The Gut Commensal Bacteria Veillonella parvula-Induced Neutrophil Activation Mediates Antitumor Activity

The Gut Commensal Bacteria Veillonella parvula-Induced Neutrophil Activation Mediates Antitumor Activity | 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 Article The Gut Commensal Bacteria Veillonella parvula-Induced Neutrophil Activation Mediates Antitumor Activity GwangPyo Ko, Sunghyun Yoon, Hyun Ju You, Jiyeon Si, Seulki Jeon, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7615171/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Recent scientific efforts have focused on identifying commensal microbiota with antitumor activity, yet most cancer studies remain limited to taxonomic associations or treatment-induced microbial changes, with only a few addressing underlying mechanisms. These mechanistic studies have focused mainly on lymphoid cells such as CD8⁺ T cells, while neutrophil-mediated mechanisms have received little attention despite their abundance and functions. Here, we screened 16 taxa linked to positive responses to cancer immunotherapy and identified Veillonella parvula as effective in reducing tumor growth in a murine model. Using single-cell transcriptomics and flow cytometry, we show that V. parvula reshapes the tumor microenvironment by promoting infiltration of neutrophil subsets that secrete reactive oxygen and nitrogen species, driving tumor cell death. We further identify Vpar0143, a ferritin Dps family protein, as the bacterial effector responsible for neutrophil activation and the antitumor effect. Together, these findings reveal a commensal–neutrophil axis harnessable for microbe-based cancer therapy. Biological sciences/Microbiology Biological sciences/Immunology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Main The gut microbiota has emerged as a key regulator of cancer immunity, influencing both tumor progression and therapeutic responses 1 , 2 . Recent evidence suggests that the efficacy of immune checkpoint inhibitors (ICIs), such as anti-PD1 therapy, is closely linked to the composition of the gut microbiota 3 . However, despite the growing interest in microbiota-immune interactions, the mechanisms by which microbiota enhance antitumor immunity remain to be fully elucidated. Given the therapeutic potential of microbiota modulation, strategies such as fecal microbiota transplantation (FMT) have been explored to improve responses in patients with poor ICI efficacy 4 , 5 . Yet, FMT presents challenges, including safety concerns, high costs, and inconsistent clinical outcomes, necessitating alternative, targeted approaches 6 , 7 . One promising strategy involves the identification and administration of specific bacterial strains with defined antitumor properties. However, the efficacy of administered bacteria, which depends on their stable colonization within host-specific intestinal environments 8 , 9 , underscores the need to identify precise molecular mechanisms and targets to ensure therapeutic consistency. While prior research has largely focused on adaptive immune responses, particularly T cell-mediated mechanisms 10 , the role of innate immune cells in microbiota-driven antitumor immunity remains underexplored. Neutrophils, as key components of the innate immune system, exhibit a paradoxical role in cancer, with both tumor-promoting and antitumor activities. Although they have been implicated in immunosuppressive functions that support tumor growth, emerging evidence indicates that neutrophils can also mediate potent antitumor effects through direct cytotoxicity, antibody-dependent mechanisms, and recruitment of additional immune effectors 11 . For instance, neutrophils can induce tumor cell death via the release of oxidative substrates such as reactive oxygen species (ROS) and nitric oxide (NO) 12 . Despite these insights, the interplay between gut microbiota and neutrophils in shaping cancer immunity necessitate further investigation. In this study, we systematically screened 16 bacterial taxa previously associated with cancer immunotherapy to identify a candidate with strong antitumor properties. Single-cell transcriptome analysis revealed the mechanisms of the selected Veillonella strain, indicating neutrophil-mediated innate immunity is central to driving downstream immune responses. Additionally, comparative proteomic analysis allowed us to uncover key antitumor molecules produced by the candidate microbe. By integrating these approaches, our study advances the understanding of microbiota-mediated tumor immunity and highlights the potential of microbial-based strategies as precision therapeutics in cancer treatment. Results Veillonella parvula Enhances Antitumor Immunity and Synergizes with Anti-PD1 Therapy To determine whether bacteria indeed have an antitumor effect related to immunotherapy response as previously reported 13 , we selected 16 bacteria and retrieved them from our in-house culture collection or commercially purchased them (Supplementary Table 1). To determine whether the beneficial component is a cellular component of bacteria or a component secreted by bacteria, a mixture of these bacteria (Mix16) was divided into a live mixture (L-mix16) and a pasteurized mixture (P-Mix16). Both L-Mix16 and P-Mix16 inhibited tumor growth when intratumorally administered to tumor-bearing mice. This indicates that bacteria associated with a positive cancer treatment response can also induce antitumor effects and that cellular components of the bacteria are the main modulators of these effects (Fig. 1 a). To identify the major taxa with tumor suppression effects, we first treated splenocytes from MC38 tumor-bearing mice with pasteurized bacteria and compared the level of IFN-γ, a typical anticancer cytokine 14 . Among the tested strains, only Akkermansia muciniphila (AM) and Veillonella parvula (VP) induced IFN-γ production. (Fig. 1 b). To determine whether these two bacteria can inhibit tumor growth in vivo , we administered the following five treatments to MC38 tumor-bearing mice: PBS, pasteurized AM (P-AM), pasteurized VP (P-VP), P-Mix16, and pasteurized Mix16 excluding AM and VP (P-Mix14). Interestingly, tumor growth inhibition was not observed in P-Mix14 (Fig. 1 c), suggesting that AM and VP were the major effector strains. Next, we isolated splenocytes from MC38 tumor-bearing mice, treated them with P-AM or P-VP, and cocultured them with MC38 tumor cells to compare the tumor suppressive effects of the two bacteria. To exclude direct bacterial cytotoxicity, MC38 tumor cells were also treated with the bacteria in the absence of splenocytes. P-AM- or P-VP-treated splenocytes displayed enhanced tumor cytotoxicity; this effect disappeared when only the tumor cells were treated with the bacteria (Fig. 1 d). Moreover, P-VP-treated splenocytes exhibited significantly higher cytotoxicity compared with P-AM-treated splenocytes. (Fig. 1 d). Ultimately, we selected VP as a final candidate. We additionally tested different administration routes (oral gavage and intraperitoneal injection), mouse strains (C57BL/6 and BALB/c), and subcutaneous tumor types (MC38, B16F10, and CT26) to compare the efficacy of VP under different conditions. The results showed comparable efficacy across administration routes, mouse strains, and tumor types, indicating that the antitumor effect of P-VP was robust across diverse conditions (Fig. 1 e). To further determine species-specific effects, we investigated the antitumor activity of pasteurized V. atypica (P-VA), another common Veillonella species, but the strain did not exhibit any antitumor effects (Fig. 1 f). Finally, the combination of P-VP with anti-PD1 antibody synergistically suppressed tumor growth compared with either treatment alone. (Fig. 1 g). Therefore, all of these results point to the potent capacity of VP to inhibit tumor growth. P-VP Treatment Reshapes Tumor Immune Landscape by Enhancing Neutrophil Infiltration Next, we performed single-cell transcriptomic analysis to profile the infiltrated immune cell composition of the tumor following intraperitoneal injection of P-VP (Fig. 2 a). After quality control and filtering, we obtained 7,707 cells from PBS-treated mice and 9,995 cells from P-VP-treated mice. Clustering analysis revealed 10 cell types in the tumors of PBS- and VP-treated mice with unique transcriptional features (Fig. 2 b). Interestingly, the myeloid cell lineage was greatly altered in the P-VP-treated tumor, with increased neutrophils and decreased macrophages and monocytes compared to PBS-treated mice. T and NK cells showed a modest apparent increase in the single-cell data, though this change was limited compared with the neutrophil expansion. Flow cytometry validation confirmed that neutrophils were the only population with a significant increase in frequency, whereas other subsets, including T and NK cells, were reduced or showed no significant changes in P-VP-treated tumors (Fig. 2 c-e and Extended Data Fig. 1 a, c, f). Functional assessment further demonstrated that P-VP treatment did not enhance T or NK cell activity, as