacturer’s instruction of BayBiopure Magnetic Stool Nucleic Acid Kit (Baybio, Guangzhou, China). The extracted DNA was quantified by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). After final precipitation, the DNA samples were resuspended in DE buffer and stored at − 80°C before further analysis. Fecal samples duplex quantitative PCR Duplex quantitative PCR (qPCR) was performed using the Applied Biosystems™ 7500 system to quantify the abundance of C. hongkongensis in fecal samples from mainland China cohort. Each 20 µL reaction contained 10.2 µL Premix Ex Taq™ (TAKARA, Tokyo, Japan), 0.4 µL each of C. hongkongensis forward and reverse primers (10 µM), 0.2 µL C. hongkongensis probe labeled with FAM (10 µM), 0.4 µL each of 16S forward and reverse primers (10 µM), 0.2 µL 16S probe labeled with VIC (10 µM), 5.8 µL nuclease-free water, and 2.0 µL template DNA (5 ng/µL). The qPCR program consisted of a pre-denaturation step at 95 ℃ for 30 s, followed by 40 cycles of denaturation at 95 ℃ for 5 s and annealing/extension at 60 ℃ for 34 s. Ct analysis was performed with a threshold of 0.07 and a baseline set between cycles 3 and 15. The sequences of the gene primers are provided in Supplementary Table 3 . Bacteria culture C. hongkongensis (DSM18959) was purchased from Leibniz Institute DSMZ-Germany Collection of Microorganisms and Cell Cultures GmbH and cultured at 37℃ in Peptone-Yeast Extract-Glucose (PYG) broth with Hungate bottles filled with nitrogen. A nonpathogenic human commensal intestinal bacterium, Escherichia coli MG1665 was used as bacterial control and cultured at the same condition as C. hongkongensis . When the absorbance of C. hongkongensis reached optical density 600 of 0.3, the conditioned medium was centrifuged and filtered with 0.22 µm pore size filter to collect C. hongkongensis conditioned medium (Chk.CM) or E. coli conditioned medium (E. coli.CM). Cell culture Normal human colon epithelial cell NCM460, human CRC cell lines HCT116 and CaCo-2 were obtained from ATCC (Rockville, MD, USA). All cell lines were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) in humidified atmosphere containing 5% CO 2 . Human CRC organoid culture CRC patient-derived organoid was obtained from Prof Nathalie Wong’s laboratory, isolating from a 75-year-old male colorectal adenocarcinoma patient. The CRC organoids were activated and embedded into Matrigel and maintained in Advanced DMEM/F12, supplied with 1X P/S, 10 mM HEPES, 1×GlutaMAX medium containing 1X N2 and B27 supplements, 100 ng/mL R-spondin-1, 100 ng/mL Noggin, 50 ng/mL EGF, 10nM Gastrin, 100 ng/mL FGF10, 1.25 mM N-acetylcysteine, 500 nM A8301, 10 µM SB202190, 10 mM Nicotinamide, 100 µg/mL Primocin and 10 µM Y27632. Treatment such as 5% (vol/vol) PYG or Chk.CM was added into the culture medium directly, which was freshly changed every three days. Surface area of organoid was measured by Image J 1.54g. Cell proliferation assay CaCo-2, HCT116 and NCM460 cells (1 × 10 3 cells per well) were seeded onto 96-well plates and treated with live E. coli (CFU = 10 6 /mL) or C. hongkongensis (CFU = 10 6 /mL), as well as 5% (vol/vol) PYG, E. coli.CM, or Chk.CM in DMEM. Cell viability was determined by Cell Counting Kit-8 (CCK8, MedChemEXpress, Monmouth Junction, NJ, USA; Hy-K0301) for 3 consecutive days. Cell apoptosis assay CaCo-2, HCT116 and NCM460 cells (5 × 10⁵ cells per well) were separately seeded in a 12-well plate and incubated until reaching approximately 80–90% confluence. Cells were infected with C. hongkongensis (MOI = 100) for 4 hours under anaerobic conditions. Following infection, cells were washed 2–3 times with 1X PBS and replaced with DMEM supplemented with antibiotics to prevent further bacterial growth. After 48 hours post-infection, cells were washed twice with cold 1X PBS and detached by adding 0.5 mL trypsin without EDTA to each well. The cell suspension was collected and centrifuged at 1 000 rpm for 5 minutes at 4°C. The resulting cell pellet was resuspended in 1X Annexin V bindng buffer to achieve a final concentration of 1 × 10⁶ cells/mL. A 100 µL aliquot of the resuspended cells (1 × 10⁵ cells) was transferred to a 5 mL flow cytometry tube. Cells were stained using the BD Pharmingen™ PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. After staining, 400 µL of 1X binding buffer was added to each tube, and samples were analyzed immediately by flow cytometry. Wound healing assay CaCo-2 and HCT116 cells (1 × 10 3 cells per well) were seeded in 6-well plates and cultured until confluence. A uniform scratch was carefully made in the cell monolayer using a sterile 200 µL pipette tip, followed by washing with PBS to remove debris. The cells were then incubated in serum-free medium under the following conditions: (1) live C. hongkongensis (MOI = 100), (2) serum-free medium (blank control for C. hongkongensis ), (3) 5% Chk.CM, and (4) 5% PYG medium (negative control for Chk.CM). Images of the scratch areas were captured at 0 h and 12 h, and scratch widths were measured using ImageJ software to assess wound closure. Colony formation assay HCT116 and CaCo-2 cells (1 × 10 3 cells per well) were seeded in 6-well plates. For live C. hongkongensis treatment, cells were co-cultured with live C. hongkongensis (MOI = 100) for 4 hours. Live E. coli MG1655 (MOI = 100) served as a negative control. After 4 hours, cells were washed three times with PBS and fresh DMEM medium was added. For C. hongkongensis treatment, cells were cultured with 5% Chk.CM, while 5% PYG medium and E. coli.CM were used as blank and negative controls, respectively. The treatment medium was refreshed every 3 days. After 10–14 days, cells from both treatments were fixed with 4% PFA and stained with 0.1% crystal violet. Colonies containing more than 50 cells were counted. All experiments were performed in triplicate and repeated three times. RNA sequencing analysis Total RNAs were extracted from the HCT116 and CaCo-2 cells after co-culturing with live C. hongkongensis (MOI = 100) or 5% Chk.CM respectively using Trizol Reagent (Thermo Fisher Scientific). RNA sequencing libraries were prepared with at least 6G raw data per sample (NovaSeq PE150, Novogene, Beijing, China). Raw sequencing data were qualified and cleaned using fastp [ 45 ] and TrimGalore. The processed reads were then aligned to the reference human genome (GRCh38) using Hisat2 [ 46 ]. Differential gene expression analysis was performed with DESeq2 [ 47 ], applying the thresholds of |Log2FoldChange|>1 and adjusted P -value < 0.05 to identify significantly differential genes. The identified differentially expressed genes were subjected to KEGG enrichment analysis using the ClusterProfiler [ 48 ]. Additionally, Gene Set Enrichment Analysis (GSEA) was conducted via the GSEA software [ 49 ] with default parameters using the KEGG pathway database. Finally, pathway visualizations of differential gene expression were generated using the Pathview [ 50 ]. RT-PCR cDNA was synthesized from total RNA by PrimeScript RT reagent Kit (RR037A, Takara, Tokyo, Japan) according to the manufacturer’s instructions. Quantitative PCR (qPCR) was conducted using TB Green® Premix Ex Taq™ II (TAKARA) on the QuantStudio 7 Flex System (Thermo Fisher Scientific). The sequences of the primers are listed in Supplementary Table 3 . Statistical analysis GraphPad Prism 10 Software and SPSS version 27.0 software were used for data analysis in this study. The experimental data are generally represented as mean ± SD, except for C. hongkongensis abundance, which is expressed as mean ± SE. Paired Student’s t-test and one-way ANOVA with Tukey’s multiple comparison test were used to evaluate the statistical significance of differences between two groups and among three groups, respectively. The graphical abstract was created with BioRender.com. Results Identification of the novel species C. hongkongensis enriched in patients with CRC To identify potential novel bacterial markers for CRC, we performed MaAsLin2 analysis on metagenomic sequencing data from three public datasets to determine differentially abundant bacterial species in fecal samples of CRC patients compared to healthy controls (Fig. 1 A). Among all identified differential bacterial species, seven CRC-enriched species were consistently observed across all three cohorts, including Ruthenibacterium lactatiformans , Alistipes shahii , Anaerotruncus colihominis , Peptostreptococcus stomatis , Parvimonas micra , Gemella morbillorum and Christensenella hongkongensis (all P < 0.05, Fig. 1 B and Fig. S1 ). Notably, C. hongkongensis demonstrated the lowest abundance in healthy controls among these seven species (Fig. 1 C). In each individual cohort, the relative abundance of C. hongkongensis was also significantly increased in CRC patients compared to healthy controls (all P < 0.05, Fig. 1 D). Furthermore, the prevalence of C. hongkongensis was consistently elevated in CRC patients compared to healthy controls across all three cohorts (Fig. 1 E). Collectively, we identified a novel bacterial species that is consistently enriched in CRC across the cohorts. The involvement of C. hongkongensis in CRC progression from adenoma to carcinoma To investigate the potential role of C. hongkongensis in CRC progression, we further quantitatively examined its abundance in an expanded cohort of fecal samples collected from healthy controls (NC, n = 194), patients with non-advanced adenomas (nAA, n = 134), patients with advanced adenomas (AA, n = 73) and those with CRC (n = 285) across five regions from mainland China (Guangzhou, Kunming, Beijing, Shanghai and Xi’an, n = 686) using our previously established duplex-qPCR platform (Fig. 2 A) [ 5 ]. Consistent with previous findings, a substantially enriched abundance of C. hongkongensis was observed in CRC patients compared to healthy controls ( P < 0.01, Fig. 2 B). More importantly, the abundance of C. hongkongensis progressively increased along with the CRC progression, from the nAA stage to the AA stage, and ultimately to carcinoma, with no significant differences detected between patients with nAA and healthy controls ( Fig. 2 B ) . Furthermore, among the CRC patients, the abundance of C. hongkongensis also demonstrated a continuous upward trend as the disease advanced from TNM stage I to TNM stage IV, reflecting a compelling correlation with tumor progression ( P < 0.01, Fig. 2 C ) . In addition to the abundance, the prevalence of C. hongkongensis also demonstrated a notable increase in CRC patients at the AA stage and those presenting with adenoma, in comparison to healthy controls and CRC patients at the nAA stage ( NC: 32.5%; nAA: 33.1%; AA: 45.9%; CRC: 44.6%, Fig. 2 D ) . However, no statistically significant differences were observed between CRC patients at the nAA stage and healthy controls ( Fig. 2 D ) . Notably, the abundance of C. hongkongensis exhibited a markedly elevated level in patients with distal CRC, compared to those with tumors located in proximal colon or rectal regions (both P < 0.05, Fig. 2 E). These compelling findings suggested the potential of C. hongkongensis as an early fecal biomarker, providing a valuable approach for the early detection of precursory lesions that may lead to CRC, particularly in patients with distal CRC. C. hongkongensis promotes CRC cell growth The progressively increased abundance of C. hongkongensis during CRC progression suggests that C. hongkongensis may play a carcinogenic role in CRC tumorigenesis. To prove this, we conducted the in vitro experiments using two CRC cell lines (CaCo-2 and HCT116) and a normal colon epithelial cell line (NCM460), co-cultured with C. hongkongensis and its conditioned medium. E. coli MG1655, a non-pathogenic strain of E. coli , was employed as a negative control. Both live C. hongkongensis and its 5% conditioned medium demonstrated the ability in enhancing the proliferation of two CRC cell lines (live C. hongkongensis : both P < 0.001; 5% conditioned medium: both P < 0.0001), yet neither exerted any influence on the growth of NCM460 cells (Fig. 3 A and 3 B). In keeping with this, the number of colonies which had formed in CaCo-2 and HCT116 cells cultured with C. hongkongensis or its conditioned medium were increased significantly compared with those treated with PBS or E. coli MG1655 ( Fig. 3 C and 3 D ) . In addition, flow cytometry utilizing dual staining with Annexin V-PE and 7-AAD was conducted to evaluate the effect of C. hongkongensis on cell apoptosis. The result exhibited an increase in the number of apoptotic cells in C. hongkongensis treated CaCo-2 cells compared with those treated with PBS (9.43 ± 0.52% vs 13.53 ± 1.01%, P < 0.01, Fig. 3 E). A similar effect was observed in C. hongkongensis cultured HCT116 cells, which demonstrated an increased proportion of cells in the apoptotic phase compared with cells co-cultured with PBS (18.69 ± 0.36% vs 20.93 ± 1.05%, P < 0.05, Fig. 3 F). Furthermore, the monolayer wound healing assay was performed to investigate the effects of C. hongkongensis on CRC cell migration. Administration of either C. hongkongensis or its conditioned medium significantly enhanced the cell migration capacity in both CaCo-2 and HCT116 cell lines ( Fig. 3 G and 3 H ) . Quantitative analyses at 12 hours post-wounding confirmed a significant increase in wound closure in cells administered with C. hongkongensis or its conditioned medium compared to control