Association of Fusobacterium nucleatum with suppressed anti-tumor T cell responses in gastric cancer

Association of Fusobacterium nucleatum with suppressed anti-tumor T cell responses in gastric cancer | 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 Association of Fusobacterium nucleatum with suppressed anti-tumor T cell responses in gastric cancer Yung-Yu Hsieh, Wen-Chun Liu, Chiu-Tzu Wang, Wen-Lin Kuo, Chin Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8216991/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background Gastric cancer is etiologically linked to pathogenic microbes. Fusobacterium nucleatum in gastric cancer-associated microbiota is associated with high tumor mutation burden and poor prognosis. F. nucleatum was also shown to suppress anti-tumor immune response in colorectal cancer. The aim of this study is to identify the F. nucleatum -induced change in the immune microenvironment of gastric cancer. Materials Resected gastric cancer specimens and endoscopic-extracted biopsies were obtained from Human Biobank of Chiayi Chang Gung Memorial Hospital. The presence of F. nucleatum in the specimens was determined by nested PCR. Resected specimens were analyzed by transcriptomic analysis. In vitro experiment using F. nucleatum -infected gastric cancer cell lines was utilized to identify deregulated genes in the cancer cells by F. nucleatum infection. Results Helicobacter pylori infection was significantly declined among gastric cancer patients in Southwestern Taiwan. Conversely, F. nucleatum was identified in nearly 50% of patients, emerging as the dominant oncogenic infection. Transcriptomic and ontological analyses of resected specimens revealed that F. nucleatum correlates with increased T cell markers, including T cell receptor constant region and CD3E . T cell receptor subunit levels correlated with CD8A , indicating cytotoxic T cell infiltration in F. nucleatum -positive lesions. However, upregulation of negative regulators LAX1 and PRDM1 , proportional to CD3E and CD8A , suggests these abundant T cells are inactive. In vitro , F. nucleatum-induced CXCL9 and CXCL10 upregulation in select gastric cancer cell lines. Correspondingly, CXCL9 and CXCL10 were elevated and highly correlated with CD3E and CD8A in a subset of F. nucleatum -positive specimens. Conclusions F. nucleatum infection in gastric cancer increased drastically in Taiwan. Higher abundance of cytotoxic T cells is associated with F. nucleatum , likely being attracted to tumor sites by F. nucleatum -induced CXCL9 and CXCL10 . However, despite being attracted to gastric tumor sites, these tumor-infiltrating cytotoxic T cells are inactive and exhibit an exhausted phenotype, indicating that F. nucleatum is associated with an immunosuppressive microenvironment. Our finding suggest that targeting F. nucleatum could promote anti-tumor immune response and subsequently the treatment efficacy. Fusobacterium nucleatum gastric cancer PRDM1 CXCL9 CXCL10 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The role of oncogenic microbial infection as a driver of cancer is well-established. Specifically for gastric cancer, Helicobacter pylori is recognized as the principal risk factor [1], and comprehensive eradication programs have demonstrated a significant impact on reducing its incidence [2, 3]. In Taiwan, the current age-standardized incidence stands at 9.81 per 100,000 person-years, with a male-female ratio of 1.71 [4]. Although a gradual decline in age-standardized incidence has been noted, the absolute number of crude cases has remained relatively stable, primarily attributable to the rapid demographic aging of the Taiwanese population. Notwithstanding advancements in chemotherapeutic regimens, a substantive improvement in the five-year survival rate has not been observed [4]. Consequently, a significant medical need persists to both mitigate the occurrence and enhance the therapeutic outcomes for gastric cancer. Fusobacterium nucleatum , an anaerobic bacillus residing within the oral microbiota [5], has been demonstrably linked to colorectal [6-8], gastric [9-11], oral [12, 13], and esophageal cancers [11, 13]. These observations collectively establish F. nucleatum infection as a prevalent event in malignancies of the gastrointestinal tract. Transmission of F. nucleatum from the oral cavity to cancer tissue, leading to colonization, can occur via either hematogenous dissemination or ingestion [14-16]. However, only strains endowed with specific genetic attributes are capable of re-establishing colonization within cancerous lesions [17]. A recent examination of F. nucleatum clinical isolates derived from colorectal cancer revealed that the cancer-colonizing strains, categorized as the C2 clade, harbor a suite of virulence factors including fadA , fap2, fplA , radD , aim1 , cmpA , and fusolisin [17]. Additionally, the putative eut and pdu operons, integral to ethanolamine and 1,2-propanediol metabolism, were also identified within the C2 clade [17]. Considering the established association of F. nucleatum with microsatellite instability in colorectal cancer [18] and high tumor mutation burden in gastric cancer [19], it is highly probable that the virulent genes of F. nucleatum are responsible for inducing these genomic changes in cancer cells. Experimental investigations have demonstrated that F. nucleatum exhibits both adhesive and invasive capabilities with respect to cancer cells [17]. In situ analyses of cancer tissue sections revealed the intracellular localization of F. nucleatum within neoplastic cells [20]. Spatial single-cell sequencing, applied to tissue sections of oral and colorectal cancers [21], further indicated that F. nucleatum is distributed extensively within the tumor mass. Moreover, clinical isolates of F. nucleatum belonging to the C2 clade possess the capacity to invade colorectal cancer cells under controlled in vitro co-culture conditions [17]. These collective observations strongly support the premise that F. nucleatum establishes an intracellular presence, likely thereby exerting a protracted influence on cancer progression. F. nucleatum exerts modulatory effects on the tumor microenvironment, thereby suppressing the anti-tumor immune response in esophageal [20, 22], breast [23], gastric [24], and colorectal cancers [25-28]. Clinically, the presence of F. nucleatum in colorectal and breast cancer correlates with enhanced therapeutic outcomes following immune checkpoint inhibitor therapy [27, 29, 30]. This phenomenon suggests that immune recognition of F. nucleatum- infected cancer cells occurs, yet the anti-cancer response is actively inhibited. Herein, we report the comprehensive profiling of F. nucleatum- infected gastric cancer tissue. Our data indicate that F. nucleatum infection promotes T cell exhaustion, notwithstanding the recruitment of an increased number of immune cells to the neoplastic tissue. Our observations therefore support the notion that F. nucleatum actively modulates the tumor microenvironment to facilitate cancer immune evasion. This finding implicates F. nucleatum in contributing to cancer progression and suggests its potential utility as a predictive biomarker for successful immunotherapy in gastric cancer . Materials and Methods Study cohort This study was approved by the Institutional Review Board of Chiayi Chang Gung Memorial Hospital (IRB approval Nos. 202102249B0 and 202202139B0). Sections of formalin-fixed, paraffin-embedded (FFPE) resected gastric cancer tissues were procured from the Human Biobank of Chiayi Chang Gung Memorial Hospital. Acquisition and subsequent use of all clinical specimens were conducted in strict accordance with the principles outlined in the Declaration of Helsinki. In total, DNA and RNA were successfully isolated from 29 surgically resected gastric cancer tissues and 45 upper endoscopy-derived biopsy gastric cancer tissues. H. pylori infection status was ascertained using the standard rapid urease test at the time of initial specimen collection. Purification of RNA and DNA was executed utilizing the RNAeasy FFPE kit and DNA FFPE Advanced UNG kit (Qiagen, Hilden, Germany), respectively. We determined the RNA integrity number of purified RNA on a TapeStation 2200 system (Agilent, Santa Clara, CA, USA), and measured the concentration of RNA and DNA using a Qubit fluorometer (Thermo Fisher, Waltham, MA, USA). We detected F. nucleatum presence in the DNA specimens by nested PCR of the NusG gene as previously described. Transcriptomic analysis Total RNA extracted from surgically resected specimens was used to prepare sequencing-ready libraries with the SureSelect XT HS2 RNA library preparation kit (Agilent). These libraries were subsequently sequenced on a NovaSeq X sequencer (Illumina, San Diego, CA, USA). Sequencing reads were then collapsed using the Trimmer package (Agilent) and imported into CLC Genomic Workbench v.25.0 (QIagen) for downstream analysis. Quality-trimmed reads were mapped and annotated against the human reference genome GRCh38.p14. The cohort was stratified into F. nucleatum -positive and F. nucleatum -negative groups according to nested PCR results. Differentially expressed genes between these two groups were identified using CLC Genomic Workbench. Ontology analysis was conducted using both the NCBI DAVID platform web service and a locally run GESA analysis program. Cell and bacterial culture The gastric cancer cell lines AGS, MKN29, and MKN45, utilized in this study, were acquired from the Bioresource Collection and Research Center (Hsinchu). MKN-28 is a well-differentiated gastric cell line, originating from an adenocarcinoma with intestinal differentiation morphology, whereas MKN-45 is derived from a poorly differentiated gastric adenocarcinoma. AGS, also established from a gastric adenocarcinoma, is characterized by a defect in peptidoglycan-recognition NOD1. All cell lines were maintained in DMEM or RPMI medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 atmosphere. F. nucleatum ATCC25586 was obtained from the Bioresource Collection and Research Center (Hsinchu) and cultured anaerobically on blood agar. Colony formation was generally observed on culture plates after 4 to 6 days of incubation at 37°C. Cellular co-culture with Fusobacterium nucleatum Prior to initiating co-culture experiments, F. nucleatum was harvested from plates by scraping and resuspended in fresh cell culture medium. Bacterial quantification was performed via direct