indicated by the frequencies of IFN-γ⁺, granzyme B (GZMB)⁺, and perforin (PFN)⁺ cells (Extended Data Fig. 1 b, d-e, g-h). Importantly, neutrophil enrichment was not observed in mice administered P-VA (Fig. 2 f). A similar increase in neutrophils was detected in the spleen and bone marrow, suggesting that VP treatment promotes systemic neutrophil expansion and recruitment into tumors through the circulation (Extended Data Fig. 2 a–e). P-VP Promotes Tumor-Suppressive Neutrophil Differentiation Through Oxidative Burst Activity To confirm whether T-cells and NK cells were involved in the antitumor effect of VP, athymic nude mice lacking T-cells 15 and NK cell-depleted mice (administered a neutralizing antibody) were transplanted with MC38 tumor cells and intraperitoneally injected with P-VP (Fig. 3 a-b). Treatment with P-VP still delayed tumor growth in both athymic nude mice and NK cell-depleted mice, indicating that its effect is independent of T and NK cells. To further investigate whether the antitumor effect of P-VP was dependent only on neutrophils, we employed the NOD-scid IL2rγ null (NSGA) mouse model, an immunodeficient mouse strain lacking T-, B- and NK cells and having defective dendritic cells (DCs) and macrophages. Neutrophils are therefore the only remaining functional immune cells in this mouse 16 (Fig. 3 c). P-VP still suppressed tumor growth in these neutrophil-functional mice. To further assess whether the tumor-suppressive potential of neutrophils could be induced by P-VP treatment, we isolated neutrophils from the spleens of MC38 tumor-bearing mice treated with PBS or P-VP and cocultured them with MC38 colon cancer cells. As a result, neutrophils from P-VP-treated mice displayed enhanced tumor cytotoxicity, indicating that VP can increase neutrophil antitumor cytotoxicity (Fig. 3 d). Given that P-VP treatment yielded antitumorigenic neutrophils, further characterization of the enriched single-cell clusters was carried out to better define functional neutrophils with antitumor effects (Fig. 3 e). When we reanalyzed the cluster defined as neutrophils, we obtained three subclusters. Cluster 0 was detected only in PBS-treated tumors, whereas clusters 1 and 2 emerged uniquely in the P-VP-treated group. Cluster 0 neutrophils expressed inflammatory and tissue-remodeling genes such as S100a8, S100a9, Mmp8 , and Mmp9 , consistent with a baseline pro-inflammatory state. By contrast, cluster 1 neutrophils were enriched for type I interferon–stimulated genes ( Ifit1, Ifit2, Rsad2, Gbp2/5 ) and metabolic regulators such as Acod1 , consistent with interferon-driven antimicrobial and oxidative effector programs. Cluster 2 neutrophils displayed a degranulation-associated signature, with high expression of Camp, Ngp, Ltf, Chil3 , and Cd177 , together with redox enzymes ( G6pdx, Sqor ), indicative of enhanced oxidative burst capacity consistent with tumor-suppressive effector functions. These findings suggest that P-VP treatment promotes the emergence of neutrophil subsets transcriptionally equipped for oxidative burst–mediated effector activity (Supplementary Table 2). Given that neutrophils exert antitumor effects by inducing oxidative bursts that are toxic to cancer cells 17 , 18 , we next performed flow cytometry analysis and confirmed the elevation of ROS- and iNOS-positive cells in the P-VP group (Fig. 3 f-g). In line with this observation, Gene Ontology (GO) analysis of the neutrophil subclusters showed upregulation of ROS metabolic processes in clusters 1 and 2, while these processes were downregulated in cluster 0 (Fig. 3 h). Taken together, these results indicated that the newly differentiated neutrophil subtypes induced by P-VP administration can suppress tumor growth through oxidative burst activity. P-VP Activates Neutrophils via TLR2 to Enhance Antitumor Cytotoxicity Next, to investigate how VP activates neutrophils, we examined variations in pattern recognition receptors (PRRs), which are critical mediators of host immune responses to microbial stimuli 19 . To this end, we analyzed the expression of multiple PRR genes in tumors, including Tlr1 , Tlr2 , Tlr4 , Tlr5 , Tlr6 , Tlr9 , Nod1 , and Nod2 , after treating tumor-bearing mice with P-VP. The results showed that Tlr2 was the only gene significantly upregulated by P-VP (Fig. 4 a). Flow cytometry analysis further confirmed this finding by showing increased TLR2 expression on neutrophils, whereas TLR4 expression was not altered (Fig. 4 b-c). Importantly, neutralization of TLR2 markedly diminished the antitumor activity of P-VP in vivo (Fig. 4 d). Consistently, neutrophils isolated from the mouse spleen treated with anti-TLR2 antibody lost their capacity to cause cytotoxicity when exposed to P-VP (Fig. 4 e). Together, these results suggest that P-VP activates neutrophils through TLR2 to promote the production of ROS and NO, resulting in the inhibition of tumor growth. Ferritin Dps Family Protein Vpar0143 Mediates VP-Induced Antitumor Neutrophil Activation Next, we tried to find the key bacterial antitumor substances. To this end, we purified the membrane fraction of VP (VP-MF) and further treated it with pronase, a mixture of nonspecific endo- and exoproteases, to eliminate the protein fraction (VP-MFP) from the membrane fraction, as it is known that bacterial proteins can affect immunity 20 , 21 . The antitumor effect was not observed in the VP-MFP-treated mice, while administration of VP-MF reduced tumor growth in mice (Fig. 5 a). Additionally, the frequency of tumor-infiltrating neutrophils was increased only in VP-MF-treated mice (Fig. 5 b and Extended Data Fig. 3 a-d), whereas VA-MF treatment failed to elicit such an effect (Extended Data Fig. 4 a-b). These results suggest that proteins within the membrane fraction are the main effector components of VP. To identify a specific antitumor protein, we performed a proteome analysis and compared the proteins from the membrane fraction of VP to those of VA based on the iBAQ intensity (Fig. 5 c and Supplementary Table 3a-b). We initially evaluated one of the most abundant proteins, an S-layer domain-related protein (Vpar0556), from the VP membrane fraction (Supplementary Table 3a). This protein was also abundant in VA and did not inhibit tumor growth or induce an increase in neutrophils infiltrating the tumor (Extended Data Fig. 5 a-b). Next, we searched for a highly abundant protein found only in the VP and selected Vpar0143, a ferritin Dps family protein (Fig. 5 d and Supplementary Table 3b). Administration of Vpar0143 to mice bearing MC38 tumors inhibited tumor growth in a dose-dependent manner (Fig. 5 e) and increased the frequency of tumor-infiltrating neutrophils (Fig. 5 f). Moreover, a cytotoxicity assay showed that neutrophils isolated from mice administered Vpar0143 had enhanced cytotoxicity compared to neutrophils isolated from PBS-treated mice (Fig. 5 g). This protein also promoted neutrophil-mediated antitumor activity through induction of ROS and NO production (Fig. 5 h-i). Consistent with the results of P-VP itself, Vpar0143 specifically upregulated TLR2 expression in neutrophils (Extended Data Fig. 6a-b). All of these findings point to important roles for the ferritin Dps family protein Vpar0143 in the antitumor activity of VP via neutrophil stimulation. Discussion Previous reports have shown that commensal bacteria are closely related to the immunotherapy response; however, the bacteria identified vary between studies. For example, Bifidobacterium and Akkermansia have previously been shown to have anticancer efficacy 22 , 23 , and we discovered that VP had the most robust antitumor effects out of the 16 selected bacteria under the same conditions. Beyond confirming the efficacy of VP, our study elucidates its underlying antitumor mechanisms. Previous studies on the antitumor mechanisms of the human microbiota have centered on improving adaptive immune responses, especially CD8 + cytotoxic T-cells, although microbiota also have important roles in the establishment of the innate immune system 24 . In the present study, single-cell transcriptome analysis showed that VP induced neutrophil subtypes with antitumor effects by upregulating ROS and NO production. The role of neutrophils in cancer remains in debate. Neutrophils have potent antitumor effects through ROS or NO production, but in different settings, these oxygen free radicals can induce host immunosuppression and tumor progression because they are also toxic to normal immune cells including cytotoxic T-cells 25 , 26 . Peishan et al. reported that these dual roles of neutrophils depend on the immune status of the host. The study found that neutrophils had a ‘net tumoricidal’ effect when NK cells were absent in the host, but they had a ‘net tumor-promoting’ effect when NK cells were present because the NK cells were able to block the increased tumoricidal activity of neutrophils. This is because neutrophils are cytotoxic not only to tumors but also to effector immune cells such as NK cells. It would therefore potentially be necessary to check the patient's immune