cells (29.22% and 26.25% in CaCo-2 cells; 30.95% and 38.15% in HCT116 cells, respectively), thereby suggesting that the migration rate of cells cultured with C. hongkongensis or its conditioned medium was significantly higher than that of the control cells. Collectively, these data suggest that C. hongkongensis and its conditioned medium exert pro-tumorigenic effects on CRC cells by enhancing proliferation, colony formation, and migration, while simultaneously inhibiting apoptosis. C. hongkongensis activates the tumorigenic signaling pathways in CRC To further elucidate the molecular mechanisms mediated by C. hongkongensis in executing its oncogenic functions, we conducted transcriptomic sequencing to identify the genes and signaling pathways regulated by live C. hongkongensis and its conditioned medium in CaCo-2 and HCT116 cells. Principal component analysis (PCA) demonstrated a clear separation among all three treatment groups in HCT116 cells (Fig. S2 A) , whereas cells treated with live C. hongkongensi s and its conditioned medium clustered closely together in CaCo-2 cells, yet remained distinctly separated from the blank controls ( Fig. S2 B ). The differentially expressed genes (DEGs) between C. hongkongensis -treated and PBS-treated CRC cells were identified using DESeq2 (Fig. 4 A). In CaCo-2 cells, co-culturing with live C. hongkongensis resulted in the upregulation of 203 genes and downregulation of 223 genes. While treatment with its conditioned medium significantly altered the expression of 939 genes, including 313 upregulated and 626 downregulated ( Fig. 4 A ) . Similarly, in HCT116 cells, treatment with live C. hongkongensis led to the upregulation of 1,960 genes and downregulation of 1,119 genes, whereas treatment with its conditioned medium resulted in 324 upregulated and 468 downregulated genes ( Fig. 4 A ) . Among all DEGs, 290 genes in CaCo-2 cells and 472 genes in HCT116 cells were consistently regulated in both live C. hongkongensis -treated and conditioned medium-treated cells compared to the control groups (Fig. S2 C and Fig. 4 B ) . Following KEGG pathway enrichment analysis, the Wnt signaling pathway and the Hippo signaling pathway were identified as activated in both CRC cell lines treated with live C. hongkongensis or its conditioned medium (Fig, 4C and Fig. S2 D) . Moreover, gene set enrichment analysis (GSEA) further confirmed that the Hippo signaling pathway and the Wnt/β-catenin signaling pathway were significantly upregulated in HCT116 cells treated with either live C. hongkongensis (Hippo signaling pathway: normalized enrichment score [NES] = 1.44, P = 0.009; Wnt/β-catenin signaling pathway: NES = 1.52, P = 0.145) or its conditioned medium (Hippo signaling pathway: NES = 1.20, P = 0.004; Wnt/β-catenin signaling pathway: NES = 1.36, P = 0.025, Fig. 4 D and 4 E). Additionally, PathView analysis visualized the gene expression changes in both Hippo signaling pathway and Wnt signaling pathway, revealing that activation of the Wnt/β-catenin signaling pathway represents a shared mechanism underlying the response in both cell lines ( Fig. S3 and S4 ). The Wnt/β-catenin signaling pathway serves as a critical regulator of intestinal homeostasis, governing essential cellular processes such as proliferation, differentiation, and migration, and has been strongly associated with the initiation and progression of CRC. We further examined the mRNA expression levels of key downstream target genes of Wnt/β-catenin signaling pathway, such as Cyclin-D1 and c-Jun . Consistent with the findings, the levels of both Cyclin-D1 and c-Jun were significantly increased in cells treated with live C. hongkongensis or its conditioned medium ( Fig. 4 F ) . These results therefore indicate that C. hongkongensis exerts its oncogenic function in CRC through the activation of Wnt/β-catenin signaling pathway. The conditioned medium of C. hongkongensis enhances growth of CRC patient-derived organoids via activation of Wnt/β-catenin signaling pathway The activation of the Wnt/β-catenin signaling pathway triggered by C. hongkongensis was further validated in CRC organoid derived from one CRC patient. The organoids were co-cultured with C. hongkongensis conditioned medium or fresh PYG medium. As shown in Fig. 5 A, the CRC organoids treated with the conditioned medium of C. hongkongensis exhibited a significantly greater size compared to the black control ( P < 0.05). Moreover, administration of the conditioned medium of C. hongkongensis significantly enhanced the proliferation rate of CRC organoids during the 5-day cultivation period ( P < 0.001), and the number of organoids was higher than that in the blank control group ( P < 0.05, Fig. 5 B and 5 C). We further validated the expression of pivotal downstream targets of the Wnt/β-catenin signaling pathway, Cyclin-D1 and c-Jun, in CRC organoids. The mRNA expression levels of both genes were significantly increased in CRC organoids treated with the conditioned medium of C. hongkongensis compared to the blank control group ( Fig. 5 D ) . These findings suggest that the secretome of C. hongkongensis is capable of enhancing the growth of CRC patient-derived organoids by activating the Wnt/β-catenin signaling pathway. Discussion Due to the high cost and invasive nature of standard diagnostic methods like colonoscopy and sigmoidoscopy, there is an urgent need to improve the sensitivity of non-invasive tests for detecting CRC [ 16 ]. Increasing evidence has revealed that alterations in gut microbiota composition are closely associated with CRC progression, suggesting that the gut microbiome represents a rich reservoir of potential biomarkers [ 17 ]. Among various approaches, shotgun metagenomic sequencing, due to its high taxonomic precision and functional resolution, has been widely employed to identify CRC associated microbial markers [ 18 ]. In this study, we analyzed fecal metagenomic sequencing data from our internal cohort and two public cohorts, identifying seven species significantly enriched in CRC patients compared to healthy controls. Among these, six species including Ruthenibacterium lactatiformans [ 19 ], Alistipes shahii [ 20 ], Anaerotruncus colihominis [ 21 ], Peptostreptococcus stomatis [ 22 ], Parvimonas micra [ 16 ] and Gemella morbillorum [ 23 ], have previously been reported as potential CRC biomarkers with varying diagnostic performances [ 24 ]. Notably, C. hongkongensis was distinguished by its lowest abundance in healthy controls and significant enrichment in CRC patients. Furthermore, another multi-omics analysis from CRC patients revealed that, at the genus level, Christensenella abundance continuously increased from early-stage colonic intramucosal carcinoma to advanced CRC stages [ 25 ]. These findings suggest the strong association of C. hongkongensis with CRC and its potential as a microbial marker. To validate the diagnostic potential of C. hongkongensis , we recruited a large cohort from mainland China and performed targeted quantitative PCR assay. Consistent with metagenomic data, the qPCR results confirmed a significant enrichment of C. hongkongensis in CRC patients compared to healthy controls, accompanied by a higher prevalence in the CRC group. Early detection during precancerous stages is critical for reducing CRC incidence and mortality [ 26 ]. However, most current biomarkers, including microbial species and genetic markers, primarily differentiate between colorectal adenomas or CRC and healthy controls, with limited ability to distinguish among precancerous stages. For example, Fusobacterium nucleatum and Peptostreptococcus anaerobius are significantly more abundant in CRC patients compared to healthy controls, but its level showed no significant differences between patients with advanced adenoma and controls, which may suggest its limited potential in detecting advanced adenoma [ 27 ]. Similarly, while the methylated gene SHOX2 has been proposed as a biomarker for CRC diagnosis, quantitative methylation analysis reveals no significant differences in SHOX2 methylation levels between nAA and AA patients [ 28 ]. Interestingly, in our recruited cohort, the abundance of C. hongkongensis progressively increased from non-advanced adenoma (nAA) to advanced adenoma (AA) and ultimately to CRC, with its levels in nAA patients significantly lower than those in AA and CRC patients. Additionally, the prevalence of C. hongkongensis was highest in AA patients, highlighting its potential as a promising biomarker for distinguishing pre-tumor stages. TNM stage remains a critical determinant of CRC survival rates [ 26 ], emphasizing the need for biomarkers capable of tracking disease progression. In our study, the abundance of C. hongkongensis positively correlated with TNM stages, suggesting its potential utility not only in early detection but also in monitoring disease advancement. Consistent with previous study showing that C. hongkongensis is enriched in left-sided CRC patients [ 29 ], we observed that the fecal levels of C. hongkongensis were significantly higher in distal CRC patients. Collectively, our findings underscore the potential of C. hongkongensis as a versatile bacterial marker, spanning early detection, disease staging, and CRC location. Currently, FIT is the most widely used non-invasive methods for CRC diagnosis [ 30 ]. Despite its widespread use, Thomas e.tal found that FIT showed lower sensitivity (23.8%) for detecting advanced precancerous lesions based on a cohort more than 9000 subjects [ 31 ]. Recent studies have demonstrated that combining FIT with bacterial biomarkers significantly enhances the ability to detect adenomas and CRC compared to using FIT alone [ 16 , 32 ]. For example, Fusobacterium nucleatum , a well-documented CRC-associated bacterium, has been shown to enhance the sensitivity and specificity of FIT when used in combination, especially for early-stage CRC detection [ 5 ]. Given the promising potential of bacterial biomarkers in complementing FIT, future studies could explore the utility of combining C. hongkongensis with FIT. Additionally, C. hongkongensis has been implicated in intestinal and biliary infections, with cases of C. hongkongensis -associated bacteremia occurring predominantly in CRC patients [ 33 ]. However, existing studies have not provided species-level evidence or functional validation for the role of C. hongkongensis in CRC progression. CRC progression involves several hallmarks, including uncontrolled cell proliferation, tumor suppressor gene inactivation, and metastasis [ 34 ]. Our in vitro studies revealed that C. hongkongensis and its conditioned medium enhance CRC cell proliferation and migration, while simultaneously inhibiting apoptosis. These findings suggest a pathogenic role for C. hongkongensis in CRC development, contrasting with the previously reported probiotic properties of the Christensenella family, whose abundance is negatively correlated with metabolic diseases such as obesity and type 2 diabetes (T2D) [ 35 ]. This discrepancy may be attributed to that C. hongkongensis represents a distinct phylogenetic branch within the Christensenella genus, suggesting that C. hongkongensis may possess unique functions that differ from other Christensenella species [ 36 ]. Metabolic profiling further supports this, revealing that C. hongkongensis produces fewer potential beneficial metabolites compared to other Christensenella species [ 36 ]. Future studies using animal models are needed to provide deeper insights into the role of C. hongkongensis in CRC progression. Additionally, identifying the specific metabolites responsible for its pathogenic effects warrants further investigation. Mechanistically, we found that C. hongkongensis promotes CRC progression by activating the Wnt/β-catenin signaling pathway. Dysregulation of this pathway, a hallmark of CRC, is characterized by hyperactivation of the proto-oncoprotein β-catenin, which drives the transcription of oncogenic target genes [ 37 ]. Cyclin-D1, a key regulators of cell cycle, can bind with cyclin-dependent kinases CDK4 and CDK6 to form a complex targeting transcriptional factor E2F1, which is responsible for inducing G1/S transition, leading to uncontrolled cell proliferation [ 38 ]. We discovered that Cyclin-D1 was upregulated in CRC cells or organoids in both treatments. Conversely, downregulated Cyclin-D1 has been shown to cause G1/S phase arrest [ 39 ]. In addition to Cyclin-D1 , c-Jun , a component of the AP-1 transcription factor complex, was also upregulated in response to C. hongkongensis or its conditioned medium. As a downstream effector of Wnt/β-catenin signaling, c-Jun promotes the transcription of genes involved in cell proliferation and survival [ 40 ]. Furthermore, activated c-Jun has been reported to enhance epithelial-mesenchymal transition (EMT), a critical process for tumor invasion and metastasis in gastric cancer [ 41 ]. While our findings suggest a strong link between C. hongkongensis and the activation of the Wnt/β-catenin pathway, further validation at the protein level is necessary to confirm pathway activation and its downstream effects. Conclusions In summary, this study characterizes a novel gut bacteria C. hongkongensis that increases in abundance from the adenoma-to-carcinoma sequence, highlighting its potential as an auxiliary marker for CRC, with potential applications in early