microscopic counting, followed by dilution to the desired concentration. The bacterial suspension was then deposited onto cultured cells at a multiplicity of infection (MOI) of 100. Following a two-day infection period, the cells were resuspended in phosphate-buffered saline, subjected to extensive washing to eliminate unattached F. nucleatum , and subsequently reseeded onto clean culture plates. Co-cultured cells were collected at 2 and 6 days after the wash step. Total RNA was extracted from these co-cultured cells using TRIZOL reagent (Thermo Fisher) and subjected to RNA sequencing. Results Eradication of H. pylori serves as a standard preventative treatment to reduce gastric cancer incidence. The cohort in this study consisted of patients who underwent gastrectomy between 2020 and 2022. The presence of F. nucleatum in the specimens was determined by nested PCR, revealing that 14 of 24 specimens (58.3%) were positive for F. nucleatum NusG (Fig. S1). H. pylori infection status, recorded in the clinical data retrieved from the Human Biobank, indicated a prevalence of only 16.7% in this cohort after excluding patients with no confirmed H. pylori status. Despite the reduced H. pylori prevalence, the majority of H. pylori -positive specimens were concurrently positive for F. nucleatum (Table 1). To further validate this observed change in microbial risk, an independent cohort of biopsy specimens collected during upper gastrointestinal endoscopic examination was also analyzed. The results from these biopsies reaffirmed the findings obtained from surgically resected tissues (Table 1). A comparison with our earlier study revealed a significant alteration in the microbial risk profile of gastric cancer in Taiwan. Patients diagnosed by 2015 in the previous investigation were predominantly H. pylori -positive [31], indicating that H. pylori remained a prevalent pathogenic factor for gastric cancer in the early 2010s (Table 1). The prevalence of F. nucleatum in that earlier cohort was 26%, and the majority of F. nucleatum -infected specimens also exhibited H. pylori infection (Table 1). By comparing the data from the earlier and most recent cohorts, it becomes evident that the prevalence of H. pylori significantly decreased in Taiwan over the past decade, concurrently with an increase in F. nucleatum prevalence during the same period. Therefore, these results signify a substantial change in the bacterial risk factors associated with gastric cancer in Taiwan. Given the observed significant epidemiological shift, this study specifically investigated the impact of F. nucleatum on H. pylori -negative gastric cancer. The pathological characteristics of the H. pylori -negative patients included in this study are detailed in Tables S1 and S2. For patients undergoing surgery, clinical and TNM staging was determined post-operatively. Within the group of resected specimens, F. nucleatum infection demonstrated no stage dependence (Table S1). In the biopsy specimens, the prevalence of F. nucleatum infection appeared higher in later-stage (17 of 29 specimens; stages III and IV) compared to early-stage (2 of 6 specimens; stages I and II) patients (Table S2); however, this observation might be attributable to the limited number of early-stage specimens. Overall, our analysis revealed that F. nucleatum infection in H. pylori -negative gastric cancer was not associated with patients’ age, sex, HER2 status, or cancer stage. F. nucleatum exhibits a complex association with colorectal cancer, correlating with poor prognosis yet also with favorable outcomes from immune checkpoint inhibitor therapy [29]. These observations indicate that F. nucleatum may promote cancer progression by actively suppressing anti-cancer immune responses. Whether F. nucleatum exerts similar effects within the gastric cancer microenvironment, however, remains to be elucidated. To investigate the impact of F. nucleatum on the anti-tumor immune response in gastric cancer, we performed transcriptome analysis of resected tissues to determine immune activity. Differentially expressed protein-coding genes in F. nucleatum -positive specimens were identified using the Mann–Whitney U test, and subsequent pathway analysis was conducted via the NCBI DAVID platform. Pathways with a p -value less than 0.05 are presented in Figure 1. The most striking deregulated functions observed included the activation of the immune response and several pathogen-response pathways, particularly those related to viral and bacterial infection. Genes associated with endocytosis were also deregulated, likely as a consequence of pathogen infection. GESA analysis also revealed the upregulation of chemokine signaling, B cell receptor signaling, T cell receptor signaling, and NK cell-mediated cytotoxicity among the deregulated pathways in F. nucleatum -positive specimens (Fig. 1B). Detailed inspection of the datasets showed an increase in immunoglobulin-encoding genes, including IgD and IgG2 heavy chain ( IGDH and IGHG2 ), and kappa and lambda light chain constant regions ( IGLC1 , IGLC2 , IGLC3 , and IGKC ). Furthermore, CD79A , a component of the B-cell receptor, and CD27 , a marker for memory B cells, were found at higher levels in multiple F. nucleatum -positive specimens (Fig. 2). Beyond B cells, an increase in T cell markers was also observed, encompassing T cell receptor β-chain constant region ( TRBC1 and TRBC2_1 ), CD8A , and CD3E . The T cell activation scaffold protein LAT also exhibited increased expression (Fig. 2). Given that CD4 showed no significant change in F. nucleatum -positive specimens (data not shown), the predominant population of infiltrating T cells in F. nucleatum- positive specimens is likely cytotoxic T cells. Conversely, FUT4 , FCGR3A , and FCGR3B showed no significant difference, and MPO levels were barely detectable across all specimens. These data suggest the presence of macrophages within the gastric cancer tumor mass but indicate minimal infiltration by neutrophils. Therefore, our analysis suggests that the effector cells of innate immunity are not specifically attracted to the tumor sites despite F. nucleatum infection. Further examination of immune cell markers revealed an association between F. nucleatum and upregulated adaptive immunity. Elevated levels of CD6 , CD7 , and CD84 in F. nucleatum -positive specimens provide additional evidence for T cell infiltration. Moreover, CD74 and CLEC10A , both implicated in antigen presentation, were also upregulated in F. nucleatum -positive specimens (Fig. 2). The expression levels of other genes broadly involved in intracellular signaling within immune cells, including PIK3CG , PTK2B , IRAG2 , and MPEG1 , were similarly elevated in F. nucleatum -positive specimens. Conversely, MPEG1 and CTSC, both intracellular effector proteins critical for pathogen destruction, were also upregulated in F. nucleatum -positive specimens. This observation suggests potential macrophage activation in response to F. nucleatum infection. Collectively, our analysis indicates that the effector cells of adaptive immunity are specifically recruited to tumor lesions by F. nucleatum . To validate the accuracy of our sequencing data, we performed a linear correlation analysis and thus determine whether the coding genes of the T cell receptor (TCR) complex were coordinately detectable in the specimens. Across the entire cohort, the TCR α chain constant region gene TRAC, β chain constant region gene TRΒC2_1 , and CD8A correlated with CD3E levels (Fig. 3A). When specimens were stratified into F. nucleatum -positive and -negative groups, TRAC showed no statistical difference in expression level between groups but maintained a correlation with CD3E within each group (data not shown). CD8A and CD6 also correlated with CD3E levels in both groups (Fig. 3B). However, the TCR β chain constant region genes TRBC1 and TRBC2_1 did not correlate with CD3E in F. nucleatum -negative specimens (Fig. 3), a discrepancy likely attributable to greater variation in calculated TPM at low expression levels. Taken together, these results indicate that CD3E levels reliably represent the relative abundance of the total T cell population in the specimens. Furthermore, the correlation between cytotoxic T cell markers CD8A and CD3E suggests that the abundance of cytotoxic T cells is proportional to the total T cell population. Furthermore, a subset of F. nucleatum-positive specimens exhibited elevated levels of TRDC and TRGC2 encoding the TCR γ and δ chain constant regions, respectively (Fig. 3C). This observation suggests the presence of γδ T cells in these samples. In addition to TCR components, DOCK2 was found at higher levels in F. nucleatum -positive specimens and showed a correlation with CD8A , CD3E , and TCR β chain (Fig. 3D). Given the critical role of DOCK2 in lymphocyte function and migration, its upregulation provides further evidence of an increased influx of cytotoxic T cells into the tumor microenvironment. Our analysis further demonstrated the upregulation of T cell negative regulators. Specifically, both LAX1 and PRDM1 exhibited elevated expression in the F. nucleatum -positive specimens (Fig. 4). Functionally, LAX1 serves as a negative regulator of B and TCR signaling, while PRDM1 is indicative of T cell exhaustion. Correlation analysis revealed a positive correlation between LAX1 and PRDM1 with CD3E when all specimens were combined for analysis (data not shown); however, this statistical significance was not maintained when specimens were stratified into F. nucleatum -negative and -positive groups (Fig. 4). Conversely, LAX1 and PRDM1 were found to correlate with CD8A specifically in the F. nucleatum -positive specimens. Therefore, despite their increased abundance, cytotoxic T cells in F. nucleatum -positive specimens appear to be functionally suppressed. PD-L1 expressed by the cancer cells is a key mechanism to suppress the anti-tumor immune response, but our data show that most F. nucleatum -positive specimens (all but two) have low CD274 (PD-L1) expression (Fig. 4), with only 8.7% of all samples exhibiting high levels. Hence, PD-L1 is activated through distinct pathway independent from F. nucleatum infection. On the other hand, TGFB1 was found to be upregulated and positively correlated with PRDM1 in F. nucleatum -positive specimens (Fig. 4), thereby implicating a direct role for TGFβ signaling in the observed immune suppression. An in vitro infection model experiment was subsequently