status prior to the clinical application of neutrophil-based cancer therapy. Neutrophils and T-cells may have a complementary relationship in terms of cancer treatment. The maturation of neutrophils to antigen-presenting cells (APCs) is promoted by IFN-γ and granulocyte–macrophage colony-stimulating factor (GM-CSF), which also activate adaptive T-cells 27 . Additionally, neutrophils polarize CD4 – CD8 – TCRαβ + double-negative unconventional T-cells (UTC αβ ) toward the type 1 immune response and produce IFN-γ by interacting with macrophages 28 . In addition, a recent study uncovered an interaction between T-cells and neutrophils in T-cell immunotherapies. T-cells mediate an initial antitumor immune response, while neutrophils are responsible for removing tumor antigen-loss variants. The complete eradication of the tumor relied on the presence of neutrophils and was partially dependent on inducible NOS 29 . Recent efforts have attempted to utilize neutrophil-activated oxidative damage for cancer treatment. A previous study demonstrated that the combined actions of tumor necrosis factor (TNF), CD40 agonist, and tumor-binding antibody activated neutrophil infiltration and induced an inflammatory cascade driving ROS production, which drove T-cell–independent tumor clearance 30 . Given that neutrophils are the first antimicrobial responders, microbes can be an promising adjuvant for neutrophil-activation therapy. Another recent study highlighted the potential applications of microbial immunotherapy in the treatment of solid tumors 31 . The therapeutic impact of checkpoint inhibitor therapy was found to be improved by intratumoral injection of Staphylococcus aureus , which increased CD8 T-cell function as well as secretion of ROS by neutrophil activation 31 . Our study also showed the potential of commensal bacteria as an adjuvant for microbe-based neutrophil-activation therapy. We further discovered a bacteria-driven antitumor protein with the potential for safe, stable and controllable therapeutic development. Ferritin Dps family protein is a molecule with iron-storage activity and is known to affect host immunity in addition to protecting bacterial DNA from oxidizing free radicals 32 . One of the most-studied bacterial ferritins is Helicobacter pylori neutrophil-activating protein (HP-NAP), a TLR2 agonist that activates neutrophils to generate oxygen free radicals and that induces other innate immune cells to secrete proinflammatory cytokines such as TNF-α, IL-6, IL-12 and IL-23 33 . Another study showed that this protein also has antitumor activity and enhances the immunotherapy response 33 , 34 . We observed a similar mechanistic link between Vpar0143 and MC38 tumors, yet the complexity of bacterial components opens the possibility of other effective bacterial antitumor compounds. Therefore, further studies are warranted on the antitumor efficacy of other constitutive components of VP. Further investigations into the antitumor mechanisms of VP or Vpar0143 using various preclinical models including metastasis tumor models and spontaneous cancer models may be essential for exploring clinical application, as we have only confirmed the antitumor efficacy of VP and Vpar0143 in subcutaneous tumor models. In conclusion, we have demonstrated that VP and its membrane protein, Vpar0143, can be effective antitumor treatments, as they were able to significantly inhibit tumor growth and promote neutrophil-mediated antitumor immunity by inducing ROS and NO production. This study provides a new perspective on the clinical potential of VP in cancer treatment and the development of a novel neutrophil-targeting cancer therapeutic. Declarations Author contributions S.Y., H.J.Y., and G.P.K. conceived and designed the study. S.Y. and H.J.Y. drafted the manuscript with input from all authors. J.S. performed the single-cell RNA-seq data analysis. S.J., J.P., and M.J. assisted with in vivo experiments and sample processing. D.H. and K.L. performed the proteomic analysis. G.P.K. supervised the project and acquired funding. H.K.L. provided critical advice and reviewed the manuscript. L.S., G.L., and J.Y. contributed to data acquisition and analysis. J.S.C. performed cloning and protein production. All authors reviewed and approved the final version of the manuscript. Acknowledgements This research was supported by the Bio&Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (RS-2022-NR067344). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (RS-2024-00336212). Ethics declarations Conflict of interest statement G.K. is a founder of KoBioLabs, Inc., a company characterizing the role of host-microbiome interaction. The other authors declare no competing interests. References Helmink, B. A., Khan, M. A. W., Hermann, A., Gopalakrishnan, V. & Wargo, J. A. The microbiome, cancer, and cancer therapy. Nat Med 25 , 377–388 (2019). Sepich-Poore, G. D. et al. The microbiome and human cancer. Science (1979) 371 , (2021). Bullman, S., Eggermont, A., Johnston, C. D. & Zitvogel, L. Harnessing the microbiome to restore immunotherapy response. Nat Cancer 2 , 1301–1304 (2021). Routy, B. et al. Fecal microbiota transplantation plus anti-PD-1 immunotherapy in advanced melanoma: a phase I trial. Nat Med 29 , 2121–2132 (2023). Kim, Y. et al. Fecal microbiota transplantation improves anti-PD-1 inhibitor efficacy in unresectable or metastatic solid cancers refractory to anti-PD-1 inhibitor. Cell Host Microbe 32 , 1380-1393.e9 (2024). Karimi, M. et al. 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The neutrophil-activating protein of Helicobacter pylori promotes Th1 immune responses. 116 , 1092–1101 (2006). Lim, M. Y. et al. The effect of heritability and host genetics on the gut microbiota and metabolic syndrome. Gut 66 , 1031–1038 (2017). Materials and methods Bacteria culture and pasteurization Brief information on the strains used in this study is listed in Supplementary Table 1. Bifidobacterium adolescentis SNUG30339, Bifidobacterium longum SNUG30192, Collinsella aerofaciens SNUG30018, Parabacteroides merdae SNUG30168, Bacteroides caccae SNUG30273, Bacteroides thetaiotamicron SNUG30057, Alistipes indistinctus SNUG30286, Enterococcus faecium SNUG10198, Enterococcus hirae SNUG10078, Eubacterium callanderi SNUG40125, Faecalibacterium prausnitzii SNUG30106, and Akkermansia muciniphila SNUG61027 were isolated from freshly collected fecal samples, or Lactobacillus crispatus SNUV206 and Veillonella atypica SNUV638 were isolated from fresh vaginal fluid samples that were obtained from healthy individuals who had not been treated with antibiotics during the preceding year 35 . Ruminococcus bromii ATCC27255 was purchased from American Type Culture Collection (ATCC), and Ruminococcus faecis KCTC5757 and Veillonella parvula KCTC5019 were purchased from the Korean Collection for Type Cultures (KCTC Seoul, Korea). Bifidobacterium adolescentis SNUG30339, Bifidobacterium longum SNUG30192, and Lactobacillus crispatus SNUV206 were cultured in MRS media (BD Difco). Veillonella parvula KCTC5019 and Veillonella atypica SNUV638 were cultured in GAM Broth supplemented with 0.75% Sodium DL-lactate, 2 mg/L putrescine. Other bacteria were cultured in BHI broth. Akkermansia muciniphila SNUG61027 was cultured in BHI broth with 16 g/l soy-peptone, 4 g/l threonine, and a mix of glucose and N-acetylglucosamine (25 mM each). All media were supplemented with 0.05% L-cysteine and all bacteria were cultured anaerobically at 37 °C. Cultures were washed and concentrated in anaerobic PBS with 25% (vol/vol) glycerol under strict anaerobic conditions. Additionally, an identical quantity of A. muciniphila grown on the synthetic medium was inactivated by pasteurization for 30 min at 70 °C. Cultures were then immediately frozen and stored at −80 °C. Bacterial viable count was performed using LIVE/DEAD™ BacLight™ Bacterial Viability and Counting Kit according to the manufacturer’s instructions (Thermo Fisher Scientific). Animal experiments Male C57BL/6 and NOD/ShiLtJ-Prkdcem1AMCIl2rgem1AMC (NSGA) mice were purchased from JA bio. Male athymic nude mice (Koat:Athymic NCr-nu/nu) were purchased from Koatech. All mice used in this study were 8 weeks old. All animal procedures were approved by Institutional Animal Care and Use Committee (IACUC) of Seoul National University, and they were performed in accordance with institutional guidelines and international law and policies (IACUC No. SNU-200820-3). To generate the mouse tumor model, MC38, B16F10, and CT26 (2.5 × 10 5 ) cells were subcutaneously (s.c.) implanted into the right flank. Tumors were measured with a caliper every 2-4 days beginning on day 7 after tumor inoculation. Tumor volume was calculated by the following equation, where L is the tumor length (larger dimension) and W is the width (smaller dimension): 𝑇𝑢𝑚𝑜𝑟 𝑣𝑜𝑙𝑢𝑚𝑒 = (𝐿 × 𝑊2)/2. Bacteria (1.6 × 10 8 cells/mouse for intratumoral injection; 5 × 10 9 cells/mouse for intraperitoneal injection) were administered intratumorally (i.t.) or intraperitoneally (i.p.) once every 3 days, starting on day 7 after tumor