detection, disease staging, and tumor localization. Mechanistically, C. hongkongensis promotes CRC progression via the Wnt/β-catenin signaling pathway, providing new insights into its pathogenic role. Future studies integrating animal models, metabolite profiling, and therapeutic interventions are warranted to fully elucidate the clinical and biological significance of C. hongkongensis in CRC. Abbreviations CRC Colorectal cancer TNM Tumor node metastasis FIT Fecal immunochemical test DEGs Differentially expressed genes GSEA Gene set enrichment analysis NES Normalized enrichment score nAA Non-advanced adenoma AA Advanced adenoma T2D Type 2 diabetes EMT Epithelial-mesenchymal transition PYG Peptone-Yeast Extract-Glucose FBS Fetal bovine serum CCK8 Cell counting kit 8 Declarations Ethic approval and consent to participate All subjects and clinical information used in this study were obtained under conditions of informed consent and with approval of the institutional review boards of each participating institute (Beijing Friendship Hospital, 2022-P2-084-01; The First Affiliated Hospital of Kunming Medical University, 2022-95-2; Renji Hospital, Ly2022-081-B; Xijing Hospital, KY2022181-C-1; and the Sixth Affiliated Hospital of Sun Yat-sen University, 2022ZSLYEC-436). Consent for publication Not applicable. Availability of data and materials The raw data of the RNA-seq analysis were deposited in the SRA database (PRJNA1280769). The data in this study are available within the article and related supplementary information files, or by inquiring the corresponding authors. Competing interests S.C.N. has served as an advisory board member for Pfizer, Ferring, Janssen and Abbvie and received honoraria as a speaker for Ferring, Tillotts, Menarini, Janssen, Abbvie and Takeda; has received research grants through her affiliated institutions from Olympus, Ferring and Abbvie; is a founder member, non-executive director, non-executive scientific advisor and shareholder of GenieBiome Ltd which is non-remunerative; is a shareholder of MicroSigX Diagnostic Holding Limited; is a founder member, non-executive Board Director, and non-executive scientific advisor of MicroSigX Biotech Diagnostic Limited, which is non-remunerative; and receives patent royalties through her affiliated institutions. FKLC serves as the Principal Investigator for the Faecal Microbiota Transplantation Service under the Hospital Authority (HA). He is a Board Director of EHealth Plus Digital Technology Ltd., an HA-owned subsidiary driving the eHealth+ programme to transform the Electronic Health Record Sharing System into a comprehensive digital healthcare platform and advance other IT initiatives within the eHealth ecosystem. Additionally, he is a Board Director of CUHK Medical Services Limited. FKLC is a shareholder of GenieBiome Holdings Limited and the co-founder, non-executive Board Chairman, and non-executive Scientific Advisor of its wholly owned subsidiary, GenieBiome Ltd. Similarly, he is a shareholder of MicroSigX Diagnostic Holding Limited and the co-founder, non-executive Board Chairman, and non-executive Scientific Advisor of its wholly owned subsidiary, MicroSigX Biotech Diagnostic Limited. He also serves as a Director of the Hong Kong Investment Corporation Limited and a member of the Steering Committee for the RAISe+ Scheme under the Innovation and Technology Commission. Furthermore, he is the Co-Director of the Microbiota I-Center (MagIC) Ltd. FKLC receives advisory fees and speaker honoraria from AstraZeneca and Comvita New Zealand Limited, as well as patent royalties through affiliated institutions for microbiome-related applications. QS is Scientists (Diagnostics) of GenieBiome Ltd. JZ is Chief Scientist (Diagnostics) of GenieBiome Ltd. The other authors declare no competing interests. Funding This study is funded by InnoHK, The Government of Hong Kong, Special Administrative Region of the People’s Republic of China, Research Grants Council–Research Impact Fund (RGC-RIF, R4030-22), New Cornerstone Science Foundation (NCI202346), Research Grants Council-General Research Fund (RGC-GRF, 14121322), Health and Medical Research Fund (10210816), National Natural Science Foundation of China (82100573), Hong Kong Research Grants Council Areas of Excellence Scheme (Ref. AoE/M-401/20) and Leona M. and Harry B. Helmsley Charitable Trust (2017PG-IBD003). Authors affiliated with MagIC are partially supported by InnoHK, The Government of Hong Kong, Special Administrative Region of the People’s Republic of China. Author’s contributions S.C.N and JZ conceived this study. HS, YS, XL, ML, and HW were responsible for recruiting colorectal adenoma and CRC patients, collecting fecal samples, and performing colonoscopies. WN provided CRC-patients derived organoid. WZ and QS analyzed metagenomic sequencing data obtained from public datasets. WZ and JZ developed the methodology and performed the experiments. WZ drafted the manuscript, while WN, JY, FKLC, JZ, and S.C.N reviewed, edited, and supervised the study. Acknowledgements The authors thank Prof. Nathalie Wong and Prof. Yujuan Dong for providing CRC patient-derived organoids, which were invaluable for this study. References Arnold M, Sierra MS, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global patterns and trends in colorectal cancer incidence and mortality. Gut. 2017 Apr 1;66(4):683–91. Morgan E, Arnold M, Gini A, Lorenzoni V, Cabasag CJ, Laversanne M, et al. Global burden of colorectal cancer in 2020 and 2040: incidence and mortality estimates from GLOBOCAN. Gut. 2023 Feb;72(2):338–44. Bresalier RS, Senore C, Young GP, Allison J, Benamouzig R, Benton S, et al. An efficient strategy for evaluating new non-invasive screening tests for colorectal cancer: the guiding principles. 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Supplementary Files SupplementaryTable1.docx SupplementaryTable2.xlsx SupplementaryTable3.docx ClincalmetadatamainlandChina.pdf Supplementaryfigures.pdf GraphicalAbstract.pdf Cite Share Download PDF Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Journal of Translational Medicine → Version 1 posted Reviewers agreed at journal 07 Sep, 2025 Reviewers invited by journal 02 Sep, 2025 Editor assigned by journal 25 Aug, 2025 First submitted to journal 22 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-7436037\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":509096187,\"identity\":\"6fd2ce92-5267-49dc-8d30-c291380e28a7\",\"order_by\":0,\"name\":\"Wenqing Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"The Chinese University of Hong Kong\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Wenqing\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":509096188,\"identity\":\"600cee8f-b907-4736-a314-925717c605d9\",\"order_by\":1,\"name\":\"Qi 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\\u003c/em\\u003e\\u003cstrong\\u003eA \\u003c/strong\\u003eWorkflow of metagenomic analysis conducted using one in-house Hong Kong dataset and two public datasets.\\u003cstrong\\u003e \\u003c/strong\\u003eDifferentially abundant species were identified across the three cohorts using MaAslin2\\u003cstrong\\u003e. B \\u003c/strong\\u003eUpset plot showing 7 identified differential species shared across 3 cohorts identified by MaAslin2 analysis. \\u003cstrong\\u003eC\\u003c/strong\\u003eComparison of the overall relative abundance of these 7 species in\\u003cem\\u003e \\u003c/em\\u003ehealthy controls across the combined data from the three cohorts. \\u003cstrong\\u003eD\\u003c/strong\\u003e Comparison of the relative abundance of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e between CRC patients and healthy controls in each individual cohort. \\u003cstrong\\u003eE\\u003c/strong\\u003e Comparison of the prevalence of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e between CRC patients and healthy controls in each cohort. Data are presented as mean±SEM, with statistical differences between groups assessed using the two-sided Mann-Whitney test. Sample sizes (n) are indicated at the base of each bar.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"fig1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7436037/v1/82fd7e604a20ad6b0651ac89.jpg\"},{\"id\":91305712,\"identity\":\"51036fe3-2bdf-4dcf-9bb2-5eaeb12d8d90\",\"added_by\":\"auto\",\"created_at\":\"2025-09-15 06:29:05\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":326145,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe involvement of \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eC. hongkongensis\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e in CRC progression from adenoma to carcinoma. A \\u003c/strong\\u003eWorkflow illustrating the quantitation of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e abundance in patients with non-advanced adenoma (nAA), advanced adenoma (AA), CRC and healthy controls (NC) from mainland China. Clinical metadata, including demographics, fecal samples, colonoscopy results, and histology, were collected for analysis. \\u003cstrong\\u003eB\\u003c/strong\\u003eQuantitative analysis of the relative abundance of \\u003cem\\u003eC. hongkongensis \\u003c/em\\u003eamong different groups (NC, nAA, AA, and CRC) from mainland China using qPCR. \\u003cstrong\\u003eC \\u003c/strong\\u003eLinear regression analysis showing a positive association between the relative abundance of \\u003cem\\u003eC. hongkongensis \\u003c/em\\u003eand TNM stages in CRC patients. \\u003cstrong\\u003eD\\u003c/strong\\u003eComparison of the prevalence of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e among nAA, AA, CRC and NC groups. \\u003cstrong\\u003eE \\u003c/strong\\u003eDistribution of fecal \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e levels across different colon locations (proximal, distal colons and rectum) in CRC patients.\\u003cstrong\\u003e \\u003c/strong\\u003eData are presented as mean±SEM, with statistical differences between groups assessed using the two-sided Mann Whitney test. (NS, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026gt;0.05). The Chi-square test was used to evaluate the prevalence of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e across groups, Simple regression analyses were used to estimate the association between \\u003cem\\u003eC. hongkongensis \\u003c/em\\u003elevels and factors of interest.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"fig2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7436037/v1/9f668395e7aa10eafe42ad6a.jpg\"},{\"id\":91306944,\"identity\":\"a9fb15da-023a-49ef-95ab-fea967dbdf32\",\"added_by\":\"auto\",\"created_at\":\"2025-09-15 06:37:05\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1151353,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003e\\u003cstrong\\u003eC. hongkongensis\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003epromotes CRC cell growth\\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003e. \\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003eA \\u003c/strong\\u003eEffects of live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e (MOI=100) on the proliferation of CRC cells (CaCo-2, HCT116) and normal colon epithelial cells (NCM460). \\u003cem\\u003eE. coli MG1655\\u003c/em\\u003ewas used as a negative control.\\u003cstrong\\u003e B \\u003c/strong\\u003eEffects of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e conditioned medium (Chk.CM, 5% \\u003cem\\u003ev/v\\u003c/em\\u003e) on CRC cell viability, compared to \\u003cem\\u003eE. coli\\u003c/em\\u003e conditioned medium (E. coli.CM) and peptone yeast extract glucose (PYG) medium as blank controls. \\u003cstrong\\u003eC and D \\u003c/strong\\u003eColony formation assays showing the effects of live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e and Chk.CM on CaCo-2 (C) and HCT116 (D) cells compared to blank controls. \\u003cstrong\\u003eE and F \\u003c/strong\\u003eCell apoptosis analysis in CaCo-2 (E) and HCT116 (F) cells using Annexin V/7-AAD staining and flow cytometry after 48 hours of co-culture with live \\u003cem\\u003eC. hongkongensis \\u003c/em\\u003e(MOI=100, 4-hour infection).\\u003cstrong\\u003e G and H \\u003c/strong\\u003eWound healing assay results showing the migration rates of CaCo-2 and HCT116 cells after 12 hours of treatment with either live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e (G) or 5% Chk.CM (H).\\u003cstrong\\u003e \\u003c/strong\\u003eData are presented as mean±SD. Statistical significance was determined by two-way ANOVA (A), one-way ANOVA with Fisher’s LSD test (B, C and D), and unpaired t-test (E, G and H). *\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.05, **\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.01, ***\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.001; NS, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026gt;0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"fig3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7436037/v1/4b0a7d1aa9a885139ce6ca61.jpg\"},{\"id\":91305713,\"identity\":\"c042a459-0266-4b47-91bf-18ddf19a4c9e\",\"added_by\":\"auto\",\"created_at\":\"2025-09-15 06:29:05\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":643653,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003e\\u003cstrong\\u003eC. hongkongensis\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e activates the tumorigenic signaling pathways in CRC. A \\u003c/strong\\u003eMulti-group volcano plot displaying significantly upregulated and downregulated genes in HCT116 and CaCo-2 cells after treatment with live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e or 5% Chk.CM\\u003cstrong\\u003e.