conducted to elucidate the underlying mechanism by which F. nucleatum promotes immune cell infiltration into tumor sites. To simulate infection, F. nucleatum ATCC 25586 was introduced to gastric cancer cell lines MKN28, MKN45, and AGS at an MOI of 100 (Fig. 6). After two days of co-culturing, the cells were detached, thoroughly washed to eliminate unattached extracellular pathogens, and reseeded onto fresh culture plates. Following an additional 2 and 6 days of culture, total RNA was extracted and analyzed by RNA sequencing. Pathway analysis indicated that different gastric cancer cell lines exhibited distinct responses to F. nucleatum infection. MKN28, a well-differentiated adenocarcinoma cell line harboring TP53 and APC mutations, showed no clear response to F. nucleatum infection. In contrast, an inflammatory response was induced in the poorly-differentiated MKN45 and moderately-differentiated AGS cell lines. In particular, CXCL10 was upregulated in MKN45 at the 4-day time point but reverted to baseline expression levels by day 8 (Fig. 5A). In AGS, the expression of both CXCL9 and CXCL10 was significantly elevated at day 4 and maintained this upregulation through day 8. Moreover, TLR3 also exhibited upregulation at day 4 (Fig. 5A). Given that both CXCL9 and CXCL10 are recognized as potent chemoattractants for activated T cells, this in vitro challenge experiment proposes a potential mechanism for F. nucleatum -mediated augmentation of T cell abundance within tumor sites. To provide corroborating data for our in vitro findings, we re-examined the expression levels of CXCL9 and CXCL10 in clinical specimens. While no statistically significant difference was observed between the two groups (Mann–Whitney U test, p >0.05), it is evident that a greater proportion of F. nucleatum -positive specimens displayed high CXCL9 or CXCL10 expression (Fig. 5B). Although a few F. nucleatum-negative specimens also exhibited high CXCL9 or CXCL10 , this expression was not correlated with CD3E levels (Fig. 5C). In comparison, within the F. nucleatum -positive specimens, CXCL9 or CXCL10 was strongly correlated with CD3E , supporting the notion that F. nucleatum induces CXCL9 and CXCL10 in a subset of gastric cancers, subsequently promoting T cell infiltration into tumor lesions. Discussion Gastric cancer continues to rank among the top ten most prevalent cancers in Taiwan. The present findings unequivocally demonstrate the efficacy of public health policy interventions in reducing H. pylori infection, evident in its prevalence decreasing from 70% to under 20% within a period of fewer than ten years. Notwithstanding this considerable decline in H. pylori prevalence, the incidence of gastric adenocarcinoma has exhibited only a marginal decrease, which strongly suggests the involvement of additional, as-yet-unmitigated risk factors. A concurrent increase in F. nucleatum infection indicates a profound shift in the pathogenic microbial landscape contributing to gastric cancer, particularly given a recent animal study demonstrating the sufficiency of F. nucleatum alone to induce gastric cancer in the absence of H. pylori . Consequently, our rigorous analysis of clinical specimens strongly implicates that F. nucleatum possesses the potential to supersede H. pylori as the predominant carcinogenic bacterial infection driving the pathogenesis of gastric cancer. A discernible increase in infiltrating cytotoxic T cells was consistently observed within F. nucleatum -positive gastric cancer. Under controlled in vitro conditions, F. nucleatum demonstrably induced the upregulation of CXCL10 in both MKN45 and AGS cell lines, and of CXCL9 in MKN45. Given that F. nucleatum has been extensively documented as detectable intracellularly in comparable in vitro experimental setups across multiple prior investigations, and the observed upregulation of TLR3 in AGS further implies an intracellular mode of F. nucleatum infection, our collective results suggest that intracellular F. nucleatum elicited an inflammatory response. This response encompassed the upregulation of CXCL9 and CXCL10, both of which function as potent chemoattractants to orchestrate the migration of T cells to sites of inflammation. In corroboration with this hypothesis, the levels of CXCL9 and CXCL10 in clinical specimens exhibited a strong correlation with the abundance of T cells. Intriguingly, IL-17, recently implicated as a key mediator in promoting T cell infiltration to gastric cancer [24], was not found to be upregulated in our clinical cohort. This discrepancy potentially indicates that F. nucleatum elicits a convergent immune response through distinct underlying pathways across various gastric cancer cell lines. It is unequivocally clear that not all gastric cancer cell lines respond equivalently to F. nucleatum , as exemplified by the complete absence of an inflammatory response in MKN28. While specific genetic aberrations could play a role in modulating the cellular response to F. nucleatum , comprehensive further analysis is warranted to identify the genes critically essential for the F. nucleatum -induced inflammatory response. A concomitant increase in the abundance of T cells was, however, correlated with the upregulation of negative immunoregulators. This finding is entirely consistent with the observed poorer prognosis but paradoxically improved clinical benefits derived from immune checkpoint inhibitors in F. nucleatum -positive cancers. Only a restricted subset of gastric cancers manifest high PD-L1 expression, but it appears to be independent from F. nucleatum infection; moreover, in the absence of combined positive score data determined by immunohistochemistry in the available clinical records, the precise correspondence between observed RNA levels and clinically relevant PD-L1 expression remains to be fully elucidated. PDCD-1 , the gene encoding PD-1, was detectable at uniformly low levels across all analyzed samples (data not shown), rendering it an impractical biomarker for predicting immunotherapy outcomes in this context. Conversely, elevated levels of CTLA-4 were detected in a minority of samples, yet these elevations showed no association with F. nucleatum presence, PRDM1 expression, or CD274 levels (data not shown). In contrast, TGFB1 was strongly upregulated in multiple F. nucleatum-positive specimens. While TGFβ signaling is known to exert a pivotal role in immune suppression, the specific cellular source responsible for the release of TGFβ within the F. nucleatum-infected microenvironment remains unclarified. Interestingly, TGFB1 expression was not stimulated by F. nucleatum in the in vitro cell line models utilized in this study, suggesting that TGFβ is released from other types of cells in the tumor microenvironment. Although the detail molecular mechanism remains unillustrated, our comprehensive analysis employing clinical specimens provides robust evidence that F. nucleatum is intricately associated with a suppressed immune response, thereby contributing to cancer progression. Declarations Ethics approval This study was approved by the Institutional Review Board of Chiayi Chang Gung Memorial Hospital (approval number IRB 202102249B0 and IRB 202202139B0). Clinical specimens used in this study was acquired from the Human BioBank of Chiayi Chang Gung Memorial Hospital. Human BioBank was established under the regulation of the Human Biobank Management Act. Informed consents were obtained from the patients before acceptance of the specimens to the Human BioBank. Subsequent use of the specimens requires approval from the Institutional Review Board, but additional informed consent from the patients is not required according to the regulation of the Human Biobank Management Act. Consent for publication Not applicable. Availability of data and materials The sequencing data is deposited to Sequence Read Archive, NCBI. The accession numbers of Bioprojects are PRJNA1302123 and PRJNA1302213. Competing interests Authors declare no conflict of interest. Funding This work was supported by the funding awarded to Y.Y.H. from Chiayi Chang Gung Memorial Hospital, Taiwan (grant number CMRPG6M0291, NMRPG6N6021) and National Science and Technology Council, R.O.C (grant number NSTC 112-2314-B-182A-139 -MY3). Author contributions Y.Y.H. recruited patients to the study and oversaw the study. W.L.K., C.T.W., and W.L.K. performed sample processing and experiments. C.L. and Y.Y.H. carried out data analysis and manuscript writing. References Duan Y, Xu Y, Dou Y, Xu D: Helicobacter pylori and gastric cancer: mechanisms and new perspectives. J Hematol Oncol 2025, 18(1):10. Chen MJ, Bair MJ, Chen PY, Lee JY, Yang TH, Fang YJ, Chen CC, Chang AT, Hsiao WD, Yu JJ et al : Declining trends of prevalence of Helicobacter pylori infection and incidence of gastric cancer in Taiwan: An updated cross-sectional survey and meta-analysis. Helicobacter 2022, 27(5):e12914. 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Wang X, Fang Y, Liang W, Wong CC, Qin H, Gao Y, Liang M, Song L, Zhang Y, Fan M et al : Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer. Cancer Cell 2024, 42(10):1729-1746 e1728. Duizer C, Salomons M, van Gogh M, Grave S, Schaafsma FA, Stok MJ, Sijbranda M, Kumarasamy Sivasamy R, Willems RJL, de Zoete MR: Fusobacterium nucleatum upregulates the immune inhibitory receptor PD-L1 in colorectal cancer cells via the activation of ALPK1. Gut Microbes 2025, 17(1):2458203. Gao Y, Bi D, Xie R, Li M, Guo J, Liu H, Guo X, Fang J, Ding T, Zhu H et al : Fusobacterium nucleatum enhances the efficacy of PD-L1 blockade in colorectal cancer. Signal Transduct Target Ther 2021, 6(1):398. Guo J, Zhu P, Li J, Xu L, Tang Y, Liu X, Guo S, Xia J: Fusobacterium nucleatum promotes PD-L1 expression in cancer cells to evade CD8(+) T cell killing in breast cancer. Hum Immunol 2024, 85(6):111168. Hsieh YY, Tung SY, Pan HY, Chang TS, Wei KL, Chen WM, Deng YF, Lu CK, Lai YH, Wu CS et al : Fusobacterium nucleatum colonization is associated with decreased survival of helicobacter pylori-positive gastric cancer patients. World J Gastroenterol 2021, 27(42):7311-7323. Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":707789,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic pathway analysis of \u003cem\u003eF. nucleatum\u003c/em\u003e-associated gene expression alterations in \u003cem\u003eH. pylori\u003c/em\u003e-negative gastric cancer. Total RNA, isolated from FFPE sections of surgically resected \u003cem\u003eH. pylori\u003c/em\u003e-negative gastric cancer specimens (\u003cem\u003eF. nucleatum\u003c/em\u003e-negative, n=10; \u003cem\u003eF. nucleatum\u003c/em\u003e-positive, n=13), was utilized for the generation of sequencing libraries. Subsequent sequencing reads were mapped and annotated against the human reference genome GRCh38.p14. (A) Identification of differentially expressed protein-coding genes was performed using the Mann–Whitney U test. Functional enrichment of cellular pathways associated with these differentially expressed genes was conducted via the NCBI DAVID service, with pathways demonstrating a \u003cem\u003ep\u003c/em\u003e-value less than 0.05 depicted. (B) The prepared dataset was further analyzed by GESA 4.4.0, with a selection of relevant immune response pathways presented.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8216991/v1/f6796f2d845ebcb6af20cbed.jpeg"},{"id":100388582,"identity":"06044990-c10a-44e2-91f3-2da7267f0eec","added_by":"auto","created_at":"2026-01-16 11:17:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1536672,"visible":true,"origin":"","legend":"\u003cp\u003eSelected immune-specific genes differentially expressed in \u003cem\u003eF. nucleatum\u003c/em\u003e-positive vs. -negative \u003cem\u003eH. pylori\u003c/em\u003e-negative gastric cancer. Differential expression is shown for selected immune-specific genes between \u003cem\u003eF. nucleatum\u003c/em\u003e-negative (n=10) and -positive (n=13) resected \u003cem\u003eH. pylori\u003c/em\u003e-negative gastric cancer specimens. \u003cem\u003eP\u003c/em\u003e-values were calculated by the Mann–Whitney U test.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8216991/v1/1c356fbb3201b7d9a3e8b021.png"},{"id":100388173,"identity":"8e79ef06-1f08-490b-8871-6722af392a20","added_by":"auto","created_at":"2026-01-16 11:17:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1971078,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelated expression patterns of TCR components and a positive migration regulator in \u003cem\u003eF. nucleatum\u003c/em\u003e-positive gastric cancer. (A) Transcriptomic analysis depicting the coordinated detection and correlated expression of \u003cem\u003eTRAC\u003c/em\u003e, \u003cem\u003eTRAB2_1\u003c/em\u003e, and \u003cem\u003eCD8A \u003c/em\u003ewith \u003cem\u003eCD3E\u003c/em\u003e. (Β) Correlation analysis of \u003cem\u003eTRBC1\u003c/em\u003e, \u003cem\u003eTRAB2_1\u003c/em\u003e, \u003cem\u003eCD8A, and CD8A, \u003c/em\u003eand\u003cem\u003e CD6 \u003c/em\u003ewith CD3E expression within the \u003cem\u003eF. nucleatum\u003c/em\u003e-negative (n=10) and -positive (n=13) specimen cohorts.Additionally, the expression level of \u003cem\u003ePRDM1 \u003c/em\u003ewas positively correlated with \u003cem\u003eCD8A \u003c/em\u003especifically\u003cem\u003e \u003c/em\u003ein the \u003cem\u003eF. nucleatum\u003c/em\u003e-positive specimens.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8216991/v1/46e0e5a106a385d6fa30200e.png"},{"id":100388537,"identity":"4c1946b3-6a3b-4156-8dde-2d1888559262","added_by":"auto","created_at":"2026-01-16 11:17:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":887461,"visible":true,"origin":"","legend":"\u003cp\u003eExpression and correlation of \u003cem\u003eLAX1\u003c/em\u003e, \u003cem\u003ePRDM1\u003c/em\u003e, \u003cem\u003eCD274\u003c/em\u003e, and \u003cem\u003eTGFB1 \u003c/em\u003ein \u003cem\u003eF. nucleatum\u003c/em\u003e-positive and -negative gastric cancer. Expression levels of \u003cem\u003eLAX1\u003c/em\u003e, \u003cem\u003ePRDM1\u003c/em\u003e, \u003cem\u003eCD274\u003c/em\u003e, and \u003cem\u003eTGFB1\u003c/em\u003e in \u003cem\u003eF. nucleatum\u003c/em\u003e-negative (n=10) and -positive (n=13) resected gastric cancer specimens. Correlations of \u003cem\u003eLAX1\u003c/em\u003e, \u003cem\u003ePRDM1\u003c/em\u003e, and \u003cem\u003eCD274 \u003c/em\u003ewith \u003cem\u003eCD3E \u003c/em\u003eand \u003cem\u003eCD8A \u003c/em\u003eare plotted. Correlation of \u003cem\u003eTGFB1\u003c/em\u003e with\u003cem\u003e PRDM1 \u003c/em\u003eis plotted.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8216991/v1/8efb28261efdd60832e8a1cc.png"},{"id":100388350,"identity":"df89b95e-5169-4f27-9b25-bb4d1e6c371c","added_by":"auto","created_at":"2026-01-16 11:17:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1142333,"visible":true,"origin":"","legend":"\u003cp\u003eUpregulation of \u003cem\u003eCXCL9 \u003c/em\u003eand \u003cem\u003eCXCL10 \u003c/em\u003eby \u003cem\u003eF. nucleatum\u003c/em\u003e. (A) Expression of \u003cem\u003eCXCL9\u003c/em\u003e, \u003cem\u003eCXCL10, \u003c/em\u003eand \u003cem\u003eTLR3 \u003c/em\u003ein MKN45 and AGS cell lines following \u003cem\u003eF. nucleatum \u003c/em\u003echallenge. (B) The expression level of \u003cem\u003eCXCL9\u003c/em\u003e, \u003cem\u003eCXCL10 \u003c/em\u003eand \u003cem\u003eTLR3 \u003c/em\u003ein resected gastric cancer specimens. (C) Correlation between \u003cem\u003eCXCL9 \u003c/em\u003eand \u003cem\u003eCXCL10 \u003c/em\u003ewith \u003cem\u003eCD3E \u003c/em\u003ein resected gastric cancer specimens.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8216991/v1/e80ba36ad42dfa3ba1a9ff3d.png"},{"id":100421648,"identity":"184ab743-af49-42b1-a232-e7cb58a0aa18","added_by":"auto","created_at":"2026-01-16 13:37:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4718824,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8216991/v1/e5d90ef5-0158-4b94-b110-2b9a05204594.pdf"},{"id":100388650,"identity":"902d4183-55be-4cac-a78c-6a376a20ca57","added_by":"auto","created_at":"2026-01-16 11:17:51","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28246921,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaloriginalgelimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8216991/v1/fcfd091448dc9d71638ab573.pdf"},{"id":100388373,"identity":"cda49ca8-99ca-466b-977a-edd805fb353e","added_by":"auto","created_at":"2026-01-16 11:17:24","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":303236,"visible":true,"origin":"","legend":"","description":"","filename":"Supplemental.docx","url":"https://assets-eu.researchsquare.com/files/rs-8216991/v1/372d890f1e054ef1b91fe9e1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Association of Fusobacterium nucleatum with suppressed anti-tumor T cell responses in gastric cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe role of oncogenic microbial infection as a driver of cancer is well-established. Specifically for gastric cancer,\u0026nbsp;\u003cem\u003eHelicobacter pylori\u0026nbsp;\u003c/em\u003eis recognized as the principal risk factor [1], and comprehensive eradication programs have demonstrated a significant impact on reducing its incidence [2, 3]. In Taiwan, the current age-standardized incidence stands at 9.81 per 100,000 person-years, with a male-female ratio of 1.71 [4]. Although a gradual decline in age-standardized incidence has been noted, the absolute number of crude cases has remained relatively stable, primarily attributable to the rapid demographic aging of the Taiwanese population. Notwithstanding advancements in chemotherapeutic regimens, a substantive improvement in the five-year survival rate has not been observed [4]. Consequently, a significant medical need persists to both mitigate the occurrence and enhance the therapeutic outcomes for gastric cancer.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFusobacterium nucleatum\u003c/em\u003e, an anaerobic bacillus residing within the oral microbiota [5], has been demonstrably linked to colorectal [6-8], gastric [9-11], oral [12, 13], and esophageal cancers [11, 13]. These observations collectively establish\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003einfection as a prevalent event in malignancies of the gastrointestinal tract. Transmission of\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003efrom the oral cavity to cancer tissue, leading to colonization, can occur via either hematogenous dissemination or ingestion\u0026nbsp;[14-16]. However, only strains endowed with specific genetic attributes are capable of re-establishing colonization\u0026nbsp;within cancerous lesions\u0026nbsp;[17]. A recent examination of\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eclinical isolates derived\u003cem\u003e\u0026nbsp;\u003c/em\u003efrom colorectal cancer revealed that the cancer-colonizing strains, categorized as the C2 clade, harbor a suite of virulence factors including\u0026nbsp;\u003cem\u003efadA\u003c/em\u003e,\u0026nbsp;\u003cem\u003efap2, fplA\u003c/em\u003e,\u0026nbsp;\u003cem\u003eradD\u003c/em\u003e,\u0026nbsp;\u003cem\u003eaim1\u003c/em\u003e,\u0026nbsp;\u003cem\u003ecmpA\u003c/em\u003e, and fusolisin\u0026nbsp;[17]. Additionally, the putative\u0026nbsp;\u003cem\u003eeut\u0026nbsp;\u003c/em\u003eand\u0026nbsp;\u003cem\u003epdu\u0026nbsp;\u003c/em\u003eoperons, integral to ethanolamine and 1,2-propanediol metabolism, were also identified within the C2 clade\u0026nbsp;[17]. Considering the established association of\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003ewith microsatellite instability in colorectal cancer\u0026nbsp;[18]\u0026nbsp;and high tumor mutation burden in gastric cancer\u0026nbsp;[19], it is highly probable that the virulent genes of\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eare responsible for\u003cem\u003e\u0026nbsp;\u003c/em\u003einducing these genomic changes in cancer cells.\u003c/p\u003e\n\u003cp\u003eExperimental investigations have demonstrated that\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eexhibits both adhesive and\u003cem\u003e\u0026nbsp;\u003c/em\u003einvasive capabilities with respect to cancer cells [17].\u0026nbsp;\u003cem\u003eIn situ\u0026nbsp;\u003c/em\u003eanalyses of cancer tissue sections revealed the intracellular localization of\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003ewithin neoplastic cells\u0026nbsp;[20]. Spatial single-cell sequencing, applied to tissue sections of oral and colorectal cancers\u0026nbsp;[21],\u0026nbsp;further indicated that\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eis distributed extensively within the tumor mass. Moreover, clinical isolates of\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003ebelonging to the\u003cem\u003e\u0026nbsp;\u003c/em\u003eC2\u0026nbsp;clade possess the capacity to invade colorectal cancer cells under controlled\u0026nbsp;\u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eco-culture conditions\u0026nbsp;[17].