inoculation. For oral treatment, Veillonella parvula (1 × 10 12 cells/mouse) was administered every other day, starting from 7 days after tumor inoculation. Membrane fractions of Veillonella species were administered every 3 days i.p. at a dose of 50 μg/mouse. For the NK cell-depletion study, the mice were i.p. administered with control IgG (Clone 2A3; Bio X Cell) or anti-NK1.1 (Clone PK136; Bio X Cell) starting on day 6 after tumor inoculation (one day before Veillonella parvula administration) at a dose of 200 µg per mouse every 3 days until the end of the experiment. To neutralize TLR2, control IgG or TLR2 neutralizing antibody (Clone C9A12; Invivogen) was administered i.p. starting one day before Veillonella parvula administration at a dose of 50 μg/mouse every other day until the end of the experiment. For checkpoint blockade immunotherapy study, a tumor was implanted into the right flank as described above. Control IgG or PD1 blocking antibody (clone RMP1-14, InVivoPlus grade, BioXCell) was administered i.p. starting on day 6 after tumor inoculation (one day before Veillonella parvula administration) at a dose of 100 µg per mouse every 3 days until the end of the experiment. Single-cell transcriptome cell preparation and sequencing MC38 cells (2.5 × 10 5 ) were s.c. implanted into the right flank of 8-week-old male C57BL/6 mice. PBS or P-VP (5 × 10 9 cells/mouse) was administered twice i.p. on days 7 and 10. On day 11, tumor tissues were harvested and dissociated into single-cell suspensions using a Mouse Tumor Dissociation Kit according to the manufacturer’s instructions (130-096-730; Miltenyi Biotec). After dissociation, cells from each group were pooled for further analysis. Next, the dissociated cells were stained with Trypan blue, and their number and viability were assessed using a Countess II Automated Cell Counter (Thermo Fisher). Following QC, the single-cell suspension was loaded onto Chromium Chip A (10X Genomics PN 230027), and GEM generation, cDNA synthesis, cDNA amplification, and library preparation of 1,700-6,400 cells were processed using the Chromium Single Cell 5′ Reagent Kit (10X Genomics PN 1000006) according to the manufacturer’s protocol. cDNA amplification included 13-16 cycles, and 11-50 ng of the material was used to prepare sequencing libraries with 14-16 cycles of PCR. Indexed libraries were pooled equimolar and sequenced on a NovaSeq 6000 in a PE28/91 run using the NovaSeq 6000 S1 Reagent Kit (100 cycles) (Illumina). An average of 160 million paired reads were generated per sample. Single -cell RNA-Seq analysis 10X datasets were further processed using Cell Ranger v2.2.0 software using default parameters (10X Genomics) to generate fastq files. The Cell Ranger pipeline took the fastq files and generated expression matrices. A feature-barcode matrix was generated after preprocessing, aligning, counting UMI, and filtering cells with default options. We used R software version 4.3.0 to perform all further analyses using the Seurat package version 4.3.0. We removed low-quality cells with 9000 detected genes per cell and a mitochondrial RNA content of >10%. Normalization of the single-cell RNA-seq data was performed with the number of variable genes set at 10,000. Low-dimensional projection was performed using UMAP based on the top 15 principal components and for preliminary clustering of cells (resolution = 0.5). Differential gene expression analysis was performed using the FindMarkers function. Automated cell-type labeling was performed on the single-cell RNA-seq counts using the R package SingleR with reference purified microarray expression profiles from the Immunological Genome Project dataset. Gene ontology biological processes To perform the GO biological process enrichment analysis, we used the gost function from the gprofiler2 package version 0.2.2 to compare the genes identified in the neutrophil subclusters against genes that were present in the whole neutrophil cluster. Upregulated and downregulated genes were analyzed separately. Adjusted p values were calculated using the Benjamin & Hochberg (BH) false discovery rate method. Flow cytometry analysis Cell analysis was performed on a BD FACSCanto™ II (BD biosciences). For cytometry analysis, tissues including tumor, spleen, and bone marrow were harvested after intraperitoneal administration of pasteurized V. parvula to MC38-bearing mice, once every 3 days for a total of 4 times. Single cell suspensions from tumors were prepared after incubation of the dissected tissue for 30 min at 37 °C in RPMI culture medium containing 40 μg/mL DNase I (Sigma) and 2 mg/mL collagenase IV (Sigma). Mouse spleens were homogenized and splenic single cell suspensions were prepared after erythrocyte lysis with red blood cell lysis buffer (Sigma). The bone marrow cells were collected by flushing the femurs and treated with red blood cell lysis buffer (Sigma). Staining for flow cytometry was conducted in round bottom 96-well plates in the dark. Viability staining was conducted with Zombie Aqua™ Fixable Viability Kit (Biolegend) for 20 min at RT, followed by a surface staining cocktail for 30 min on ice. For intracellular cytokine staining, isolated cells were stimulated with 50 ng/mL phorbol-12-myristate-13-acetate (Sigma), 1 μg/mL ionomycin (Sigma) and 1 μL/mL GolgiPlug (BD biosciences) for 3 h at 37 °C prior to the addition of antibodies. After surface staining, cells were fixed and permeabilized for 20 min on ice with Cytofix/Cytoperm and washed with Perm/Wash buffer (BD Biosciences). Intracellular staining was then conducted using antibodies diluted in Perm/Wash buffer for 30 min on ice, followed by washing in Perm/Wash buffer. To determine intracellular levels of ROS, single cell suspensions from tumors were stained with antibodies against surface markers and then were stained with CellROX™ Green Flow Cytometry Assay Kit (Thermo Fisher). Data analysis was performed using FlowJo (Tree Star) software. The following antibodies were used for flow cytometry analysis: anti-mouse CD45-BV421 (clone 30-F11, Biolegend), anti-mouse CD45-FITC (clone 30-F11, Biolegend), anti-mouse CD3-BV421 (clone 17A2, Biolegend), anti-mouse CD3-FITC (clone 17A2, Biolegend), anti-mouse CD3-PerCP/Cyanine5.5 (clone 17A2, Biolegend), anti-mouse CD4-BV421 (clone GK1.5, Biolegend), anti-mouse CD4-FITC (clone GK1.5, Biolegend), anti-mouse CD8a-PE/Cyanine7 (clone 53-6.7, Biolegend), anti-mouse NK1.1-PE (clone PK136, Biolegend), anti-mouse/human CD11b-PE/Cyanine7 (clone M1/70, Biolegend), anti-mouse F4/80-FITC (clone BM8, Biolegend), anti-mouse F4/80-PerCP/Cyanine5.5 (clone BM8, Biolegend), anti-mouse F4/80-PE (clone BM8, Biolegend), anti-mouse Ly6C-PE (clone HK1.4, Biolegend), anti-mouse Ly6G-APC (clone 1A8, Biolegend), anti-mouse CD54 (clone YN1/1.7.4, Biolegend), anti-mouse CD16-PerCP/Cyanine5.5 (clone S17014E, Biolegend), anti-mouse CD170 (Siglec-F)-PE (clone S17007L, Biolegend), anti-mouse Nos2-PE (iNOS; clone W16030C, Biolegend), anti-mouse IFN-γ-APC (clone XMG1.2, Biolegend), anti-mouse/human Granzyme B-FITC (clone GB11, Biolegend), anti-mouse Perforin-APC (clone S16009B, Biolegend), anti-mouse CD282 (TLR2)-PE (clone CB225, Biolegend), anti-mouse CD284 (TLR4)-PE (clone SA15-21, Biolegend). The following markers were used for identifying different immune cell subsets: CD45+CD3+ for T-cells, CD45+CD3+CD4+ for CD4+ T-cells, CD45+CD3+CD8+ for CD8+ T-cells, CD45+CD3-NK1.1+ for NK cells, CD45+CD11b+ for myeloid cells, CD45+CD11b+F4/80+ for macrophages, CD45+CD11b+Ly6CloLy6G+ for neutrophils, and CD45+CD11b+Ly6ChiLy6G- for monocytes. The flow cytometry gating strategies are shown in Extended Data Fig. 7. Mouse cancer cell culture MC38 (murine colon adenocarcinoma cell line) was purchased from Kerafast. B16F10 (murine melanoma cell line) and CT26 (murine colorectal carcinoma cell line) were obtained from KCLB (Korean Cell Line Bank). Cancer cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 IU/mL penicillin/streptomycin (Sigma) in a 5% CO2 humidified incubator at 37 °C. Isolation of neutrophils To isolate neutrophils for ex vivo coculture experiments, splenocytes were isolated as described under ‘Flow cytometry analysis’. Neutrophils were purified using anti-Ly6G magnetic beads (Miltenyi Biotech) according to the manufacturer’s instructions. Purity of the magnetically isolated populations was > 90%. Measurement of cytokines IFN-γ were measured by ELISA MAX™ Deluxe Set (Biolegend) following the manufacturer’s instructions. Splenocytes were isolated from MC38-bearing mice as described under ‘Flow cytometry analysis’. Splenocytes were treated with the pasteurized mix of 16 bacteria described under ‘Bacteria culture and pasteurization’ at 1:100 = splenocyte:bacteria cells for 16 h, and then cultured supernatant was harvested and stored at -80 °C until measurement by ELISA. Cytotoxicity assay MC38 colon carcinoma cells were incubated in 96-well plate at 5 × 10 3 cells per plate one day before the cytotoxicity assay. Cytotoxicity was measured using CyQUANT LDH Cytotoxicity Assay (Invitrogen) according to the manufacturer’s