\\u003c/strong\\u003e \\u003cstrong\\u003eB \\u003c/strong\\u003eVenn diagram showing the intersecting differentially expressed genes (DEGs) between live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e and 5% Chk.CM treatments in HCT116 cells. \\u003cstrong\\u003eC \\u003c/strong\\u003eBubble plot illustrating KEGG pathway enrichment of these intersecting DEGs in HCT116 cells. \\u003cstrong\\u003eD and E\\u003c/strong\\u003e GSEA enrichment plots for HCT116 cells co-cultured with live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e(D) or 5% Chk.CM (E), showing significant enrichment of the Hippo and Wnt signaling pathway. Normalized Enrichment Scores (NES) and p-values are indicated. \\u003cstrong\\u003eF \\u003c/strong\\u003eThe mRNA expression level of Wnt/β-catenin signaling pathway (\\u003cem\\u003ec-Jun, and Cyclin-D1\\u003c/em\\u003e) in HCT116 and CaCo-2 cells after different treatments. (n=3). Data are presented as mean±SD. One-way ANOVA with the Turkey’s post hoc test was performed.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"fig4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7436037/v1/259207c83fa58bc6c9e93630.jpg\"},{\"id\":91305715,\"identity\":\"1fa0ba22-2ba0-477a-9be7-fc66b22d21d7\",\"added_by\":\"auto\",\"created_at\":\"2025-09-15 06:29:05\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":285585,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe conditioned medium of \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eC. hongkongensis\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e enhances growth of CRC patient-derived organoids \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003evia\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e activation of Wnt/β-catenin signaling pathway. A \\u003c/strong\\u003eRepresentative images of CRC organoids co-cultured with \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e conditioned medium (Chk.CM, 5% \\u003cem\\u003ev/v\\u003c/em\\u003e) at Day 0 (D0) and Day 14 (D14). Organoid size was quantified at Day 14 (right panel). \\u003cstrong\\u003eB \\u003c/strong\\u003eProliferation curve showing the growth rate of CRC organoids treated with Chk.CM over 5 days. \\u003cstrong\\u003eC \\u003c/strong\\u003eQuantification of organoid number after treatment with Chk.CM at Day 14. \\u003cstrong\\u003eD \\u003c/strong\\u003eThe mRNA expression level of genes involved in Wnt/β-catenin signaling pathway (\\u003cem\\u003ec-Jun\\u003c/em\\u003e, and \\u003cem\\u003eCyclin-D1\\u003c/em\\u003e) in CRC organoids after co-culture with Chk.CM (n=3).\\u003cstrong\\u003e \\u003c/strong\\u003eData are presented as mean±SD. Statistical significance was determined by two-way ANOVA (B) and unpaired t-test was performed (A, C, D). ***\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"fig5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7436037/v1/1f48f75717f2f2c75869e533.jpg\"},{\"id\":103765540,\"identity\":\"0b449162-c0f0-429f-82b1-dc2dc3438dbd\",\"added_by\":\"auto\",\"created_at\":\"2026-03-02 16:03:42\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":4285710,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7436037/v1/c91efc73-4939-40fe-9fb2-a6f39d6832ac.pdf\"},{\"id\":91303916,\"identity\":\"21146a6c-9931-41bc-a39b-61b9ae0ecea4\",\"added_by\":\"auto\",\"created_at\":\"2025-09-15 06:21:05\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":17624,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryTable1.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7436037/v1/60243f8a5ad3c197e763051a.docx\"},{\"id\":91303911,\"identity\":\"6ff444de-4612-4d2c-b97e-4bf6d5e4eaee\",\"added_by\":\"auto\",\"created_at\":\"2025-09-15 06:21:05\",\"extension\":\"xlsx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":13342,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryTable2.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7436037/v1/7f99f73f3812bbdaf8ab9a50.xlsx\"},{\"id\":91303913,\"identity\":\"b3c59a45-e850-4516-ab5a-88558f62ad92\",\"added_by\":\"auto\",\"created_at\":\"2025-09-15 06:21:05\",\"extension\":\"docx\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":17208,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryTable3.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7436037/v1/ece07b65fe2262e1082aae92.docx\"},{\"id\":91303917,\"identity\":\"224df3ed-21d0-4f55-b1aa-090fcb10c521\",\"added_by\":\"auto\",\"created_at\":\"2025-09-15 06:21:05\",\"extension\":\"pdf\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":127512,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"ClincalmetadatamainlandChina.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7436037/v1/176080b62e342775c56c6919.pdf\"},{\"id\":91303925,\"identity\":\"b3d87002-563b-4dff-bf96-b58da532a61e\",\"added_by\":\"auto\",\"created_at\":\"2025-09-15 06:21:05\",\"extension\":\"pdf\",\"order_by\":5,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1587126,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementaryfigures.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7436037/v1/b04d12ac0dd81dca63d7a0e5.pdf\"},{\"id\":91303919,\"identity\":\"e0f10e9d-f167-458f-8277-311d2ba8e332\",\"added_by\":\"auto\",\"created_at\":\"2025-09-15 06:21:05\",\"extension\":\"pdf\",\"order_by\":6,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":293471,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"GraphicalAbstract.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7436037/v1/2837c7e47c96ef51b198fd5d.pdf\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Discovery and characterization of Christensenella hongkongensis as a novel bacterium in the adenoma-carcinoma progression\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eColorectal cancer (CRC) is the third most common malignancy and the second leading cause of cancer-related mortality worldwide, with its incidence continuing to rise and imposing substantial medical and economic burdens [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. Most cases of CRC originate from precursor lesions, including non-advanced adenomas, advanced adenomas, and serrated polyps, each carrying distinct risks of progression to cancer [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. Among these, advanced adenomas present a significantly higher risk of developing into CRC compared to non-advanced adenomas [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. Early detection and endoscopic removal of precursor lesions are effective strategies for markedly reducing CRC risk [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Although the fecal immunochemical test (FIT) is the most widely used non-invasive method for early CRC detection, its sensitivity for identifying advanced adenomas remains suboptimal, ranging from 25 to 42% [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. Consequently, there is an urgent need for novel biomarkers with greater accuracy to improve the detection of precancerous lesions and further reduce the incidence and mortality of CRC [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eAccumulating evidence, including findings from animal studies, has linked dysregulation of the gut microbiota to the progression of CRC, which is characterized by reduced microbial diversity and expansion of pathobiont [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. Microbial depletion, achieved either through germ-free conditions [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e] or antibiotic intervention [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e], significantly reduces colorectal tumor formation compared to conventional microbiota. Pathogenic bacteria such as \\u003cem\\u003eFusobacterium nucleatum\\u003c/em\\u003e (\\u003cem\\u003eF. nucleatum\\u003c/em\\u003e) [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e], \\u003cem\\u003ePeptostreptococcus stomatis\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e], \\u003cem\\u003epks\\u0026thinsp;+\\u0026thinsp;Escherichia coli\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e], and \\u003cem\\u003eEnterotoxigenic Bacteroides fragilis\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e] are enriched in CRC patients relative to healthy controls. These pathobionts contribute to CRC initiation and progression through multiple mechanisms, including the production of carcinogenic genotoxins and tumorigenic metabolites, bacterial adhesin-host cell receptor interactions, genetic and epigenetic modifications, and the induction of inflammation [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. These findings underscore the crucial role of the gut microbiota in CRC pathogenesis and highlights its potential as a potential biomarker for non-invasive CRC diagnosis.\\u003c/p\\u003e\\u003cp\\u003eStool-based bacterial markers have been shown to serve as non-invasive diagnostic tools for early CRC detection \\u003cem\\u003evia\\u003c/em\\u003e targeted quantification using quantitative polymerase chain reaction (PCR) [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. For example, \\u003cem\\u003eF. nucleatum\\u003c/em\\u003e and the novel gene marker \\u0026ldquo;m3\\u0026rdquo; from \\u003cem\\u003eLachnoclostrium sp\\u003c/em\\u003e. are significantly enriched during the progression from colorectal adenoma to CRC, showing good diagnostic performance for CRC [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. However, their accuracy for adenoma diagnosis decreases significantly to 48%, and \\u0026ldquo;m3\\u0026rdquo; does not significantly distinguish between non-advanced and advanced adenomas [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e].These findings highlight the current limitations on biomarkers discovery for effective colorectal adenoma detection.\\u003c/p\\u003e\\u003cp\\u003eHence, this study aims to identify potential bacterial marker for early CRC detection through metagenomic sequencing analysis and qPCR validation, and to explore its molecular mechanisms in promoting CRC development \\u003cem\\u003evia in vitro\\u003c/em\\u003e experiments. The findings of this study are expected to contribute to the development of new diagnostic tool for tracking the adenoma-to-carcinoma progression in colorectal cancer and provide valuable insights for its prevention and treatment.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eData download and metagenomic analysis\\u003c/h2\\u003e\\u003cp\\u003eA total of 1 656 fecal metagenomic sequencing samples from colorectal cancer patients and healthy controls, were collected from three datasets conducted in Hong Kong and Japan. The Hong Kong cohort (174 CRC, 893 controls) derived from our previous study was used as the discovery cohort. Two public cohorts from Japan were designated as validation cohort 1 (40 CRC, 40 controls) and validation cohort 2 (251 CRC, 258 controls). Bacterial taxonomic profiles were generated using MetaPhlAn 3 [\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e] and accessed by the \\u003cem\\u003ecurated Metagenomic Data\\u003c/em\\u003e package [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e] in R. Differential species between CRC patients and controls were identified by MaAslin2 across all cohorts. The abundance of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e was specifically compared using Mann-Whitney test. The characteristics of all datasets are provided in \\u003cb\\u003eSupplementary Table\\u0026nbsp;1\\u003c/b\\u003e.\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eHuman subject recruitment\\u003c/h3\\u003e\\n\\u003cp\\u003eParticipants were included if they were between 40 and 75 years old and had undergone a standard colonoscopy. Participants were excluded from the study if they had active gastrointestinal or had used antibiotics within the past month. Healthy controls are individuals with normal colorectal mucosa. Subjects with colorectal adenoma and CRC were diagnosed through colonoscopy and confirmed by histological examination. The TNM stage for CRC patients was assessed by the AJCC (American Joint Committee on Cancer) TNM system.\\u003c/p\\u003e\\u003cp\\u003eFinally, we recruited 686 consecutive subjects, including 285 CRC, 73 advanced adenomas, 133 non-advanced adenomas and 194 healthy controls) from five different sites (Guangzhou, Beijing, Shanghai, Kunming and Xi\\u0026rsquo;an) of mainland China. Advanced adenomas (AA) were defined as adenomas with any of the following criteria: size\\u0026thinsp;\\u0026gt;\\u0026thinsp;10 mm, more than three adenomas, the presence of a tubulovillous or villous component, or high-grade intraepithelial dysplasia. Non-advanced adenomas were defined as 1\\u0026ndash;2 adenomas, each \\u0026lt;\\u0026thinsp;10 mm in size [\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e]. Tumor anatomic locations were typically divided into three categories: rectum, proximal colon, and distal colon. The rectum refers to the terminal segment of the larger intestine. The proximal colon includes the ileocecal junction, ascending colon, hepatic flexure and transverse colon. The distal colon includes the splenic flexure, descending colon and sigmoid colon. Clinical characteristics of healthy subjects and colorectal adenoma/cancer patients are shown in \\u003cb\\u003eSupplementary Table\\u0026nbsp;2\\u003c/b\\u003e.