\u0026nbsp;These collective observations strongly support the premise that\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eestablishes an\u003cem\u003e\u0026nbsp;\u003c/em\u003eintracellular presence,\u0026nbsp;likely thereby exerting a protracted influence on cancer progression.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eexerts modulatory effects on\u003cem\u003e\u0026nbsp;\u003c/em\u003ethe tumor microenvironment, thereby suppressing the anti-tumor immune response in esophageal [20, 22], breast [23], gastric [24], and colorectal cancers [25-28]. Clinically, the presence of\u003cem\u003e\u0026nbsp;F. nucleatum\u0026nbsp;\u003c/em\u003ein colorectal and breast cancer correlates with enhanced therapeutic outcomes following immune checkpoint inhibitor therapy\u0026nbsp;[27, 29, 30]. This phenomenon suggests that immune recognition of\u0026nbsp;\u003cem\u003eF. nucleatum-\u003c/em\u003einfected cancer cells occurs, yet the anti-cancer response is actively inhibited. Herein, we report the comprehensive profiling of\u0026nbsp;\u003cem\u003eF. nucleatum-\u003c/em\u003einfected gastric cancer tissue. Our data indicate that\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003einfection promotes T cell exhaustion, notwithstanding the recruitment of an increased number of immune cells to the neoplastic tissue. Our observations therefore support the notion that\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eactively\u003cem\u003e\u0026nbsp;\u003c/em\u003emodulates the tumor microenvironment to facilitate cancer immune evasion. This finding implicates\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003ein contributing to cancer progression and suggests its potential utility as a predictive biomarker for successful immunotherapy in gastric cancer\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eStudy cohort\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Review Board of Chiayi Chang Gung Memorial Hospital (IRB approval Nos. 202102249B0 and 202202139B0). Sections of formalin-fixed, paraffin-embedded (FFPE) resected gastric cancer tissues were procured from the Human Biobank of Chiayi Chang Gung Memorial Hospital. Acquisition and subsequent use of all clinical specimens were conducted in strict accordance with the principles outlined in the Declaration of Helsinki. In total, DNA and RNA were successfully isolated from 29 surgically resected gastric cancer tissues and 45 upper endoscopy-derived biopsy gastric cancer tissues. \u003cem\u003eH. pylori\u0026nbsp;\u003c/em\u003einfection status was ascertained using the standard rapid urease test at the time of initial specimen collection. Purification of RNA and DNA was executed utilizing the RNAeasy FFPE kit and DNA FFPE Advanced UNG kit (Qiagen, Hilden, Germany), respectively. We determined the RNA integrity number of purified RNA on a TapeStation 2200 system (Agilent, Santa Clara, CA, USA), and measured the concentration of RNA and DNA using a Qubit fluorometer (Thermo Fisher, Waltham, MA, USA). We detected \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003epresence\u003cem\u003e\u0026nbsp;\u003c/em\u003ein the DNA specimens by nested PCR of the \u003cem\u003eNusG\u0026nbsp;\u003c/em\u003egene as previously described.\u003c/p\u003e\n\u003cp\u003eTranscriptomic analysis\u003c/p\u003e\n\u003cp\u003eTotal RNA extracted from surgically resected specimens was used to prepare sequencing-ready libraries with the SureSelect XT HS2 RNA library preparation kit (Agilent). These libraries were subsequently sequenced on a NovaSeq X sequencer (Illumina, San Diego, CA, USA). Sequencing reads were then collapsed using the Trimmer package (Agilent) and imported into CLC Genomic Workbench v.25.0 (QIagen) for downstream analysis. Quality-trimmed reads were mapped and annotated against the human reference genome GRCh38.p14. The cohort was stratified into \u003cem\u003eF. nucleatum\u003c/em\u003e-positive and \u003cem\u003eF. nucleatum\u003c/em\u003e-negative groups according to nested PCR results. Differentially expressed genes between these two groups were identified using CLC Genomic Workbench. Ontology analysis was conducted using both the NCBI DAVID platform web service and a locally run GESA analysis program.\u003c/p\u003e\n\u003cp\u003eCell\u0026nbsp;and\u0026nbsp;bacterial\u003cem\u003e\u0026nbsp;\u003c/em\u003eculture\u003c/p\u003e\n\u003cp\u003eThe gastric cancer cell lines AGS, MKN29, and MKN45, utilized in this study, were acquired from the Bioresource Collection and Research Center (Hsinchu). MKN-28 is a well-differentiated gastric cell line, originating from an adenocarcinoma with intestinal differentiation morphology, whereas MKN-45 is derived from a poorly differentiated gastric adenocarcinoma. AGS, also established from a gastric adenocarcinoma, is characterized by a defect in peptidoglycan-recognition NOD1. All cell lines were maintained in DMEM or RPMI medium supplemented with 10% fetal bovine serum at 37\u0026deg;C in a 5% CO2 atmosphere. \u003cem\u003eF. nucleatum ATCC25586\u0026nbsp;\u003c/em\u003ewas obtained from the Bioresource Collection and Research Center (Hsinchu) and cultured anaerobically on blood agar. Colony formation was generally observed on culture plates after 4 to 6 days of incubation at 37\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eCellular co-culture with \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePrior to initiating co-culture experiments, \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003ewas harvested from plates by scraping and resuspended in fresh cell culture medium. Bacterial quantification was performed via direct microscopic counting, followed by dilution to the desired concentration. The bacterial suspension was then deposited onto cultured cells at a multiplicity of infection (MOI) of 100. Following a two-day infection period, the cells were resuspended in phosphate-buffered saline, subjected to extensive washing to eliminate unattached \u003cem\u003eF. nucleatum\u003c/em\u003e, and subsequently reseeded onto clean culture plates. Co-cultured cells were collected at 2 and 6 days after the wash step. Total RNA was extracted from these co-cultured cells using TRIZOL reagent (Thermo Fisher) and subjected to RNA sequencing.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eEradication of \u003cem\u003eH. pylori\u003c/em\u003e serves as a standard preventative treatment to reduce gastric cancer incidence. The cohort in this study consisted of patients who underwent gastrectomy between 2020 and 2022. The presence of \u003cem\u003eF. nucleatum\u003c/em\u003e in the specimens was determined by nested PCR, revealing that 14 of 24 specimens (58.3%) were positive for \u003cem\u003eF. nucleatum NusG\u003c/em\u003e (Fig. S1). \u003cem\u003eH. pylori\u0026nbsp;\u003c/em\u003einfection status, recorded in the clinical data retrieved from the Human Biobank, indicated a prevalence of only 16.7% in this cohort after excluding patients with no confirmed \u003cem\u003eH. pylori\u0026nbsp;\u003c/em\u003estatus. Despite the reduced \u003cem\u003eH. pylori\u003c/em\u003e prevalence, the majority of \u003cem\u003eH. pylori\u003c/em\u003e-positive specimens were concurrently positive for \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003e(Table 1). To further validate this observed change in microbial risk, an independent cohort of biopsy specimens collected during upper gastrointestinal endoscopic examination was also analyzed. The results from these biopsies reaffirmed the findings obtained from surgically resected tissues (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"697\" height=\"165\" src=\"https://myfiles.space/user_files/58895_8739fc6c57c1c19a/58895_custom_files/img1768315572.jpg\" alt=\"Image 1\"\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;A comparison with our earlier study revealed a significant alteration in the microbial risk profile of gastric cancer in Taiwan. Patients diagnosed by 2015 in the previous investigation were predominantly \u003cem\u003eH. pylori\u003c/em\u003e-positive [31], indicating that \u003cem\u003eH. pylori\u0026nbsp;\u003c/em\u003eremained a prevalent pathogenic factor for gastric cancer in the early 2010s (Table 1). The prevalence of \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003ein that earlier cohort was 26%, and the majority of \u003cem\u003eF. nucleatum\u003c/em\u003e-infected specimens also exhibited \u003cem\u003eH. pylori\u0026nbsp;\u003c/em\u003einfection (Table 1). By comparing the data from the earlier and most recent cohorts, it becomes evident that the prevalence of \u003cem\u003eH. pylori\u0026nbsp;\u003c/em\u003esignificantly decreased in Taiwan over the past decade, concurrently with an increase in \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eprevalence\u003cem\u003e\u0026nbsp;\u003c/em\u003eduring the same period. Therefore, these results signify\u0026nbsp;a substantial change in the bacterial risk factors associated with gastric cancer in Taiwan.\u003c/p\u003e\n\u003cp\u003eGiven the observed significant epidemiological shift, this study specifically investigated the impact of \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eon \u003cem\u003eH. pylori\u003c/em\u003e-negative gastric cancer. The pathological characteristics of the \u003cem\u003eH. pylori\u003c/em\u003e-negative patients included in this study are detailed in Tables S1 and S2. For patients undergoing surgery, clinical and TNM staging was determined post-operatively. Within the group of resected specimens, \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003einfection demonstrated no stage dependence (Table S1). In the biopsy specimens, the prevalence of \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003einfection appeared higher in later-stage (17 of 29 specimens; stages III and IV) compared to early-stage (2 of 6 specimens; stages I and II) patients (Table S2); however, this observation might be attributable to the limited number of early-stage specimens. Overall, our analysis revealed that \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003einfection in \u003cem\u003eH. pylori\u003c/em\u003e-negative gastric cancer was not associated with patients\u0026rsquo; age, sex, HER2 status, or cancer stage.