instructions. For ex vivo experiments, splenic neutrophils isolated from mice treated with PBS or pasteurized V. parvula /Vpar0143 were cocultured with MC38 cells at 1:10, 1:50, and 1:100 (MC38 cells:neutrophils) for 16 h. For in vitro experiments, MC38 cells were cocultured with pasteurized V. parvula at 1:10000 (MC38 cells:bacteria cells) with splenocytes at 1:100 (MC38 cells:splenocytes) or without splenocytes. For TLR2 blocking experiment, splenic neutrophils were treated with 20μg/mL of anti-mouse TLR2 antibody (Clone C9A12; Invivogen) for 4 h before pasteurized V. parvula treatment, and then cocultured with MC38 cells (1:100 = MC38 cells:neutrophils) and pasteurized V. parvula (1:100 = neutrophils:bacteria cells) in a 5% CO 2 humidified incubator at 37 °C. RNA isolation and quantitative real time PCR (RT‒qPCR) RNA was extracted from tumor tissues using the Easy-spin Total RNA extraction kit (iNtRON Biotechnology). Complementary DNA was synthesized using the High-Capacity RNA-to-cDNA Kit according to the manufacturer’s instructions (Applied Biosystems). RT‒qPCR was performed by using THUNDERBIRD™ SYBR® qPCR Master Mix (TOYOBO) and QuantStudio™ 6 Flex Real-Time PCR System (Thermo Fisher Scientific). A list of the sequences of the primers used for RT–qPCR is provided in Supplementary Table 4. Relative mRNA expression was calculated using the comparative (2 −ΔΔCT ) method and normalized to β-actin housekeeping gene. Membrane fraction extraction of Veillonella species Veillonella species grown anaerobically were harvested by centrifugation at 5,000 × g for 20 min at 4 °C and resuspended in 0.1 M pH 7.3 Tris-HCl, and then the culture suspension was frozen at -80 °C overnight. Cells were thawed and lysed by sonication in an ice water bath in cycles of 15 sec sonication followed by 45 sec cooling until the lysate changes from an opaque solution into a less turbid solution. Cellular debris were pelleted by centrifugation at 6,500 × g for 20 min at 4 °C. The supernatant was collected and the pellet was discarded. This step was repeated an additional time. The supernatant was ultracentrifuged at 120,000 × g for 1 h at 4 °C and the pellet washed in Tris-HCl (0.1 M pH 7.3) and spun at 85,000 × g for 20 min at 4 °C. The wash was repeated twice. The pellet was resuspended in PBS pH 7 for in vivo experiments or ddH2O for LC‒MS/MS analysis. Pronase (Sigma) was also added to suspended membrane pellet at 200 µg/mL, which was incubated at 37 °C overnight, and then the enzyme activity was heat-inactivated at 95 °C for 10 min. The BCA Protein Assay kit (Thermo Fisher Scientific) was used to determine the protein concentration according to the manufacturer’s instructions. LC–MS/MS analysis of membrane fraction proteins The cellular proteins of V. parvula were isolated by carbonate extraction method. The extracted membrane proteins were sent to the Proteomics Core Facility at Seoul National University Hospital in Seoul, Korea for protein identification and analysis. The samples were analyzed using an LC–MS instrument consisting of an Ultimate 3000 RSLC system (Dionex), coupled via a nanoelectrospray ion source (Thermo Fisher Scientific) to a Q Exactive Plus Orbitrap (Thermo Fisher Scientific), according to previously described procedures. MS raw files were searched using MaxQuant (version 1.6.1.0) and the Andromeda search engine against the UniProt reference proteome database ( V. parvula ; UP000007968 and V. atypica ; UP000005942). The false-discovery rate was set to 0.01 for both proteins and peptides with a minimum length of six amino acids, and was determined by searching a reverse database. Plasmid constructs and protein expression The homologous protein to Vpar0143 or Vpar0556 was identified in E. coli DH5α (EcPrc) in the UniProt database (https://www.uniprot.org/). Each of the corresponding cDNAs was cloned into the pET-28a plasmid (Novagen) digested with Nde Ⅰ /Xho Ⅰ via Gibson assembly (NEB), which contains an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter. A list of the primer sequences used to generate the construct is provided in Supplementary Table 5. A His tag was added to the C-terminus of each protein for subsequent purification. The correct sequences of the resulting plasmids were then confirmed. These vectors were transformed into BL21 Escherichia coli (RRID: WBHT115(DE3)) and grown in LB broth containing kanamycin (100 μg/mL), into which 1.0 mM IPTG was added during the mid-exponential growth stage, after which a further 4 h was provided for protein expression. The cells were pelleted by centrifuging for 10 min at 5,000 × g and the cell pellets were stored at −80 °C until lysis. The cell pellets of BL21 (DE3) E. coli expressing Vpar0143 were resuspended and lysed by sonication Vibra-Cell™ VCX500 (Sonics & Materials). Vpar0556 expressing BL21 (DE3) E. coli pellets were resuspended with 8 M urea buffer and incubated for 1 h at 37 °C. After incubation, the resuspended BL21 (DE3) was centrifuged 10 min at 20,000 × g and the supernatant was used for protein purification. For protein purification, Ni-NTA agarose resin (QIAGEN) and the HisTALON buffer kit (Takara Bio) were used according to the manufacturer’s instructions, with minor modifications. The purified proteins were then dialyzed against endotoxin-free dialysis buffer. The purification of each protein was confirmed using SDS–polyacrylamide gel electrophoresis and Coomassie blue staining by the presence of a single band at the expected size. Statistical analysis Results are presented as the means ± SEM. Data were analyzed by two-tailed Student’s t test or Mann-Whitney U test as appropriate. Multiple-group comparisons were performed using two-way ANOVA with Tukey’s or Sidak’s multiple comparisons test. Statistical analyses were performed with GraphPad Prism software (GraphPad Inc.) and statistical significance was set at p < 0.05. Additional Declarations Yes there is potential Competing Interest. GwangPyo Ko is a founder and board member of KoBioLabs. Supplementary Files SupplementaryTable1.xlsx Dataset 1 SupplementaryTable2.xlsx Dataset 2 SupplementaryTable3a.xlsx Dataset 3A SupplementaryTable3b.xlsx Dataset 3B SupplementaryTable4.xlsx Dataset 4 SupplementaryTable5.xlsx Dataset 5 ExtendedDataFigs.docx Cite Share Download PDF Status: Under Review Version 1 posted 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. 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05:48:18","extension":"html","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":149368,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7615171/v1/07791623e9ecf98ee1e65205.html"},{"id":93651040,"identity":"f4f87065-53a6-4a13-addb-f51bcaacd249","added_by":"auto","created_at":"2025-10-16 05:48:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3236229,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eVeillonella parvula \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eis the major effector taxon and inhibits tumor growth in syngeneic mouse models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Experimental scheme (top): wild-type C57BL/6 mice were subcutaneously (s.c.) inoculated with MC38 murine colon adenocarcinoma cells on day 0. Beginning on day 7 after tumor implantation, PBS or bacterial samples (L-Mix16 or P-Mix16) were administered intratumorally (i.t.) every 3 days. Tumor growth curves (bottom) illustrate changes in tumor volume following treatment. \u003cem\u003en\u003c/em\u003e = 8 per group. \u003cstrong\u003eb,\u003c/strong\u003e Experimental scheme (top): splenocytes were isolated from MC38 tumor–bearing mice and stimulated \u003cem\u003ein vitro\u003c/em\u003e with pasteurized individual bacterial strains from the 16-member consortium. Interferon-γ (IFN-γ) concentrations in culture supernatants were measured by ELISA. Bars (bottom) indicate cytokine production in response to each bacterium. \u003cstrong\u003ec,\u003c/strong\u003e Experimental scheme (top): wild-type C57BL/6 mice were s.c. implanted with MC38 tumors on day 0 and received intratumoral injections of PBS, P-Mix14, P-Mix16, P-AM, or P-VP every 3 days starting on day 7. Tumor growth curves (bottom) show changes in tumor volume. \u003cem\u003en\u003c/em\u003e= 6 per group. \u003cstrong\u003ed,\u003c/strong\u003e LDH-mediated cytotoxicity assay of cocultured MC38 cells with splenocytes isolated from tumor-bearing mice (tumor + SP) or MC38 tumor cells alone (tumoronly) treated with PBS, P-VP, or P-AM. \u003cem\u003en\u003c/em\u003e = 5 for tumor + SP and \u003cem\u003en\u003c/em\u003e= 3 for tumor only \u003cstrong\u003ee,\u003c/strong\u003e Experimental scheme (left): wild-type C57BL/6 mice implanted with MC38 colon adenocarcinoma or B16F10 melanoma cells, and wild-type BALB/c mice implanted with CT26 colon carcinoma cells on day 0. Beginning on day 7, PBS or P-VP was administered intraperitoneally (i.p.) (every 3 days) or orally (daily). Tumor growth curves (right) illustrate changes in tumor volume following treatment. \u003cem\u003en\u003c/em\u003e = 6 for MC38-i.p. and CT26-i.p., \u003cem\u003en\u003c/em\u003e = 