\\u003c/p\\u003e\\n\\u003ch3\\u003eFecal sample collection\\u003c/h3\\u003e\\n\\u003cp\\u003eFecal samples were collected within one month prior to the scheduled colonoscopy and before any bowel preparation. The fresh fecal sample was collected from each participant using a sealed, sterile collection tube containing preservative media (Norgen Biotek Corp, Thorold, Ontario Canada). All samples were kept capped when not in use to prevent cross-contamination. Following collection, samples were sent to the lab within 24 h and stored at -80\\u0026deg;C until analysis.\\u003c/p\\u003e\\n\\u003ch3\\u003eFecal DNA extraction\\u003c/h3\\u003e\\n\\u003cp\\u003eFor mainland China cohort, fecal DNA was extracted following manufacturer\\u0026rsquo;s instruction of BayBiopure Magnetic Stool Nucleic Acid Kit (Baybio, Guangzhou, China). The extracted DNA was quantified by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). After final precipitation, the DNA samples were resuspended in DE buffer and stored at \\u0026minus;\\u0026thinsp;80\\u0026deg;C before further analysis.\\u003c/p\\u003e\\n\\u003ch3\\u003eFecal samples duplex quantitative PCR\\u003c/h3\\u003e\\n\\u003cp\\u003eDuplex quantitative PCR (qPCR) was performed using the Applied Biosystems\\u0026trade; 7500 system to quantify the abundance of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e in fecal samples from mainland China cohort. Each 20 \\u0026micro;L reaction contained 10.2 \\u0026micro;L Premix Ex Taq\\u0026trade; (TAKARA, Tokyo, Japan), 0.4 \\u0026micro;L each of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e forward and reverse primers (10 \\u0026micro;M), 0.2 \\u0026micro;L \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e probe labeled with FAM (10 \\u0026micro;M), 0.4 \\u0026micro;L each of 16S forward and reverse primers (10 \\u0026micro;M), 0.2 \\u0026micro;L 16S probe labeled with VIC (10 \\u0026micro;M), 5.8 \\u0026micro;L nuclease-free water, and 2.0 \\u0026micro;L template DNA (5 ng/\\u0026micro;L). The qPCR program consisted of a pre-denaturation step at 95 ℃ for 30 s, followed by 40 cycles of denaturation at 95 ℃ for 5 s and annealing/extension at 60 ℃ for 34 s. Ct analysis was performed with a threshold of 0.07 and a baseline set between cycles 3 and 15. The sequences of the gene primers are provided in \\u003cb\\u003eSupplementary Table\\u0026nbsp;3\\u003c/b\\u003e.\\u003c/p\\u003e\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eBacteria culture\\u003c/h2\\u003e\\u003cp\\u003e\\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e (DSM18959) was purchased from Leibniz Institute DSMZ-Germany Collection of Microorganisms and Cell Cultures GmbH and cultured at 37℃ in Peptone-Yeast Extract-Glucose (PYG) broth with Hungate bottles filled with nitrogen. A nonpathogenic human commensal intestinal bacterium, \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e MG1665 was used as bacterial control and cultured at the same condition as \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e. When the absorbance of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e reached optical density 600 of 0.3, the conditioned medium was centrifuged and filtered with 0.22 \\u0026micro;m pore size filter to collect \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e conditioned medium (Chk.CM) or \\u003cem\\u003eE. coli\\u003c/em\\u003e conditioned medium (E. coli.CM).\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eCell culture\\u003c/h3\\u003e\\n\\u003cp\\u003eNormal human colon epithelial cell NCM460, human CRC cell lines HCT116 and CaCo-2 were obtained from ATCC (Rockville, MD, USA). All cell lines were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) in humidified atmosphere containing 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e.\\u003c/p\\u003e\\n\\u003ch3\\u003eHuman CRC organoid culture\\u003c/h3\\u003e\\n\\u003cp\\u003e CRC patient-derived organoid was obtained from Prof Nathalie Wong\\u0026rsquo;s laboratory, isolating from a 75-year-old male colorectal adenocarcinoma patient. The CRC organoids were activated and embedded into Matrigel and maintained in Advanced DMEM/F12, supplied with 1X P/S, 10 mM HEPES, 1\\u0026times;GlutaMAX medium containing 1X N2 and B27 supplements, 100 ng/mL R-spondin-1, 100 ng/mL Noggin, 50 ng/mL EGF, 10nM Gastrin, 100 ng/mL FGF10, 1.25 mM N-acetylcysteine, 500 nM A8301, 10 \\u0026micro;M SB202190, 10 mM Nicotinamide, 100 \\u0026micro;g/mL Primocin and 10 \\u0026micro;M Y27632. Treatment such as 5% (vol/vol) PYG or Chk.CM was added into the culture medium directly, which was freshly changed every three days. Surface area of organoid was measured by Image J 1.54g.\\u003c/p\\u003e\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eCell proliferation assay\\u003c/h2\\u003e\\u003cp\\u003eCaCo-2, HCT116 and NCM460 cells (1 \\u0026times; 10\\u003csup\\u003e3\\u003c/sup\\u003e cells per well) were seeded onto 96-well plates and treated with live \\u003cem\\u003eE. coli\\u003c/em\\u003e (CFU\\u0026thinsp;=\\u0026thinsp;10\\u003csup\\u003e6\\u003c/sup\\u003e/mL) or \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e (CFU\\u0026thinsp;=\\u0026thinsp;10\\u003csup\\u003e6\\u003c/sup\\u003e/mL), as well as 5% (vol/vol) PYG, E. coli.CM, or Chk.CM in DMEM. Cell viability was determined by Cell Counting Kit-8 (CCK8, MedChemEXpress, Monmouth Junction, NJ, USA; Hy-K0301) for 3 consecutive days.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eCell apoptosis assay\\u003c/h2\\u003e\\u003cp\\u003eCaCo-2, HCT116 and NCM460 cells (5 \\u0026times; 10⁵ cells per well) were separately seeded in a 12-well plate and incubated until reaching approximately 80\\u0026ndash;90% confluence. Cells were infected with \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e (MOI\\u0026thinsp;=\\u0026thinsp;100) for 4 hours under anaerobic conditions. Following infection, cells were washed 2\\u0026ndash;3 times with 1X PBS and replaced with DMEM supplemented with antibiotics to prevent further bacterial growth. After 48 hours post-infection, cells were washed twice with cold 1X PBS and detached by adding 0.5 mL trypsin without EDTA to each well. The cell suspension was collected and centrifuged at 1 000 rpm for 5 minutes at 4\\u0026deg;C. The resulting cell pellet was resuspended in 1X Annexin V bindng buffer to achieve a final concentration of 1 \\u0026times; 10⁶ cells/mL. A 100 \\u0026micro;L aliquot of the resuspended cells (1 \\u0026times; 10⁵ cells) was transferred to a 5 mL flow cytometry tube. Cells were stained using the BD Pharmingen\\u0026trade; PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) according to the manufacturer\\u0026rsquo;s protocol. After staining, 400 \\u0026micro;L of 1X binding buffer was added to each tube, and samples were analyzed immediately by flow cytometry.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eWound healing assay\\u003c/h2\\u003e\\u003cp\\u003eCaCo-2 and HCT116 cells (1 \\u0026times; 10\\u003csup\\u003e3\\u003c/sup\\u003e cells per well) were seeded in 6-well plates and cultured until confluence. A uniform scratch was carefully made in the cell monolayer using a sterile 200 \\u0026micro;L pipette tip, followed by washing with PBS to remove debris. The cells were then incubated in serum-free medium under the following conditions: (1) live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e (MOI\\u0026thinsp;=\\u0026thinsp;100), (2) serum-free medium (blank control for \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e), (3) 5% Chk.CM, and (4) 5% PYG medium (negative control for Chk.CM). Images of the scratch areas were captured at 0 h and 12 h, and scratch widths were measured using ImageJ software to assess wound closure.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eColony formation assay\\u003c/h2\\u003e\\u003cp\\u003eHCT116 and CaCo-2 cells (1 \\u0026times; 10\\u003csup\\u003e3\\u003c/sup\\u003e cells per well) were seeded in 6-well plates. For live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e treatment, cells were co-cultured with live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e (MOI\\u0026thinsp;=\\u0026thinsp;100) for 4 hours. Live \\u003cem\\u003eE. coli\\u003c/em\\u003e MG1655 (MOI\\u0026thinsp;=\\u0026thinsp;100) served as a negative control. After 4 hours, cells were washed three times with PBS and fresh DMEM medium was added. For \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e treatment, cells were cultured with 5% Chk.CM, while 5% PYG medium and E. coli.CM were used as blank and negative controls, respectively. The treatment medium was refreshed every 3 days. After 10\\u0026ndash;14 days, cells from both treatments were fixed with 4% PFA and stained with 0.1% crystal violet. Colonies containing more than 50 cells were counted. All experiments were performed in triplicate and repeated three times.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eRNA sequencing analysis\\u003c/h2\\u003e\\u003cp\\u003eTotal RNAs were extracted from the HCT116 and CaCo-2 cells after co-culturing with live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e (MOI\\u0026thinsp;=\\u0026thinsp;100) or 5% Chk.CM respectively using Trizol Reagent (Thermo Fisher Scientific). RNA sequencing libraries were prepared with at least 6G raw data per sample (NovaSeq PE150, Novogene, Beijing, China). Raw sequencing data were qualified and cleaned using fastp [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e] and TrimGalore. The processed reads were then aligned to the reference human genome (GRCh38) using Hisat2 [\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e]. Differential gene expression analysis was performed with DESeq2 [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e], applying the thresholds of |Log2FoldChange|\\u0026gt;1 and adjusted \\u003cem\\u003eP\\u003c/em\\u003e-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 to identify significantly differential genes. The identified differentially expressed genes were subjected to KEGG enrichment analysis using the ClusterProfiler [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e]. Additionally, Gene Set Enrichment Analysis (GSEA) was conducted \\u003cem\\u003evia\\u003c/em\\u003e the GSEA software [\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e] with default parameters using the KEGG pathway database. Finally, pathway visualizations of differential gene expression were generated using the Pathview [\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e].\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eRT-PCR\\u003c/h2\\u003e\\u003cp\\u003ecDNA was synthesized from total RNA by PrimeScript RT reagent Kit (RR037A, Takara, Tokyo, Japan) according to the manufacturer\\u0026rsquo;s instructions. Quantitative PCR (qPCR) was conducted using TB Green\\u0026reg; Premix Ex Taq\\u0026trade; II (TAKARA) on the QuantStudio 7 Flex System (Thermo Fisher Scientific). The sequences of the primers are listed in \\u003cb\\u003eSupplementary Table\\u0026nbsp;3\\u003c/b\\u003e.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e\\u003cp\\u003eGraphPad Prism 10 Software and SPSS version 27.0 software were used for data analysis in this study. The experimental data are generally represented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD, except for \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e abundance, which is expressed as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SE. Paired Student\\u0026rsquo;s t-test and one-way ANOVA with Tukey\\u0026rsquo;s multiple comparison test were used to evaluate the statistical significance of differences between two groups and among three groups, respectively. The graphical abstract was created with BioRender.com.\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eIdentification of the novel species C. hongkongensis enriched in patients with CRC\\u003c/h2\\u003e\\u003cp\\u003eTo identify potential novel bacterial markers for CRC, we performed MaAsLin2 analysis on metagenomic sequencing data from three public datasets to determine differentially abundant bacterial species in fecal samples of CRC patients compared to healthy controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). Among all identified differential bacterial species, seven CRC-enriched species were consistently observed across all three cohorts, including \\u003cem\\u003eRuthenibacterium lactatiformans\\u003c/em\\u003e, \\u003cem\\u003eAlistipes shahii\\u003c/em\\u003e, \\u003cem\\u003eAnaerotruncus colihominis\\u003c/em\\u003e, \\u003cem\\u003ePeptostreptococcus stomatis\\u003c/em\\u003e, \\u003cem\\u003eParvimonas micra\\u003c/em\\u003e, \\u003cem\\u003eGemella morbillorum\\u003c/em\\u003e and \\u003cem\\u003eChristensenella hongkongensis\\u003c/em\\u003e (all \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB and \\u003cb\\u003eFig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e\\u003c/b\\u003e). Notably, \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e demonstrated the lowest abundance in healthy controls among these seven species (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC). In each individual cohort, the relative abundance of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e was also significantly increased in CRC patients compared to healthy controls (all \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD). Furthermore, the prevalence of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e was consistently elevated in CRC patients compared to healthy controls across all three cohorts (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE). Collectively, we identified a novel bacterial species that is consistently enriched in CRC across the cohorts.