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eexhibits a complex association with colorectal cancer, correlating with poor prognosis yet also with favorable outcomes from immune checkpoint inhibitor therapy [29]. These observations indicate that \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003emay\u003cem\u003e\u0026nbsp;\u003c/em\u003epromote cancer progression by actively suppressing anti-cancer immune responses. Whether \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eexerts similar effects within the gastric cancer microenvironment, however, remains to be elucidated. To investigate the impact of \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eon the anti-tumor immune response in gastric cancer, we performed transcriptome analysis of resected tissues to determine immune activity. Differentially expressed protein-coding genes in \u003cem\u003eF. nucleatum\u003c/em\u003e-positive specimens were identified using the Mann\u0026ndash;Whitney U test, and subsequent pathway analysis was conducted via the NCBI DAVID platform. Pathways with a \u003cem\u003ep\u003c/em\u003e-value less than 0.05 are presented in Figure 1. The most striking deregulated functions observed included the activation of the immune response and several pathogen-response pathways, particularly those related to viral and bacterial infection. Genes associated with endocytosis were also deregulated, likely as a consequence of pathogen infection. GESA analysis also revealed the upregulation of chemokine signaling, B cell receptor signaling, T cell receptor signaling, and NK cell-mediated cytotoxicity among the deregulated pathways in \u003cem\u003eF. nucleatum\u003c/em\u003e-positive specimens (Fig. 1B).\u003c/p\u003e\n\u003cp\u003eDetailed inspection of the datasets showed an increase in immunoglobulin-encoding genes, including IgD and IgG2 heavy chain (\u003cem\u003eIGDH\u0026nbsp;\u003c/em\u003eand \u003cem\u003eIGHG2\u003c/em\u003e), and kappa and lambda light chain constant regions (\u003cem\u003eIGLC1\u003c/em\u003e, \u003cem\u003eIGLC2\u003c/em\u003e, \u003cem\u003eIGLC3\u003c/em\u003e, and \u003cem\u003eIGKC\u003c/em\u003e). Furthermore, \u003cem\u003eCD79A\u003c/em\u003e, a component of the B-cell receptor, and \u003cem\u003eCD27\u003c/em\u003e, a marker for memory B cells, were found at higher levels in multiple \u003cem\u003eF. nucleatum\u003c/em\u003e-positive specimens (Fig. 2).\u0026nbsp;Beyond B cells, an increase in T cell markers was also observed, encompassing T cell receptor \u0026beta;-chain constant region (\u003cem\u003eTRBC1\u003c/em\u003e and \u003cem\u003eTRBC2_1\u003c/em\u003e), \u003cem\u003eCD8A\u003c/em\u003e, and \u003cem\u003eCD3E\u003c/em\u003e. The T cell activation scaffold protein LAT also exhibited increased expression (Fig. 2). Given that \u003cem\u003eCD4\u0026nbsp;\u003c/em\u003eshowed no significant change in \u003cem\u003eF. nucleatum\u003c/em\u003e-positive specimens (data not shown), the predominant population of infiltrating T cells in \u003cem\u003eF. nucleatum-\u003c/em\u003epositive specimens is likely cytotoxic T cells. Conversely, \u003cem\u003eFUT4\u003c/em\u003e, \u003cem\u003eFCGR3A\u003c/em\u003e, and \u003cem\u003eFCGR3B\u0026nbsp;\u003c/em\u003eshowed no significant difference, and \u003cem\u003eMPO\u0026nbsp;\u003c/em\u003elevels were barely detectable across all specimens. These data suggest the presence of macrophages within the gastric cancer tumor mass but indicate minimal infiltration by neutrophils. Therefore, our analysis suggests that the effector cells of innate immunity are not specifically attracted to the tumor sites despite \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003einfection.\u003c/p\u003e\n\u003cp\u003eFurther examination of immune cell markers revealed an association between \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eand upregulated adaptive immunity. Elevated levels of \u003cem\u003eCD6\u003c/em\u003e, \u003cem\u003eCD7\u003c/em\u003e, and \u003cem\u003eCD84\u0026nbsp;\u003c/em\u003ein \u003cem\u003eF. nucleatum\u003c/em\u003e-positive specimens provide additional evidence for T cell infiltration. Moreover, \u003cem\u003eCD74\u0026nbsp;\u003c/em\u003eand \u003cem\u003eCLEC10A\u003c/em\u003e, both implicated in antigen presentation, were also upregulated in \u003cem\u003eF. nucleatum\u003c/em\u003e-positive specimens (Fig. 2). The expression levels of other genes broadly involved in intracellular signaling within immune cells, including \u003cem\u003ePIK3CG\u003c/em\u003e, \u003cem\u003ePTK2B\u003c/em\u003e, \u003cem\u003eIRAG2\u003c/em\u003e, and \u003cem\u003eMPEG1\u003c/em\u003e, were similarly elevated in \u003cem\u003eF. nucleatum\u003c/em\u003e-positive specimens. Conversely, \u003cem\u003eMPEG1\u0026nbsp;\u003c/em\u003eand \u003cem\u003eCTSC,\u0026nbsp;\u003c/em\u003eboth intracellular effector proteins critical for pathogen destruction, were also upregulated in \u003cem\u003eF. nucleatum\u003c/em\u003e-positive specimens. This observation suggests potential macrophage activation in response to \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003einfection. Collectively, our analysis indicates that the effector cells of adaptive immunity are specifically recruited to tumor lesions by \u003cem\u003eF. nucleatum\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTo validate the accuracy of our sequencing data, we performed a linear correlation analysis and thus determine whether the coding genes of the T cell receptor (TCR) complex were coordinately detectable in the specimens. Across the entire cohort, the TCR \u0026alpha; chain constant region gene \u003cem\u003eTRAC,\u0026nbsp;\u003c/em\u003e\u0026beta; chain constant region gene \u003cem\u003eTR\u0026Beta;C2_1\u003c/em\u003e, and \u003cem\u003eCD8A\u0026nbsp;\u003c/em\u003ecorrelated\u003cem\u003e\u0026nbsp;\u003c/em\u003ewith \u003cem\u003eCD3E\u003c/em\u003e levels (Fig. 3A). When specimens were stratified into \u003cem\u003eF.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003enucleatum\u003c/em\u003e-positive and -negative groups, \u003cem\u003eTRAC\u0026nbsp;\u003c/em\u003eshowed no statistical difference in expression level between groups but maintained a correlation with \u003cem\u003eCD3E\u0026nbsp;\u003c/em\u003ewithin each group (data not shown). \u003cem\u003eCD8A\u0026nbsp;\u003c/em\u003eand \u003cem\u003eCD6\u0026nbsp;\u003c/em\u003ealso correlated with \u003cem\u003eCD3E\u0026nbsp;\u003c/em\u003elevels in both groups (Fig. 3B). However, the TCR \u0026beta; chain constant region genes \u003cem\u003eTRBC1\u0026nbsp;\u003c/em\u003eand \u003cem\u003eTRBC2_1\u003c/em\u003e did not correlate with \u003cem\u003eCD3E\u0026nbsp;\u003c/em\u003ein \u003cem\u003eF. nucleatum\u003c/em\u003e-negative specimens (Fig. 3), a discrepancy likely attributable to greater variation in calculated TPM at low expression levels. Taken together, these results indicate that \u003cem\u003eCD3E\u0026nbsp;\u003c/em\u003elevels reliably represent the relative abundance of the total T cell population in the specimens.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, the correlation between cytotoxic T cell markers \u003cem\u003eCD8A\u0026nbsp;\u003c/em\u003eand \u003cem\u003eCD3E\u003c/em\u003e suggests that the abundance of cytotoxic T cells is proportional to the total T cell population. Furthermore, a subset of \u003cem\u003eF. nucleatum-positive\u003c/em\u003e specimens exhibited elevated levels of \u003cem\u003eTRDC\u0026nbsp;\u003c/em\u003eand \u003cem\u003eTRGC2\u0026nbsp;\u003c/em\u003eencoding the TCR \u0026gamma; and \u0026delta; chain constant regions, respectively (Fig. 3C). This observation suggests the presence of \u0026gamma;\u0026delta; T cells in these samples. In addition to TCR components, \u003cem\u003eDOCK2\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas found at higher levels in \u003cem\u003eF.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003enucleatum\u003c/em\u003e-positive specimens and showed a correlation with \u003cem\u003eCD8A\u003c/em\u003e, \u003cem\u003eCD3E\u003c/em\u003e, and TCR \u0026beta; chain (Fig. 3D). Given the critical role of \u003cem\u003eDOCK2\u0026nbsp;\u003c/em\u003ein lymphocyte function and migration, its upregulation provides further evidence of an increased influx of cytotoxic T cells into the tumor microenvironment.\u003c/p\u003e\n\u003cp\u003eOur analysis further demonstrated the upregulation of T cell negative regulators. Specifically, both \u003cem\u003eLAX1\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePRDM1\u0026nbsp;\u003c/em\u003eexhibited elevated expression in the \u003cem\u003eF. nucleatum\u003c/em\u003e-positive specimens (Fig. 4). Functionally, \u003cem\u003eLAX1\u0026nbsp;\u003c/em\u003eserves as a negative regulator of B and TCR signaling, while \u003cem\u003ePRDM1\u0026nbsp;\u003c/em\u003eis indicative of\u003cem\u003e\u0026nbsp;\u003c/em\u003eT cell exhaustion. Correlation analysis revealed a positive correlation between \u003cem\u003eLAX1\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePRDM1\u0026nbsp;\u003c/em\u003ewith \u003cem\u003eCD3E\u0026nbsp;\u003c/em\u003ewhen all specimens were combined for analysis (data not shown); however, this statistical significance was not maintained when specimens were stratified into \u003cem\u003eF. nucleatum\u003c/em\u003e-negative and -positive groups (Fig. 4). Conversely, \u003cem\u003eLAX1\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePRDM1\u0026nbsp;\u003c/em\u003ewere found\u003cem\u003e\u0026nbsp;\u003c/em\u003eto correlate with \u003cem\u003eCD8A\u0026nbsp;\u003c/em\u003especifically\u003cem\u003e\u0026nbsp;\u003c/em\u003ein the \u003cem\u003eF. nucleatum\u003c/em\u003e-positive specimens. Therefore, despite their increased abundance, cytotoxic T cells in \u003cem\u003eF. nucleatum\u003c/em\u003e-positive specimens appear to be functionally suppressed. PD-L1 expressed by the cancer cells is a key mechanism to suppress the anti-tumor immune response, but our data show that most \u003cem\u003eF. nucleatum\u003c/em\u003e-positive specimens (all but two) have low \u003cem\u003eCD274\u0026nbsp;\u003c/em\u003e(PD-L1) expression (Fig. 4), with only 8.7% of all samples exhibiting high levels. Hence, PD-L1 is activated through distinct pathway independent from \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003einfection. On the other hand, \u003cem\u003eTGFB1\u0026nbsp;\u003c/em\u003ewas found to be\u003cem\u003e\u0026nbsp;\u003c/em\u003eupregulated and positively correlated with \u003cem\u003ePRDM1\u0026nbsp;\u003c/em\u003ein \u003cem\u003eF. nucleatum\u003c/em\u003e-positive specimens (Fig. 4), thereby implicating a direct role for TGF\u0026beta; signaling in the observed immune suppression.