5 for B16F10-i.p., and \u003cem\u003en\u003c/em\u003e = 8 for MC38-oral. \u003cstrong\u003ef,\u003c/strong\u003e Experimental scheme (top): wild-type C57BL/6 mice were treated i.p. with PBS, P-VP, or P-VA every 3 days beginning on day 7 after s.c. MC38 tumor implantation. Tumor growth curves (left) and final tumor weights (right) of MC38 tumors are shown. \u003cem\u003en\u003c/em\u003e = 8 for PBS and P-VP groups, and \u003cem\u003en\u003c/em\u003e= 7 for the P-VA group. \u003cstrong\u003eg,\u003c/strong\u003e Experimental scheme (top): wild-type C57BL/6 mice implanted s.c. with MC38 tumors and treated i.p. with PBS or P-VP every 3 days starting on day 7. Mice additionally received intraperitoneal injections of control IgG or anti-PD1 antibody one day before each P-VP administration. Tumor growth curves (left) and final tumor weights (right) of MC38 tumors are shown. \u003cem\u003en\u003c/em\u003e = 6; IgG+PBS-, IgG+P-VP-, or αPD1+PBS-treated group, \u003cem\u003en\u003c/em\u003e = 5; αPD1+VP-treated group. For all graphs, data are shown as the mean ± s.e.m. ; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001; ns, not significant. P values were calculated using two-way ANOVA with Tukey’s test (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ec, left of f and g\u003c/strong\u003e) or Sidak’s test (\u003cstrong\u003ee\u003c/strong\u003e) for multiple comparisons or two-sided unpaired t test (\u003cstrong\u003ed\u003c/strong\u003e) or Kruskal–Wallis test with Dunn’s post-hoc correction (\u003cstrong\u003eright of f and g\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7615171/v1/26bd7735196c90f83d2d76a9.png"},{"id":93651764,"identity":"c86b0a68-7dea-4991-aebf-09d6390561de","added_by":"auto","created_at":"2025-10-16 05:56:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6206799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe administration of Pasteurized \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVeillonella parvula\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e changes the immune cell composition of tumors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Schematic of the experimental workflow. For single-cell RNA-seq, PBS or P-VP was administered i.p. on days 7 and 10, and tumors were collected on day 11. For flow cytometry validation, PBS or P-VP was administered i.p. every 3 days for four doses, and tumors were analyzed on day 17. \u003cstrong\u003eb,\u003c/strong\u003e Uniform Manifold Approximation and Projection (UMAP) plot (\u003cstrong\u003eleft\u003c/strong\u003e) and proportional representation (\u003cstrong\u003eright\u003c/strong\u003e) of cell clusters in tumors of mice treated with PBS or P-VP. \u003cstrong\u003ec-e\u003c/strong\u003e, Frequency of infiltrated myeloid cells in tumor tissues from mice treated i.p. with PBS or P-VP. \u003cem\u003en\u003c/em\u003e = 5 per group. \u003cstrong\u003ef,\u003c/strong\u003e Percentage of tumor-infiltrating neutrophils in PBS-, P-VP-, or P-VA-treated mice. \u003cem\u003en\u003c/em\u003e = 8 for PBS and P-VA groups, \u003cem\u003en\u003c/em\u003e = 7 for the P-VP group. For all graphs, data are shown as the mean ± s.e.m. ; *P \u0026lt; 0.05, **P \u0026lt; 0.01; ns, not significant. P values were calculated using a two-sided unpaired Mann–Whitney U test (\u003cstrong\u003ec-e\u003c/strong\u003e) and Kruskal–Wallis test with Dunn’s post-hoc correction (\u003cstrong\u003ef\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7615171/v1/a2eebd24b110a173ce46d29f.png"},{"id":93651041,"identity":"1642fb5a-8b67-4647-b376-9a8ca7724cf5","added_by":"auto","created_at":"2025-10-16 05:48:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3160455,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe antitumor effect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVeillonella parvula \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eis dependent on neutrophils\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Experimental scheme (left): athymic nude mice s.c. implanted with MC38 tumors on day 0 and treated i.p. with PBS or P-VP every 3 days starting on day 7. Tumor growth curves (middle) and final tumor weights (right) of MC38-bearing nude mice are shown. \u003cem\u003en\u003c/em\u003e = 6 per group. \u003cstrong\u003eb,\u003c/strong\u003e Experimental scheme (left): wild-type C57BL/6 mice s.c. implanted with MC38 tumors on day 0 and treated i.p. with PBS or P-VP every 3 days starting on day 7. Mice additionally received intraperitoneal injections of control IgG or anti-NK1.1 antibody one day before each PBS or P-VP treatment. Tumor growth curves (right) of s.c. MC38 tumors in NK1.1-depleted mice are shown. \u003cem\u003en\u003c/em\u003e = 6 per group. \u003cstrong\u003ec,\u003c/strong\u003e Experimental scheme (top): NSG mice s.c. implanted with MC38 tumors on day 0 and treated i.p. with PBS or P-VP every 3 days starting on day 7. Tumor growth curves (left) and final tumor weights (right) of MC38-bearing NSG mice are shown. \u003cem\u003en\u003c/em\u003e = 7 for PBS group, \u003cem\u003en\u003c/em\u003e = 8 for P-VP group. \u003cstrong\u003ed,\u003c/strong\u003e LDH-mediated cytotoxicity assay of cocultured MC38 cells with neutrophils isolated from spleens of tumor-bearing mice treated i.p. with PBS or P-VP. Treatments were administered twice at 3-day intervals before neutrophil isolation. \u003cem\u003en\u003c/em\u003e = 3 per group.\u003cstrong\u003e e, \u003c/strong\u003eUMAP plot (\u003cstrong\u003eleft\u003c/strong\u003e) and relative proportions (\u003cstrong\u003eright\u003c/strong\u003e) of subclusters 0-2 of tumor-infiltrating neutrophils in mice treated with PBS or P-VP. \u003cstrong\u003ef,\u003c/strong\u003e Mean fluorescence intensity (MFI) of ROS in tumor-infiltrating neutrophils. \u003cem\u003en\u003c/em\u003e = 3 per group. PBS or P-VP was administered i.p. every 3 days for four doses, and tumors were analyzed on day 17. \u003cstrong\u003eg,\u003c/strong\u003e Flow cytometry analysis of the percentage of iNOS\u003csup\u003e+\u003c/sup\u003e neutrophils in tumors. \u003cem\u003en\u003c/em\u003e = 5 per group. PBS or P-VP was administered i.p. every 3 days for four doses, and tumors were analyzed on day 17. \u003cstrong\u003eh, \u003c/strong\u003eBubble plot showing Gene Ontology (GO) terms related to reactive oxygen species (ROS) metabolic processes and cellular responses to ROS in each neutrophil subcluster. For all graphs, data are shown as the mean ± s.e.m. ; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ****P \u0026lt; 0.0001; ns, not significant. P values were calculated using two-way ANOVA with Sidak’s test (\u003cstrong\u003emiddle of a and left of c\u003c/strong\u003e) or Tukey’s test (\u003cstrong\u003eb\u003c/strong\u003e) for multiple comparisons, two-sided unpaired Mann–Whitney U test (\u003cstrong\u003eright of a and c\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e), and two-sided unpaired t test (\u003cstrong\u003ef, g\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7615171/v1/a52f21960b83c315fa93e4ab.png"},{"id":93651045,"identity":"6c0de27b-30ca-47ab-b8bd-2b2848582bbe","added_by":"auto","created_at":"2025-10-16 05:48:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2034400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe antitumor effect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVeillonella parvula\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e is mediated through TLR2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Relative expression of pattern recognition receptor (PRR) genes in tumors from MC38-bearing mice treated i.p. with PBS or P-VP twice at 3-day intervals, measured by qPCR. \u003cem\u003en\u003c/em\u003e = 6 per group. \u003cstrong\u003eb-c,\u003c/strong\u003e Mean fluorescence intensity (MFI) of TLR2 (\u003cstrong\u003eb\u003c/strong\u003e) or TLR4 (\u003cstrong\u003ec\u003c/strong\u003e) in tumor-infiltrating neutrophils from mice treated i.p. with PBS or P-VP for four doses. \u003cem\u003en\u003c/em\u003e = 6 per group in \u003cstrong\u003eb\u003c/strong\u003e; \u003cem\u003en\u003c/em\u003e = 5 per group in \u003cstrong\u003ec\u003c/strong\u003e. \u003cstrong\u003ed,\u003c/strong\u003eExperimental scheme (top): wild-type C57BL/6 mice s.c. implanted with MC38 tumors on day 0 and treated i.p. with PBS or P-VP every 3 days starting on day 7. Mice additionally received daily i.p. injections of control IgG or anti-TLR2 antibody, beginning one day before PBS or P-VP treatment. Tumor growth curves (left) and final tumor weights (right) of s.c. MC38 tumors are shown. \u003cem\u003en\u003c/em\u003e = 5 per group. \u003cstrong\u003ee,\u003c/strong\u003e LDH-mediated cytotoxicity assay of cocultured MC38 cells with neutrophils isolated from the spleens of tumor-bearing mice. Anti-TLR2 antibody was administered 4 h before treatment with PBS or P-VP. For all graphs, data are shown as the mean ± s.e.m. ; *P \u0026lt; 0.05, **P \u0026lt; 0.01; ns, not significant. P values were calculated using two-way ANOVA with Tukey’s test for multiple comparisons (\u003cstrong\u003eleft