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eThe involvement of C. hongkongensis in CRC progression from adenoma to carcinoma\\u003c/h2\\u003e\\u003cp\\u003eTo investigate the potential role of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e in CRC progression, we further quantitatively examined its abundance in an expanded cohort of fecal samples collected from healthy controls (NC, n\\u0026thinsp;=\\u0026thinsp;194), patients with non-advanced adenomas (nAA, n\\u0026thinsp;=\\u0026thinsp;134), patients with advanced adenomas (AA, n\\u0026thinsp;=\\u0026thinsp;73) and those with CRC (n\\u0026thinsp;=\\u0026thinsp;285) across five regions from mainland China (Guangzhou, Kunming, Beijing, Shanghai and Xi\\u0026rsquo;an, n\\u0026thinsp;=\\u0026thinsp;686) using our previously established duplex-qPCR platform (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA) [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eConsistent with previous findings, a substantially enriched abundance of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e was observed in CRC patients compared to healthy controls (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). More importantly, the abundance of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e progressively increased along with the CRC progression, from the nAA stage to the AA stage, and ultimately to carcinoma, with no significant differences detected between patients with nAA and healthy controls \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB\\u003cb\\u003e)\\u003c/b\\u003e. Furthermore, among the CRC patients, the abundance of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e also demonstrated a continuous upward trend as the disease advanced from TNM stage I to TNM stage IV, reflecting a compelling correlation with tumor progression \\u003cb\\u003e(\\u003c/b\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC\\u003cb\\u003e)\\u003c/b\\u003e. In addition to the abundance, the prevalence of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e also demonstrated a notable increase in CRC patients at the AA stage and those presenting with adenoma, in comparison to healthy controls and CRC patients at the nAA stage \\u003cb\\u003e(\\u003c/b\\u003eNC: 32.5%; nAA: 33.1%; AA: 45.9%; CRC: 44.6%, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD\\u003cb\\u003e)\\u003c/b\\u003e. However, no statistically significant differences were observed between CRC patients at the nAA stage and healthy controls \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD\\u003cb\\u003e)\\u003c/b\\u003e. Notably, the abundance of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e exhibited a markedly elevated level in patients with distal CRC, compared to those with tumors located in proximal colon or rectal regions (both \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE). These compelling findings suggested the potential of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e as an early fecal biomarker, providing a valuable approach for the early detection of precursory lesions that may lead to CRC, particularly in patients with distal CRC.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eC. hongkongensis promotes CRC cell growth\\u003c/h2\\u003e\\u003cp\\u003eThe progressively increased abundance of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e during CRC progression suggests that \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e may play a carcinogenic role in CRC tumorigenesis. To prove this, we conducted the \\u003cem\\u003ein vitro\\u003c/em\\u003e experiments using two CRC cell lines (CaCo-2 and HCT116) and a normal colon epithelial cell line (NCM460), co-cultured with \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e and its conditioned medium. \\u003cem\\u003eE. coli\\u003c/em\\u003e MG1655, a non-pathogenic strain of \\u003cem\\u003eE. coli\\u003c/em\\u003e, was employed as a negative control. Both live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e and its 5% conditioned medium demonstrated the ability in enhancing the proliferation of two CRC cell lines (live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e: both \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001; 5% conditioned medium: both \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001), yet neither exerted any influence on the growth of NCM460 cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA and \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB). In keeping with this, the number of colonies which had formed in CaCo-2 and HCT116 cells cultured with \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e or its conditioned medium were increased significantly compared with those treated with PBS or \\u003cem\\u003eE. coli\\u003c/em\\u003e MG1655 \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC and \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD\\u003cb\\u003e)\\u003c/b\\u003e.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eIn addition, flow cytometry utilizing dual staining with Annexin V-PE and 7-AAD was conducted to evaluate the effect of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e on cell apoptosis. The result exhibited an increase in the number of apoptotic cells in \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e treated CaCo-2 cells compared with those treated with PBS (9.43\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.52% vs 13.53\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.01%, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE). A similar effect was observed in \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e cultured HCT116 cells, which demonstrated an increased proportion of cells in the apoptotic phase compared with cells co-cultured with PBS (18.69\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.36% vs 20.93\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.05%, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF).\\u003c/p\\u003e\\u003cp\\u003eFurthermore, the monolayer wound healing assay was performed to investigate the effects of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e on CRC cell migration. Administration of either \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e or its conditioned medium significantly enhanced the cell migration capacity in both CaCo-2 and HCT116 cell lines \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eG and \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eH\\u003cb\\u003e)\\u003c/b\\u003e. Quantitative analyses at 12 hours post-wounding confirmed a significant increase in wound closure in cells administered with \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e or its conditioned medium compared to control cells (29.22% and 26.25% in CaCo-2 cells; 30.95% and 38.15% in HCT116 cells, respectively), thereby suggesting that the migration rate of cells cultured with \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e or its conditioned medium was significantly higher than that of the control cells. Collectively, these data suggest that \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e and its conditioned medium exert pro-tumorigenic effects on CRC cells by enhancing proliferation, colony formation, and migration, while simultaneously inhibiting apoptosis.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eC. hongkongensis activates the tumorigenic signaling pathways in CRC\\u003c/h2\\u003e\\u003cp\\u003eTo further elucidate the molecular mechanisms mediated by \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e in executing its oncogenic functions, we conducted transcriptomic sequencing to identify the genes and signaling pathways regulated by live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e and its conditioned medium in CaCo-2 and HCT116 cells. Principal component analysis (PCA) demonstrated a clear separation among all three treatment groups in HCT116 cells \\u003cb\\u003e(Fig. \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003eA)\\u003c/b\\u003e, whereas cells treated with live \\u003cem\\u003eC. hongkongensi\\u003c/em\\u003es and its conditioned medium clustered closely together in CaCo-2 cells, yet remained distinctly separated from the blank controls (\\u003cb\\u003eFig. \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003eB\\u003c/b\\u003e).\\u003c/p\\u003e\\u003cp\\u003eThe differentially expressed genes (DEGs) between \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e-treated and PBS-treated CRC cells were identified using DESeq2 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). In CaCo-2 cells, co-culturing with live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e resulted in the upregulation of 203 genes and downregulation of 223 genes. While treatment with its conditioned medium significantly altered the expression of 939 genes, including 313 upregulated and 626 downregulated \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA\\u003cb\\u003e)\\u003c/b\\u003e. Similarly, in HCT116 cells, treatment with live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e led to the upregulation of 1,960 genes and downregulation of 1,119 genes, whereas treatment with its conditioned medium resulted in 324 upregulated and 468 downregulated genes \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA\\u003cb\\u003e)\\u003c/b\\u003e. Among all DEGs, 290 genes in CaCo-2 cells and 472 genes in HCT116 cells were consistently regulated in both live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e-treated and conditioned medium-treated cells compared to the control groups \\u003cb\\u003e(Fig. \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003eC and\\u003c/b\\u003e Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB\\u003cb\\u003e)\\u003c/b\\u003e. Following KEGG pathway enrichment analysis, the Wnt signaling pathway and the Hippo signaling pathway were identified as activated in both CRC cell lines treated with live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e or its conditioned medium \\u003cb\\u003e(Fig, 4C and Fig. \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003eD)\\u003c/b\\u003e. Moreover, gene set enrichment analysis (GSEA) further confirmed that the Hippo signaling pathway and the Wnt/β-catenin signaling pathway were significantly upregulated in HCT116 cells treated with either live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e (Hippo signaling pathway: normalized enrichment score [NES]\\u0026thinsp;=\\u0026thinsp;1.44, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.009; Wnt/β-catenin signaling pathway: NES\\u0026thinsp;=\\u0026thinsp;1.52, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.145) or its conditioned medium (Hippo signaling pathway: NES\\u0026thinsp;=\\u0026thinsp;1.20, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.004; Wnt/β-catenin signaling pathway: NES\\u0026thinsp;=\\u0026thinsp;1.36, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.025, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD and \\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eE). Additionally, PathView analysis visualized the gene expression changes in both Hippo signaling pathway and Wnt signaling pathway, revealing that activation of the Wnt/β-catenin signaling pathway represents a shared mechanism underlying the response in both cell lines (\\u003cb\\u003eFig. \\u003cspan refid=\\\"MOESM3\\\" class=\\\"InternalRef\\\"\\u003eS3\\u003c/span\\u003e and S4\\u003c/b\\u003e). The Wnt/β-catenin signaling pathway serves as a critical regulator of intestinal homeostasis, governing essential cellular processes such as proliferation, differentiation, and migration, and has been strongly associated with the initiation and progression of CRC. We further examined the mRNA expression levels of key downstream target genes of Wnt/β-catenin signaling pathway, such as \\u003cem\\u003eCyclin-D1\\u003c/em\\u003e and \\u003cem\\u003ec-Jun\\u003c/em\\u003e. Consistent with the findings, the levels of both \\u003cem\\u003eCyclin-D1\\u003c/em\\u003e and \\u003cem\\u003ec-Jun\\u003c/em\\u003e were significantly increased in cells treated with live \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e or its conditioned medium \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eF\\u003cb\\u003e)\\u003c/b\\u003e. These results therefore indicate that \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e exerts its oncogenic function in CRC through the activation of Wnt/β-catenin signaling pathway.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eThe conditioned medium of C. hongkongensis enhances growth of CRC patient-derived organoids via activation of Wnt/β-catenin signaling pathway\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe activation of the Wnt/β-catenin signaling pathway triggered by \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e was further validated in CRC organoid derived from one CRC patient. The organoids were co-cultured with \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e conditioned medium or fresh PYG medium. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA, the CRC organoids treated with the conditioned medium of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e exhibited a significantly greater size compared to the black control (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Moreover, administration of the conditioned medium of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e significantly enhanced the proliferation rate of CRC organoids during the 5-day cultivation period (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001), and the number of organoids was higher than that in the blank control group (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB and \\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC). We further validated the expression of pivotal downstream targets of the Wnt/β-catenin signaling pathway, Cyclin-D1 and c-Jun, in CRC organoids. The mRNA expression levels of both genes were significantly increased in CRC organoids treated with the conditioned medium of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e compared to the blank control group \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD\\u003cb\\u003e)\\u003c/b\\u003e. These findings suggest that the secretome of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e is capable of enhancing the growth of CRC patient-derived organoids by activating the Wnt/β-catenin signaling pathway.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eDue to the high cost and invasive nature of standard diagnostic methods like colonoscopy and sigmoidoscopy, there is an urgent need to improve the sensitivity of non-invasive tests for detecting CRC [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. Increasing evidence has revealed that alterations in gut microbiota composition are closely associated with CRC progression, suggesting that the gut microbiome represents a rich reservoir of potential biomarkers [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. Among various approaches, shotgun metagenomic sequencing, due to its high taxonomic precision and functional resolution, has been widely employed to identify CRC associated microbial markers [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. In this study, we analyzed fecal metagenomic sequencing data from our internal cohort and two public cohorts, identifying seven species significantly enriched in CRC patients compared to healthy controls. Among these, six species including \\u003cem\\u003eRuthenibacterium lactatiformans\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e], \\u003cem\\u003eAlistipes shahii\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e], \\u003cem\\u003eAnaerotruncus colihominis\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e], \\u003cem\\u003ePeptostreptococcus stomatis\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e], \\u003cem\\u003eParvimonas micra\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e] and \\u003cem\\u003eGemella morbillorum\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e], have previously been reported as potential CRC biomarkers with varying diagnostic performances [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. Notably, \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e was distinguished by its lowest abundance in healthy controls and significant enrichment in CRC patients. Furthermore, another multi-omics analysis from CRC patients revealed that, at the genus level, \\u003cem\\u003eChristensenella\\u003c/em\\u003e abundance continuously increased from early-stage colonic intramucosal carcinoma to advanced CRC stages [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. These findings suggest the strong association of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e with CRC and its potential as a microbial marker.\\u003c/p\\u003e\\u003cp\\u003eTo validate the diagnostic potential of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e, we recruited a large cohort from mainland China and performed targeted quantitative PCR assay. Consistent with metagenomic data, the qPCR results confirmed a significant enrichment of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e in CRC patients compared to healthy controls, accompanied by a higher prevalence in the CRC group. Early detection during precancerous stages is critical for reducing CRC incidence and mortality [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. However, most current biomarkers, including microbial species and genetic markers, primarily differentiate between colorectal adenomas or CRC and healthy controls, with limited ability to distinguish among precancerous stages. For example, \\u003cem\\u003eFusobacterium nucleatum\\u003c/em\\u003e and \\u003cem\\u003ePeptostreptococcus anaerobius\\u003c/em\\u003e are significantly more abundant in CRC patients compared to healthy controls, but its level showed no significant differences between patients with advanced adenoma and controls, which may suggest its limited potential in detecting advanced adenoma [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. Similarly, while the methylated gene SHOX2 has been proposed as a biomarker for CRC diagnosis, quantitative methylation analysis reveals no significant differences in SHOX2 methylation levels between nAA and AA patients [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. Interestingly, in our recruited cohort, the abundance of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e progressively increased from non-advanced adenoma (nAA) to advanced adenoma (AA) and ultimately to CRC, with its levels in nAA patients significantly lower than those in AA and CRC patients. Additionally, the prevalence of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e was highest in AA patients, highlighting its potential as a promising biomarker for distinguishing pre-tumor stages.\\u003c/p\\u003e\\u003cp\\u003eTNM stage remains a critical determinant of CRC survival rates [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e], emphasizing the need for biomarkers capable of tracking disease progression. In our study, the abundance of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e positively correlated with TNM stages, suggesting its potential utility not only in early detection but also in monitoring disease advancement. Consistent with previous study showing that \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e is enriched in left-sided CRC patients [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e], we observed that the fecal levels of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e were significantly higher in distal CRC patients. Collectively, our findings underscore the potential of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e as a versatile bacterial marker, spanning early detection, disease staging, and CRC location.\\u003c/p\\u003e\\u003cp\\u003eCurrently, FIT is the most widely used non-invasive methods for CRC diagnosis [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]. Despite its widespread use, Thomas e.tal found that FIT showed lower sensitivity (23.8%) for detecting advanced precancerous lesions based on a cohort more than 9000 subjects [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. Recent studies have demonstrated that combining FIT with bacterial biomarkers significantly enhances the ability to detect adenomas and CRC compared to using FIT alone [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. For example, \\u003cem\\u003eFusobacterium nucleatum\\u003c/em\\u003e, a well-documented CRC-associated bacterium, has been shown to enhance the sensitivity and specificity of FIT when used in combination, especially for early-stage CRC detection [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Given the promising potential of bacterial biomarkers in complementing FIT, future studies could explore the utility of combining \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e with FIT.\\u003c/p\\u003e\\u003cp\\u003eAdditionally, \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e has been implicated in intestinal and biliary infections, with cases of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e-associated bacteremia occurring predominantly in CRC patients [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. However, existing studies have not provided species-level evidence or functional validation for the role of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e in CRC progression. CRC progression involves several hallmarks, including uncontrolled cell proliferation, tumor suppressor gene inactivation, and metastasis [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e]. Our \\u003cem\\u003ein vitro\\u003c/em\\u003e studies revealed that \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e and its conditioned medium enhance CRC cell proliferation and migration, while simultaneously inhibiting apoptosis. These findings suggest a pathogenic role for \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e in CRC development, contrasting with the previously reported probiotic properties of the Christensenella family, whose abundance is negatively correlated with metabolic diseases such as obesity and type 2 diabetes (T2D) [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. This discrepancy may be attributed to that \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e represents a distinct phylogenetic branch within the \\u003cem\\u003eChristensenella\\u003c/em\\u003e genus, suggesting that \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e may possess unique functions that differ from other \\u003cem\\u003eChristensenella\\u003c/em\\u003e species [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e]. Metabolic profiling further supports this, revealing that \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e produces fewer potential beneficial metabolites compared to other \\u003cem\\u003eChristensenella\\u003c/em\\u003e species [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e]. Future studies using animal models are needed to provide deeper insights into the role of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e in CRC progression. Additionally, identifying the specific metabolites responsible for its pathogenic effects warrants further investigation.\\u003c/p\\u003e\\u003cp\\u003eMechanistically, we found that \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e promotes CRC progression by activating the Wnt/β-catenin signaling pathway. Dysregulation of this pathway, a hallmark of CRC, is characterized by hyperactivation of the proto-oncoprotein β-catenin, which drives the transcription of oncogenic target genes [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. Cyclin-D1, a key regulators of cell cycle, can bind with cyclin-dependent kinases CDK4 and CDK6 to form a complex targeting transcriptional factor E2F1, which is responsible for inducing G1/S transition, leading to uncontrolled cell proliferation [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]. We discovered that \\u003cem\\u003eCyclin-D1\\u003c/em\\u003e was upregulated in CRC cells or organoids in both treatments. Conversely, downregulated Cyclin-D1 has been shown to cause G1/S phase arrest [\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e]. In addition to \\u003cem\\u003eCyclin-D1\\u003c/em\\u003e, \\u003cem\\u003ec-Jun\\u003c/em\\u003e, a component of the AP-1 transcription factor complex, was also upregulated in response to \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e or its conditioned medium. As a downstream effector of Wnt/β-catenin signaling, c-Jun promotes the transcription of genes involved in cell proliferation and survival [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e]. Furthermore, activated c-Jun has been reported to enhance epithelial-mesenchymal transition (EMT), a critical process for tumor invasion and metastasis in gastric cancer [\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e]. While our findings suggest a strong link between \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e and the activation of the Wnt/β-catenin pathway, further validation at the protein level is necessary to confirm pathway activation and its downstream effects.\\u003c/p\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eIn summary, this study characterizes a novel gut bacteria \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e that increases in abundance from the adenoma-to-carcinoma sequence, highlighting its potential as an auxiliary marker for CRC, with potential applications in early detection, disease staging, and tumor localization. Mechanistically, \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e promotes CRC progression \\u003cem\\u003evia\\u003c/em\\u003e the Wnt/β-catenin signaling pathway, providing new insights into its pathogenic role. Future studies integrating animal models, metabolite profiling, and therapeutic interventions are warranted to fully elucidate the clinical and biological significance of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e in CRC.