\u003c/p\u003e\n\u003cp\u003eAn \u003cem\u003ein vitro\u003c/em\u003e infection model experiment was subsequently conducted to elucidate the underlying mechanism by which \u003cem\u003eF. nucleatum\u003c/em\u003e promotes immune cell infiltration into tumor sites. To simulate infection, \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eATCC 25586 was introduced to gastric cancer cell lines MKN28, MKN45, and AGS at an MOI of 100 (Fig. 6). After two days of co-culturing, the cells were detached, thoroughly washed to eliminate unattached extracellular pathogens, and reseeded onto fresh culture plates. Following an additional 2 and 6 days of culture, total RNA was extracted and analyzed by RNA sequencing. Pathway analysis indicated that different gastric cancer cell lines exhibited distinct responses to \u003cem\u003eF. nucleatum\u003c/em\u003e infection. MKN28, a well-differentiated adenocarcinoma cell line harboring \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003eAPC\u0026nbsp;\u003c/em\u003emutations, showed no clear response to F. nucleatum infection. In contrast, an inflammatory response was induced in the poorly-differentiated MKN45 and moderately-differentiated AGS cell lines. In particular, \u003cem\u003eCXCL10\u0026nbsp;\u003c/em\u003ewas upregulated in MKN45 at the 4-day time point but reverted to baseline expression levels by day 8 (Fig. 5A). In AGS, the expression of both \u003cem\u003eCXCL9\u003c/em\u003e and \u003cem\u003eCXCL10\u003c/em\u003e was significantly elevated at day 4 and maintained this upregulation through day 8. Moreover, \u003cem\u003eTLR3\u0026nbsp;\u003c/em\u003ealso exhibited upregulation at day 4 (Fig. 5A). Given that both \u003cem\u003eCXCL9\u0026nbsp;\u003c/em\u003eand \u003cem\u003eCXCL10\u0026nbsp;\u003c/em\u003eare recognized as potent chemoattractants for activated T cells, this \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003echallenge experiment proposes a potential mechanism for \u003cem\u003eF. nucleatum\u003c/em\u003e-mediated augmentation of\u003cem\u003e\u0026nbsp;\u003c/em\u003eT cell abundance within tumor sites.\u003c/p\u003e\n\u003cp\u003eTo provide corroborating data for our \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003efindings, we re-examined the expression levels of \u003cem\u003eCXCL9\u0026nbsp;\u003c/em\u003eand \u003cem\u003eCXCL10\u0026nbsp;\u003c/em\u003ein clinical specimens. While no statistically significant difference was observed between the two groups (Mann\u0026ndash;Whitney U test, \u003cem\u003ep\u003c/em\u003e\u0026gt;0.05), it is evident that a greater proportion of \u003cem\u003eF. nucleatum\u003c/em\u003e-positive\u003cem\u003e\u0026nbsp;\u003c/em\u003especimens displayed high \u003cem\u003eCXCL9\u0026nbsp;\u003c/em\u003eor \u003cem\u003eCXCL10\u0026nbsp;\u003c/em\u003eexpression (Fig. 5B). Although a few \u003cem\u003eF. nucleatum-negative\u0026nbsp;\u003c/em\u003especimens also exhibited high \u003cem\u003eCXCL9\u0026nbsp;\u003c/em\u003eor \u003cem\u003eCXCL10\u003c/em\u003e, this expression was not correlated with \u003cem\u003eCD3E\u0026nbsp;\u003c/em\u003elevels (Fig. 5C). In comparison, within the \u003cem\u003eF. nucleatum\u003c/em\u003e-positive\u003cem\u003e\u0026nbsp;\u003c/em\u003especimens, \u003cem\u003eCXCL9\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eor \u003cem\u003eCXCL10\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas strongly correlated with \u003cem\u003eCD3E\u003c/em\u003e,\u0026nbsp;supporting\u0026nbsp;the\u0026nbsp;notion\u0026nbsp;that\u003cem\u003e\u0026nbsp;F. nucleatum\u0026nbsp;\u003c/em\u003einduces \u003cem\u003eCXCL9\u0026nbsp;\u003c/em\u003eand \u003cem\u003eCXCL10\u0026nbsp;\u003c/em\u003ein a subset of gastric cancers, subsequently promoting T cell infiltration into tumor lesions.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGastric cancer continues to rank among the top ten most prevalent cancers in Taiwan. The present findings unequivocally demonstrate the efficacy of public health policy interventions in reducing\u0026nbsp;\u003cem\u003eH. pylori\u003c/em\u003e infection, evident in its prevalence decreasing from 70% to under 20% within a period of fewer than ten years. Notwithstanding this considerable decline in\u0026nbsp;\u003cem\u003eH. pylori\u003c/em\u003e prevalence, the incidence of\u0026nbsp;gastric adenocarcinoma has exhibited only a marginal decrease, which strongly suggests the involvement of additional, as-yet-unmitigated risk factors. A concurrent increase in\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003einfection indicates a profound shift in the pathogenic microbial landscape contributing to gastric cancer, particularly given a recent animal study demonstrating the sufficiency of\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003ealone to induce gastric cancer in the absence of\u0026nbsp;\u003cem\u003eH. pylori\u003c/em\u003e. Consequently, our rigorous analysis of clinical specimens strongly implicates that\u0026nbsp;\u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003epossesses the potential to supersede\u003cem\u003e\u0026nbsp;H. pylori\u0026nbsp;\u003c/em\u003eas the predominant carcinogenic bacterial infection driving the pathogenesis of gastric cancer.\u003c/p\u003e\n\u003cp\u003eA discernible increase in infiltrating cytotoxic T cells was consistently observed within \u003cem\u003eF. nucleatum\u003c/em\u003e-positive gastric cancer. Under controlled \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003econditions, \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003edemonstrably induced the upregulation of \u003cem\u003eCXCL10\u0026nbsp;\u003c/em\u003ein both MKN45 and AGS cell lines, and of \u003cem\u003eCXCL9\u0026nbsp;\u003c/em\u003ein MKN45. Given that \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003ehas been extensively documented as detectable intracellularly in comparable \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eexperimental setups across multiple prior investigations, and the observed upregulation of \u003cem\u003eTLR3\u0026nbsp;\u003c/em\u003ein AGS further implies an intracellular mode of \u003cem\u003eF. nucleatum\u003c/em\u003e infection, our collective results suggest that intracellular \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eelicited\u003cem\u003e\u0026nbsp;\u003c/em\u003ean inflammatory response. This response encompassed the upregulation of \u003cem\u003eCXCL9\u0026nbsp;\u003c/em\u003eand \u003cem\u003eCXCL10,\u0026nbsp;\u003c/em\u003eboth of which function as potent chemoattractants to orchestrate the migration of T cells to sites of inflammation. In corroboration with this hypothesis, the levels of \u003cem\u003eCXCL9\u0026nbsp;\u003c/em\u003eand \u003cem\u003eCXCL10\u0026nbsp;\u003c/em\u003ein clinical specimens exhibited a strong correlation with the abundance of T cells. Intriguingly, IL-17, recently implicated as a key mediator in promoting T cell infiltration to gastric cancer [24], was not found to be upregulated in our clinical cohort. This discrepancy potentially indicates that \u003cem\u003eF. nucleatum\u0026nbsp;\u003c/em\u003eelicits a convergent\u003cem\u003e\u0026nbsp;\u003c/em\u003eimmune response through distinct underlying pathways across various gastric cancer cell lines. It is unequivocally clear that not all gastric cancer cell lines respond equivalently to \u003cem\u003eF.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003enucleatum\u003c/em\u003e, as exemplified by the complete absence of an inflammatory response in MKN28. While specific genetic aberrations could play a role in modulating the cellular response to \u003cem\u003eF. nucleatum\u003c/em\u003e, comprehensive further analysis is warranted to identify the genes critically essential for the \u003cem\u003eF. nucleatum\u003c/em\u003e-induced inflammatory response.\u003c/p\u003e\n\u003cp\u003eA concomitant increase in the abundance of T cells was, however, correlated with the upregulation of negative immunoregulators. This finding is entirely consistent with the observed poorer prognosis but paradoxically improved clinical benefits derived from immune checkpoint inhibitors in \u003cem\u003eF. nucleatum\u003c/em\u003e-positive cancers. Only a restricted subset of gastric cancers manifest high PD-L1 expression, but it appears to be independent from \u003cem\u003eF. nucleatum\u003c/em\u003e infection; moreover, in the absence of combined positive score data determined by immunohistochemistry in the available clinical records, the precise correspondence between observed RNA levels and clinically relevant PD-L1 expression remains to be fully elucidated. \u003cem\u003ePDCD-1\u003c/em\u003e, the gene encoding PD-1, was detectable at uniformly low levels across all analyzed samples (data not shown), rendering it an impractical biomarker for predicting immunotherapy outcomes in this context. Conversely, elevated levels of \u003cem\u003eCTLA-4\u003c/em\u003e were detected in a minority of samples, yet these elevations showed no association with F. nucleatum presence, \u003cem\u003ePRDM1\u003c/em\u003e expression, or \u003cem\u003eCD274\u003c/em\u003e levels (data not shown). In contrast, \u003cem\u003eTGFB1\u003c/em\u003e was strongly upregulated in multiple F. nucleatum-positive specimens. While TGF\u0026beta; signaling is known to exert a pivotal role in immune suppression, the specific cellular source responsible for the release of TGF\u0026beta; within the F. nucleatum-infected microenvironment remains unclarified. Interestingly, \u003cem\u003eTGFB1\u003c/em\u003e expression was not stimulated by F. nucleatum in the in vitro cell line models utilized in this study, suggesting that TGF\u0026beta; is released from other types of cells in the tumor microenvironment. Although the detail molecular mechanism remains unillustrated, our comprehensive analysis employing clinical specimens provides robust evidence that F. nucleatum is intricately associated with a suppressed immune response, thereby contributing to cancer progression.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Review Board of Chiayi Chang Gung Memorial Hospital (approval number IRB 202102249B0 and IRB 202202139B0). Clinical specimens used in this study was acquired from the Human BioBank of Chiayi Chang Gung Memorial Hospital. Human BioBank was established under the regulation of the Human Biobank Management Act. Informed consents were obtained from the patients before acceptance of the specimens to the Human BioBank. Subsequent use of the specimens requires approval from the Institutional Review Board, but additional informed consent from the patients is not required according to the regulation of the Human Biobank Management Act.