of d\u003c/strong\u003e), two-sided unpaired Mann–Whitney U test (\u003cstrong\u003ea-c\u003c/strong\u003eand \u003cstrong\u003eright of d\u003c/strong\u003e), or two-sided unpaired t test (\u003cstrong\u003ee\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7615171/v1/761efa14744dc23b51b28a68.png"},{"id":93651047,"identity":"a063fd4e-78dd-458f-a0f2-dd043a7351f0","added_by":"auto","created_at":"2025-10-16 05:48:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3462334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFerritin Dps family protein Vpar0143 from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVeillonella parvula\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e is involved in the antitumor effect and increased neutrophil frequency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Experimental scheme (left): wild-type C57BL/6 mice s.c. implanted with MC38 tumors on day 0 and treated i.p. with PBS, VP-MF, or pronase-treated VP-MF (VP-MFP) every 3 days starting on day 7. Tumor growth curves (middle) and final tumor weights (right) of MC38 tumors are shown. For tumor growth curves: \u003cem\u003en\u003c/em\u003e = 8 for the PBS group, \u003cem\u003en\u003c/em\u003e = 7 for the VP-MF and VP-MFP groups. For tumor weights: \u003cem\u003en\u003c/em\u003e = 6 per group. \u003cstrong\u003eb,\u003c/strong\u003e Frequency of neutrophils in the tumors of mice treated with PBS, VP-MF, or VP-MFP. \u003cem\u003en\u003c/em\u003e = 6 per group. \u003cstrong\u003ec, \u003c/strong\u003eWorkflow of comparative proteomics from the membrane fraction between VP and VA by LC‒MS/MS. \u003cstrong\u003ed,\u003c/strong\u003e iBAQ intensities of the top 10 most abundant proteins (\u0026gt;30% sequence coverage). \u003cstrong\u003ee,\u003c/strong\u003e Experimental scheme (left): wild-type C57BL/6 mice s.c. implanted with MC38 tumors on day 0 and treated i.p. with PBS, 100 μg Vpar0143, or 300 μg Vpar0143 every 2-3 days starting on day 7. Tumor growth curves (middle) and final tumor weights (right) of MC38 tumors are shown. \u003cem\u003en\u003c/em\u003e = 6 per group. \u003cstrong\u003ef,\u003c/strong\u003e Frequency of neutrophils in the tumors of mice treated with PBS or Vpar0143. \u003cem\u003en\u003c/em\u003e = 6 per group. \u003cstrong\u003eg,\u003c/strong\u003e LDH-mediated cytotoxicity assay of cocultured MC38 cells with neutrophils isolated from spleens of tumor-bearing mice treated i.p. with PBS or Vpar0143. Treatments were administered twice at 3-day intervals before neutrophil isolation. \u003cem\u003en\u003c/em\u003e = 3 per group. \u003cstrong\u003eh,\u003c/strong\u003e Mean fluorescence intensity (MFI) of ROS in tumor-infiltrating neutrophils. PBS or Vpar0143 was administered i.p. every 3 days for four doses, and tumors were analyzed on day 17. \u003cem\u003en\u003c/em\u003e = 7 per group. \u003cstrong\u003ei\u003c/strong\u003e, Flow cytometry analysis of the percentage of iNOS\u003csup\u003e+\u003c/sup\u003e neutrophils in tumors. PBS or Vpar0143 was administered i.p. every 3 days for four doses, and tumors were analyzed on day 17. \u003cem\u003en\u003c/em\u003e = 5 per group. For all graphs, data are shown as the mean ± s.e.m. ; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001. P values were calculated using two-way ANOVA with Tukey’s test for multiple comparisons (\u003cstrong\u003emiddle of a and e\u003c/strong\u003e), Kruskal‒Wallis test (\u003cstrong\u003eright of a and e, b\u003c/strong\u003e), two-sided unpaired t test (\u003cstrong\u003eg\u003c/strong\u003e), and two-sided unpaired Mann–Whitney U test (\u003cstrong\u003ef, h-i\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7615171/v1/9ab7a2a0990db6ad170ca101.png"},{"id":93652554,"identity":"a7b86db6-2d66-4ff6-9f04-f4f3ad87985a","added_by":"auto","created_at":"2025-10-16 06:12:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21794418,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7615171/v1/364ca77b-0a82-409e-ab86-3ba033bd23b9.pdf"},{"id":93651762,"identity":"66bd8082-e3be-4d30-916f-2493e39aad59","added_by":"auto","created_at":"2025-10-16 05:56:17","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10203,"visible":true,"origin":"","legend":"Dataset 1","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7615171/v1/4bb6b265b2060c71a3a588af.xlsx"},{"id":93651046,"identity":"ba82144f-90f0-4df0-9850-05ca1f55eaa5","added_by":"auto","created_at":"2025-10-16 05:48:17","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":301383,"visible":true,"origin":"","legend":"Dataset 2","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7615171/v1/e365c9f8314c309a710c4dbe.xlsx"},{"id":93651068,"identity":"afecc1f9-0992-4bd2-ac39-318c6464a700","added_by":"auto","created_at":"2025-10-16 05:48:18","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":48695487,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 3A\u003c/p\u003e","description":"","filename":"SupplementaryTable3a.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7615171/v1/8e2b4b9bb17917d99a93c66a.xlsx"},{"id":93651061,"identity":"92fc1974-4480-4a71-97b2-8a466ba61ee1","added_by":"auto","created_at":"2025-10-16 05:48:18","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":21744408,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 3B\u003c/p\u003e","description":"","filename":"SupplementaryTable3b.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7615171/v1/8e7b0815a1eee651f286a03f.xlsx"},{"id":93651048,"identity":"957d4390-7895-4691-9253-510a93b30536","added_by":"auto","created_at":"2025-10-16 05:48:17","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10813,"visible":true,"origin":"","legend":"Dataset 4","description":"","filename":"SupplementaryTable4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7615171/v1/e303cd962627d40940368e16.xlsx"},{"id":93651765,"identity":"e8b7410c-5c10-4ec7-939c-e3e529a53574","added_by":"auto","created_at":"2025-10-16 05:56:17","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":9651,"visible":true,"origin":"","legend":"Dataset 5","description":"","filename":"SupplementaryTable5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7615171/v1/ee5b23f854409554afa911b0.xlsx"},{"id":93651919,"identity":"61795b98-f195-4e60-ad85-f39709d4d9ad","added_by":"auto","created_at":"2025-10-16 06:04:17","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":10089179,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFigs.docx","url":"https://assets-eu.researchsquare.com/files/rs-7615171/v1/7e828cc18424c93706a1a07b.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nGwangPyo Ko is a founder and board member of KoBioLabs.","formattedTitle":"The Gut Commensal Bacteria Veillonella parvula-Induced Neutrophil Activation Mediates Antitumor Activity","fulltext":[{"header":"Main","content":"\u003cp\u003eThe gut microbiota has emerged as a key regulator of cancer immunity, influencing both tumor progression and therapeutic responses\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Recent evidence suggests that the efficacy of immune checkpoint inhibitors (ICIs), such as anti-PD1 therapy, is closely linked to the composition of the gut microbiota\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, despite the growing interest in microbiota-immune interactions, the mechanisms by which microbiota enhance antitumor immunity remain to be fully elucidated. Given the therapeutic potential of microbiota modulation, strategies such as fecal microbiota transplantation (FMT) have been explored to improve responses in patients with poor ICI efficacy\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Yet, FMT presents challenges, including safety concerns, high costs, and inconsistent clinical outcomes, necessitating alternative, targeted approaches\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOne promising strategy involves the identification and administration of specific bacterial strains with defined antitumor properties. However, the efficacy of administered bacteria, which depends on their stable colonization within host-specific intestinal environments\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, underscores the need to identify precise molecular mechanisms and targets to ensure therapeutic consistency. While prior research has largely focused on adaptive immune responses, particularly T cell-mediated mechanisms\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, the role of innate immune cells in microbiota-driven antitumor immunity remains underexplored.\u003c/p\u003e\u003cp\u003eNeutrophils, as key components of the innate immune system, exhibit a paradoxical role in cancer, with both tumor-promoting and antitumor activities. Although they have been implicated in immunosuppressive functions that support tumor growth, emerging evidence indicates that neutrophils can also mediate potent antitumor effects through direct cytotoxicity, antibody-dependent mechanisms, and recruitment of additional immune effectors\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. For instance, neutrophils can induce tumor cell death via the release of oxidative substrates such as reactive oxygen species (ROS) and nitric oxide (NO)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Despite these insights, the interplay between gut microbiota and neutrophils in shaping cancer immunity necessitate further investigation.