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cdiv class=\\\"DefinitionList\\\"\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003e\\u003cb\\u003eCRC\\u003c/b\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eColorectal cancer\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003e\\u003cb\\u003eTNM\\u003c/b\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eTumor node metastasis\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003e\\u003cb\\u003eFIT\\u003c/b\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eFecal immunochemical test\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003e\\u003cb\\u003eDEGs\\u003c/b\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eDifferentially expressed genes\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003e\\u003cb\\u003eGSEA\\u003c/b\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eGene set enrichment analysis\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003e\\u003cb\\u003eNES\\u003c/b\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eNormalized enrichment score\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003e\\u003cb\\u003enAA\\u003c/b\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eNon-advanced adenoma\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003e\\u003cb\\u003eAA\\u003c/b\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eAdvanced adenoma\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003e\\u003cb\\u003eT2D\\u003c/b\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eType 2 diabetes\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003e\\u003cb\\u003eEMT\\u003c/b\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eEpithelial-mesenchymal transition\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003e\\u003cb\\u003ePYG\\u003c/b\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003ePeptone-Yeast Extract-Glucose\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003e\\u003cb\\u003eFBS\\u003c/b\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eFetal bovine serum\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003e\\u003cb\\u003eCCK8\\u003c/b\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eCell counting kit 8\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003eEthic approval and consent to participate\\u003c/p\\u003e\\n\\u003cp\\u003eAll subjects and clinical information used in this study were obtained under conditions of informed consent and with approval of the institutional review boards of each participating institute (Beijing Friendship Hospital, 2022-P2-084-01; The First Affiliated Hospital of Kunming Medical University, 2022-95-2; Renji Hospital, Ly2022-081-B; Xijing Hospital, KY2022181-C-1; and the Sixth Affiliated Hospital of Sun Yat-sen University, 2022ZSLYEC-436).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eConsent for publication\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003eAvailability of data and materials\\u003c/p\\u003e\\n\\u003cp\\u003eThe raw data of the RNA-seq analysis were deposited in the SRA database (PRJNA1280769). The data in this study are available within the article and related supplementary information files, or by inquiring the corresponding authors.\\u003c/p\\u003e\\n\\u003cp\\u003eCompeting interests\\u003c/p\\u003e\\n\\u003cp\\u003eS.C.N. has served as an advisory board member for Pfizer, Ferring, Janssen and Abbvie and received honoraria as a speaker for Ferring, Tillotts, Menarini, Janssen, Abbvie and Takeda; has received research grants through her affiliated institutions from Olympus, Ferring and Abbvie; is a founder member, non-executive director, non-executive scientific advisor and shareholder of GenieBiome Ltd which is non-remunerative; is a shareholder of MicroSigX Diagnostic Holding Limited; is a founder member, non-executive Board Director, and non-executive scientific advisor of MicroSigX Biotech Diagnostic Limited, which is non-remunerative; and receives patent royalties through her affiliated institutions. FKLC serves as the Principal Investigator for the Faecal Microbiota Transplantation Service under the Hospital Authority (HA). He is a Board Director of EHealth Plus Digital Technology Ltd., an HA-owned subsidiary driving the eHealth+ programme to transform the Electronic Health Record Sharing System into a comprehensive digital healthcare platform and advance other IT initiatives within the eHealth ecosystem. Additionally, he is a Board Director of CUHK Medical Services Limited. FKLC is a shareholder of GenieBiome Holdings Limited and the co-founder, non-executive Board Chairman, and non-executive Scientific Advisor of its wholly owned subsidiary, GenieBiome Ltd. Similarly, he is a shareholder of MicroSigX Diagnostic Holding Limited and the co-founder, non-executive Board Chairman, and non-executive Scientific Advisor of its wholly owned subsidiary, MicroSigX Biotech Diagnostic Limited.\\u0026nbsp;He also serves as a Director of the Hong Kong Investment Corporation Limited and a member of the Steering Committee for the RAISe+ Scheme under the Innovation and Technology Commission. Furthermore, he is the Co-Director of the Microbiota I-Center (MagIC) Ltd. FKLC receives advisory fees and speaker honoraria from AstraZeneca and Comvita New Zealand Limited, as well as patent royalties through affiliated institutions for microbiome-related applications. QS is Scientists (Diagnostics) of GenieBiome Ltd. JZ is Chief Scientist (Diagnostics) of GenieBiome Ltd. The other authors declare no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003eFunding\\u003c/p\\u003e\\n\\u003cp\\u003eThis study is funded by InnoHK, The Government of Hong Kong, Special Administrative Region of the People’s Republic of China, Research Grants Council–Research Impact Fund (RGC-RIF, R4030-22), New Cornerstone Science Foundation (NCI202346), Research Grants Council-General Research Fund (RGC-GRF, 14121322), Health and Medical Research Fund (10210816), National Natural Science Foundation of China (82100573), Hong Kong Research Grants Council Areas of Excellence Scheme (Ref. AoE/M-401/20) and Leona M. and Harry B. Helmsley Charitable Trust (2017PG-IBD003). Authors affiliated with MagIC are partially supported by InnoHK, The Government of Hong Kong, Special Administrative Region of the People’s Republic of China.\\u003c/p\\u003e\\n\\u003cp\\u003eAuthor’s contributions\\u003c/p\\u003e\\n\\u003cp\\u003eS.C.N and JZ conceived this study. HS, YS, XL, ML, and HW were responsible for recruiting colorectal adenoma and CRC patients, collecting fecal samples, and performing colonoscopies. WN provided CRC-patients derived organoid. WZ and QS analyzed metagenomic sequencing data obtained from public datasets. WZ and JZ developed the methodology and performed the experiments. WZ drafted the manuscript, while WN, JY, FKLC, JZ, and S.C.N reviewed, edited, and supervised the study.\\u003c/p\\u003e\\n\\u003cp\\u003eAcknowledgements\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors thank Prof. Nathalie Wong and Prof. Yujuan Dong for providing CRC patient-derived organoids, which were invaluable for this study.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eArnold M, Sierra MS, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global patterns and trends in colorectal cancer incidence and mortality. 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Bioinforma Oxf Engl. 2018 Sep 1;34(17):i884\\u0026ndash;90. \\u003c/li\\u003e\\n\\u003cli\\u003eKim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019 Aug;37(8):907\\u0026ndash;15. \\u003c/li\\u003e\\n\\u003cli\\u003eLove MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. \\u003c/li\\u003e\\n\\u003cli\\u003eYu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. Omics J Integr Biol. 2012 May;16(5):284\\u0026ndash;7. \\u003c/li\\u003e\\n\\u003cli\\u003eSubramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005 Oct 25;102(43):15545\\u0026ndash;50. \\u003c/li\\u003e\\n\\u003cli\\u003eLuo W, Brouwer C. Pathview: an R/Bioconductor package for pathway-based data integration and visualization. Bioinforma Oxf Engl. 2013 Jul 15;29(14):1830\\u0026ndash;1. \\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":true,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-translational-medicine\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"jtrm\",\"sideBox\":\"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/jtrm/default.aspx\",\"title\":\"Journal of Translational Medicine\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Colorectal adenoma, biomarker, gut microbiota, Christensenella hongkongensis\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7436037/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7436037/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e\\u003cp\\u003eColorectal cancer (CRC) is one of the most prevalent malignancies worldwide and commonly starts from a pre-cancerous stage. This study aimed to identify potential fecal bacterial candidates associated with progression of CRC from the adenoma-carcinoma sequence and to explore underlying mechanisms of carcinogenesis.\\u003c/p\\u003e\\u003ch2\\u003eMethods\\u003c/h2\\u003e\\u003cp\\u003ePublicly metagenomic datasets were analyzed using MaAsLin2 to identify bacterial species enriched in CRC patients compared to healthy controls. Additionally, we established a large cohort in mainland China, consisting of 686 subjects (285 CRC patients, 73 advanced adenoma patients, 134 non-advanced adenoma patients, and 194 healthy controls). Fecal samples from this cohort were analyzed by duplex quantitative polymerase chain reaction (qPCR) to validate the abundance of key bacterial candidate and its association with tumor node metastasis (TNM) stages. \\u003cem\\u003eIn vitro\\u003c/em\\u003e experiments and transcriptome sequencing were performed to explore the effects of \\u003cem\\u003eChristensenella hongkongensis\\u003c/em\\u003e (\\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e) and its mechanisms in CRC progression.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e\\u003cp\\u003eMaAsLin2 analysis identified seven bacterial species were significantly more abundant in fecal samples of CRC patients than in healthy controls (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Among them, \\u003cem\\u003eChristensenella hongkongensis\\u003c/em\\u003e, an obligately anaerobic, catalase-positive, motile, non-sporulating, gram-positive coccobacillus was distinguished by its lowest abundance in healthy controls and significant enrichment in CRC patients. Validation in our recruited cohort showed that the abundance of \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e progressively increased from non-advanced adenomas to advanced adenomas and CRC. Linear regression analysis revealed a significant positive association between \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e and TNM stages in CRC. \\u003cem\\u003eIn vitro\\u003c/em\\u003e experiments showed that \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e promoted CRC cell proliferation, inhibited apoptosis, and enhanced the growth of patient-derived CRC organoids. RNA-seq analysis identified activation of the Wnt/β-catenin signaling pathway, which was further validated by the upregulation of downstream targets such as c-Jun and Cyclin-D1.\\u003c/p\\u003e\\u003ch2\\u003eConclusions\\u003c/h2\\u003e\\u003cp\\u003eOur findings suggest that \\u003cem\\u003eC. hongkongensis\\u003c/em\\u003e promotes colorectal tumorigenesis \\u003cem\\u003evia\\u003c/em\\u003e Wnt/β-catenin activation, and highlights its potential as a novel non-invasive bacteria marker for early detection and monitoring of CRC progression.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Discovery and characterization of Christensenella hongkongensis as a novel bacterium in the adenoma-carcinoma progression\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-09-15 06:21:00\",\"doi\":\"10.21203/rs.3.rs-7436037/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"reviewerAgreed\",\"content\":\"\",\"date\":\"2025-09-07T08:38:25+00:00\",\"index\":0,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-09-02T12:33:52+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-08-25T14:54:47+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Journal of Translational Medicine\",\"date\":\"2025-08-22T11:53:27+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-translational-medicine\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"jtrm\",\"sideBox\":\"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/jtrm/default.aspx\",\"title\":\"Journal of Translational Medicine\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"cfa0d002-51f1-44da-9316-d0e4f5cd96cc\",\"owner\":[],\"postedDate\":\"September 15th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-03-02T16:01:20+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-7436037\",\"link\":\"https://doi.org/10.1186/s12967-026-07886-9\",\"journal\":{\"identity\":\"journal-of-translational-medicine\",\"isVorOnly\":false,\"title\":\"Journal of Translational Medicine\"},\"publishedOn\":\"2026-02-28 15:58:06\",\"publishedOnDateReadable\":\"February 28th, 2026\"},\"versionCreatedAt\":\"2025-09-15 06:21:00\",\"video\":\"\",\"vorDoi\":\"10.1186/s12967-026-07886-9\",\"vorDoiUrl\":\"https://doi.org/10.1186/s12967-026-07886-9\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7436037\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7436037\",\"identity\":\"rs-7436037\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}