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequencing data is deposited to Sequence Read Archive, NCBI. The accession numbers of Bioprojects are PRJNA1302123 and PRJNA1302213.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the funding awarded to Y.Y.H. from Chiayi Chang Gung Memorial Hospital, Taiwan (grant number CMRPG6M0291, NMRPG6N6021) and National Science and Technology Council, R.O.C (grant number NSTC 112-2314-B-182A-139 -MY3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.Y.H. recruited patients to the study and oversaw the study. W.L.K., C.T.W., and W.L.K. performed sample processing and experiments. C.L. and Y.Y.H. carried out data analysis and manuscript writing.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDuan Y, Xu Y, Dou Y, Xu D: Helicobacter pylori and gastric cancer: mechanisms and new perspectives. \u003cem\u003eJ Hematol Oncol \u003c/em\u003e2025, 18(1):10.\u003c/li\u003e\n\u003cli\u003eChen MJ, Bair MJ, Chen PY, Lee JY, Yang TH, Fang YJ, Chen CC, Chang AT, Hsiao WD, Yu JJ\u003cem\u003e et al\u003c/em\u003e: Declining trends of prevalence of Helicobacter pylori infection and incidence of gastric cancer in Taiwan: An updated cross-sectional survey and meta-analysis. \u003cem\u003eHelicobacter \u003c/em\u003e2022, 27(5):e12914.\u003c/li\u003e\n\u003cli\u003eChen YC, Malfertheiner P, Yu HT, Kuo CL, Chang YY, Meng FT, Wu YX, Hsiao JL, Chen MJ, Lin KP\u003cem\u003e et al\u003c/em\u003e: Global Prevalence of Helicobacter pylori Infection and Incidence of Gastric Cancer Between 1980 and 2022. \u003cem\u003eGastroenterology 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\u003c/em\u003e2023, 14(1):5788.\u003c/li\u003e\n\u003cli\u003eGaleano Nino JL, Wu H, LaCourse KD, Kempchinsky AG, Baryiames A, Barber B, Futran N, Houlton J, Sather C, Sicinska E\u003cem\u003e et al\u003c/em\u003e: Effect of the intratumoral microbiota on spatial and cellular heterogeneity in cancer. \u003cem\u003eNature \u003c/em\u003e2022, 611(7937):810-817.\u003c/li\u003e\n\u003cli\u003eKosumi K, Baba Y, Yamamura K, Nomoto D, Okadome K, Yagi T, Toihata T, Kiyozumi Y, Harada K, Eto K\u003cem\u003e et al\u003c/em\u003e: Intratumour Fusobacterium nucleatum and immune response to oesophageal cancer. \u003cem\u003eBr J Cancer \u003c/em\u003e2023, 128(6):1155-1165.\u003c/li\u003e\n\u003cli\u003eParhi L, Alon-Maimon T, Sol A, Nejman D, Shhadeh A, Fainsod-Levi T, Yajuk O, Isaacson B, Abed J, Maalouf N\u003cem\u003e et al\u003c/em\u003e: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression. \u003cem\u003eNat Commun \u003c/em\u003e2020, 11(1):3259.\u003c/li\u003e\n\u003cli\u003eZhang T, Li Y, Zhai E, Zhao R, Qian Y, Huang Z, Liu Y, Zhao Z, Xu X, Liu J\u003cem\u003e et al\u003c/em\u003e: Intratumoral Fusobacterium nucleatum Recruits Tumor-Associated Neutrophils to Promote Gastric Cancer Progression and Immune Evasion. \u003cem\u003eCancer Res \u003c/em\u003e2025, 85(10):1819-1841.\u003c/li\u003e\n\u003cli\u003eLee JA, Yoo SY, Oh HJ, Jeong S, Cho NY, Kang GH, Kim JH: Differential immune microenvironmental features of microsatellite-unstable colorectal cancers according to Fusobacterium nucleatum status. \u003cem\u003eCancer Immunol Immunother \u003c/em\u003e2021, 70(1):47-59.\u003c/li\u003e\n\u003cli\u003eShigematsu Y, Saito R, Amori G, Kanda H, Takahashi Y, Takeuchi K, Takahashi S, Inamura K: Fusobacterium nucleatum, immune responses, and metastatic organ diversity in colorectal cancer liver metastasis. \u003cem\u003eCancer Sci \u003c/em\u003e2024, 115(10):3248-3255.\u003c/li\u003e\n\u003cli\u003eWang X, Fang Y, Liang W, Wong CC, Qin H, Gao Y, Liang M, Song L, Zhang Y, Fan M\u003cem\u003e et al\u003c/em\u003e: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer. \u003cem\u003eCancer Cell \u003c/em\u003e2024, 42(10):1729-1746 e1728.\u003c/li\u003e\n\u003cli\u003eDuizer C, Salomons M, van Gogh M, Grave S, Schaafsma FA, Stok MJ, Sijbranda M, Kumarasamy Sivasamy R, Willems RJL, de Zoete MR: Fusobacterium nucleatum upregulates the immune inhibitory receptor PD-L1 in colorectal cancer cells via the activation of ALPK1. \u003cem\u003eGut Microbes \u003c/em\u003e2025, 17(1):2458203.\u003c/li\u003e\n\u003cli\u003eGao Y, Bi D, Xie R, Li M, Guo J, Liu H, Guo X, Fang J, Ding T, Zhu H\u003cem\u003e et al\u003c/em\u003e: Fusobacterium nucleatum enhances the efficacy of PD-L1 blockade in colorectal cancer. \u003cem\u003eSignal Transduct Target Ther \u003c/em\u003e2021, 6(1):398.\u003c/li\u003e\n\u003cli\u003eGuo J, Zhu P, Li J, Xu L, Tang Y, Liu X, Guo S, Xia J: Fusobacterium nucleatum promotes PD-L1 expression in cancer cells to evade CD8(+) T cell killing in breast cancer. \u003cem\u003eHum Immunol \u003c/em\u003e2024, 85(6):111168.\u003c/li\u003e\n\u003cli\u003eHsieh YY, Tung SY, Pan HY, Chang TS, Wei KL, Chen WM, Deng YF, Lu CK, Lai YH, Wu CS\u003cem\u003e et al\u003c/em\u003e: Fusobacterium nucleatum colonization is associated with decreased survival of helicobacter pylori-positive gastric cancer patients. \u003cem\u003eWorld J Gastroenterol \u003c/em\u003e2021, 27(42):7311-7323.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Fusobacterium nucleatum, gastric cancer, PRDM1, CXCL9, CXCL10","lastPublishedDoi":"10.21203/rs.3.rs-8216991/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8216991/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground\u003c/p\u003e\n\u003cp\u003eGastric cancer is etiologically linked to pathogenic microbes. \u003cem\u003eFusobacterium nucleatum \u003c/em\u003ein gastric cancer-associated microbiota is associated with high tumor mutation burden and poor prognosis. \u003cem\u003eF. nucleatum \u003c/em\u003ewas also shown to suppress anti-tumor immune response in colorectal cancer. The aim of this study is to identify the \u003cem\u003eF. nucleatum\u003c/em\u003e-induced change in the immune microenvironment of gastric cancer.\u003c/p\u003e\n\u003cp\u003eMaterials\u003c/p\u003e\n\u003cp\u003eResected gastric cancer specimens and endoscopic-extracted biopsies were obtained from Human Biobank of Chiayi Chang Gung Memorial Hospital. The presence of \u003cem\u003eF. nucleatum\u003c/em\u003e in the specimens was determined by nested PCR. Resected specimens were analyzed by transcriptomic analysis. \u003cem\u003eIn vitro \u003c/em\u003eexperiment using \u003cem\u003eF. nucleatum\u003c/em\u003e-infected gastric cancer cell lines was utilized to identify deregulated genes in the cancer cells by \u003cem\u003eF. nucleatum \u003c/em\u003einfection.\u003c/p\u003e\n\u003cp\u003eResults\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHelicobacter pylori \u003c/em\u003einfection was significantly declined among gastric cancer patients in Southwestern Taiwan. Conversely, \u003cem\u003eF. nucleatum\u003c/em\u003e was identified in nearly 50% of patients, emerging as the dominant oncogenic infection. Transcriptomic and ontological analyses of resected specimens revealed that \u003cem\u003eF. nucleatum correlates \u003c/em\u003ewith increased T cell markers, including T cell receptor constant region and \u003cem\u003eCD3E\u003c/em\u003e. T cell receptor subunit levels correlated with \u003cem\u003eCD8A\u003c/em\u003e, indicating cytotoxic T cell infiltration in \u003cem\u003eF. nucleatum\u003c/em\u003e-positive lesions. However, upregulation of negative regulators \u003cem\u003eLAX1 \u003c/em\u003eand \u003cem\u003ePRDM1\u003c/em\u003e, proportional to \u003cem\u003eCD3E \u003c/em\u003eand \u003cem\u003eCD8A\u003c/em\u003e, suggests these abundant T cells are inactive. \u003cem\u003eIn vitro\u003c/em\u003e, \u003cem\u003eF. nucleatum-induced\u003c/em\u003e \u003cem\u003eCXCL9 \u003c/em\u003eand \u003cem\u003eCXCL10 \u003c/em\u003eupregulation in select gastric cancer cell lines. Correspondingly, \u003cem\u003eCXCL9 \u003c/em\u003eand \u003cem\u003eCXCL10 \u003c/em\u003ewere elevated and highly correlated with \u003cem\u003eCD3E \u003c/em\u003eand \u003cem\u003eCD8A \u003c/em\u003ein a subset of \u003cem\u003eF. nucleatum\u003c/em\u003e-positive specimens.\u003c/p\u003e\n\u003cp\u003eConclusions\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eF. nucleatum infection \u003c/em\u003ein gastric cancer increased drastically in Taiwan. Higher abundance of cytotoxic T cells is associated with \u003cem\u003eF. nucleatum\u003c/em\u003e, likely being attracted to tumor sites by \u003cem\u003eF. nucleatum\u003c/em\u003e-induced \u003cem\u003eCXCL9 \u003c/em\u003eand \u003cem\u003eCXCL10\u003c/em\u003e. However, despite being attracted to gastric tumor sites, these tumor-infiltrating cytotoxic T cells are inactive and exhibit an exhausted phenotype, indicating that \u003cem\u003eF. nucleatum \u003c/em\u003eis associated with an immunosuppressive microenvironment. Our finding suggest that targeting \u003cem\u003eF. nucleatum\u003c/em\u003e could promote anti-tumor immune response and subsequently the treatment efficacy.\u003c/p\u003e","manuscriptTitle":"Association of Fusobacterium nucleatum with suppressed anti-tumor T cell responses in gastric cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-16 06:22:01","doi":"10.21203/rs.3.rs-8216991/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-02-10T21:56:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-11T04:20:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"206270798584118718882190622085776264131","date":"2026-01-10T12:13:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"256346812031035612967330984106746715748","date":"2026-01-10T04:53:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-09T00:53:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-17T01:53:25+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-08T10:57:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-08T05:19:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2025-12-08T04:53:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"afc55e26-dd18-410f-bdea-bc15a33104b0","owner":[],"postedDate":"January 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-16T06:22:01+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-16 06:22:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8216991","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8216991","identity":"rs-8216991","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}