\u003c/p\u003e\u003cp\u003eIn this study, we systematically screened 16 bacterial taxa previously associated with cancer immunotherapy to identify a candidate with strong antitumor properties. Single-cell transcriptome analysis revealed the mechanisms of the selected \u003cem\u003eVeillonella\u003c/em\u003e strain, indicating neutrophil-mediated innate immunity is central to driving downstream immune responses. Additionally, comparative proteomic analysis allowed us to uncover key antitumor molecules produced by the candidate microbe. By integrating these approaches, our study advances the understanding of microbiota-mediated tumor immunity and highlights the potential of microbial-based strategies as precision therapeutics in cancer treatment.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eVeillonella parvula\u003c/b\u003e \u003cb\u003eEnhances Antitumor Immunity and Synergizes with Anti-PD1 Therapy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine whether bacteria indeed have an antitumor effect related to immunotherapy response as previously reported \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, we selected 16 bacteria and retrieved them from our in-house culture collection or commercially purchased them (Supplementary Table\u0026nbsp;1). To determine whether the beneficial component is a cellular component of bacteria or a component secreted by bacteria, a mixture of these bacteria (Mix16) was divided into a live mixture (L-mix16) and a pasteurized mixture (P-Mix16). Both L-Mix16 and P-Mix16 inhibited tumor growth when intratumorally administered to tumor-bearing mice. This indicates that bacteria associated with a positive cancer treatment response can also induce antitumor effects and that cellular components of the bacteria are the main modulators of these effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo identify the major taxa with tumor suppression effects, we first treated splenocytes from MC38 tumor-bearing mice with pasteurized bacteria and compared the level of IFN-γ, a typical anticancer cytokine\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Among the tested strains, only \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e (AM) and \u003cem\u003eVeillonella parvula\u003c/em\u003e (VP) induced IFN-γ production. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). To determine whether these two bacteria can inhibit tumor growth \u003cem\u003ein vivo\u003c/em\u003e, we administered the following five treatments to MC38 tumor-bearing mice: PBS, pasteurized AM (P-AM), pasteurized VP (P-VP), P-Mix16, and pasteurized Mix16 excluding AM and VP (P-Mix14). Interestingly, tumor growth inhibition was not observed in P-Mix14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), suggesting that AM and VP were the major effector strains.\u003c/p\u003e\u003cp\u003eNext, we isolated splenocytes from MC38 tumor-bearing mice, treated them with P-AM or P-VP, and cocultured them with MC38 tumor cells to compare the tumor suppressive effects of the two bacteria. To exclude direct bacterial cytotoxicity, MC38 tumor cells were also treated with the bacteria in the absence of splenocytes. P-AM- or P-VP-treated splenocytes displayed enhanced tumor cytotoxicity; this effect disappeared when only the tumor cells were treated with the bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Moreover, P-VP-treated splenocytes exhibited significantly higher cytotoxicity compared with P-AM-treated splenocytes. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Ultimately, we selected VP as a final candidate. We additionally tested different administration routes (oral gavage and intraperitoneal injection), mouse strains (C57BL/6 and BALB/c), and subcutaneous tumor types (MC38, B16F10, and CT26) to compare the efficacy of VP under different conditions. The results showed comparable efficacy across administration routes, mouse strains, and tumor types, indicating that the antitumor effect of P-VP was robust across diverse conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). To further determine species-specific effects, we investigated the antitumor activity of pasteurized \u003cem\u003eV. atypica\u003c/em\u003e (P-VA), another common \u003cem\u003eVeillonella\u003c/em\u003e species, but the strain did not exhibit any antitumor effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Finally, the combination of P-VP with anti-PD1 antibody synergistically suppressed tumor growth compared with either treatment alone. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Therefore, all of these results point to the potent capacity of VP to inhibit tumor growth.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eP-VP Treatment Reshapes Tumor Immune Landscape by Enhancing Neutrophil Infiltration\u003c/h2\u003e\u003cp\u003eNext, we performed single-cell transcriptomic analysis to profile the infiltrated immune cell composition of the tumor following intraperitoneal injection of P-VP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). After quality control and filtering, we obtained 7,707 cells from PBS-treated mice and 9,995 cells from P-VP-treated mice. Clustering analysis revealed 10 cell types in the tumors of PBS- and VP-treated mice with unique transcriptional features (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Interestingly, the myeloid cell lineage was greatly altered in the P-VP-treated tumor, with increased neutrophils and decreased macrophages and monocytes compared to PBS-treated mice. T and NK cells showed a modest apparent increase in the single-cell data, though this change was limited compared with the neutrophil expansion. Flow cytometry validation confirmed that neutrophils were the only population with a significant increase in frequency, whereas other subsets, including T and NK cells, were reduced or showed no significant changes in P-VP-treated tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-e and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, c, f). Functional assessment further demonstrated that P-VP treatment did not enhance T or NK cell activity, as indicated by the frequencies of IFN-γ⁺, granzyme B (GZMB)⁺, and perforin (PFN)⁺ cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, d-e, g-h). Importantly, neutrophil enrichment was not observed in mice administered P-VA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). A similar increase in neutrophils was detected in the spleen and bone marrow, suggesting that VP treatment promotes systemic neutrophil expansion and recruitment into tumors through the circulation (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eP-VP Promotes Tumor-Suppressive Neutrophil Differentiation Through Oxidative Burst Activity\u003c/h3\u003e\n\u003cp\u003eTo confirm whether T-cells and NK cells were involved in the antitumor effect of VP, athymic nude mice lacking T-cells\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e and NK cell-depleted mice (administered a neutralizing antibody) were transplanted with MC38 tumor cells and intraperitoneally injected with P-VP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b). Treatment with P-VP still delayed tumor growth in both athymic nude mice and NK cell-depleted mice, indicating that its effect is independent of T and NK cells. To further investigate whether the antitumor effect of P-VP was dependent only on neutrophils, we employed the NOD-scid IL2rγ\u003csup\u003enull\u003c/sup\u003e (NSGA) mouse model, an immunodeficient mouse strain lacking T-, B- and NK cells and having defective dendritic cells (DCs) and macrophages. Neutrophils are therefore the only remaining functional immune cells in this mouse\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). P-VP still suppressed tumor growth in these neutrophil-functional mice. To further assess whether the tumor-suppressive potential of neutrophils could be induced by P-VP treatment, we isolated neutrophils from the spleens of MC38 tumor-bearing mice treated with PBS or P-VP and cocultured them with MC38 colon cancer cells. As a result, neutrophils from P-VP-treated mice displayed enhanced tumor cytotoxicity, indicating that VP can increase neutrophil antitumor cytotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven that P-VP treatment yielded antitumorigenic neutrophils, further characterization of the enriched single-cell clusters was carried out to better define functional neutrophils with antitumor effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). When we reanalyzed the cluster defined as neutrophils, we obtained three subclusters. Cluster 0 was detected only in PBS-treated tumors, whereas clusters 1 and 2 emerged uniquely in the P-VP-treated group. Cluster 0 neutrophils expressed inflammatory and tissue-remodeling genes such as \u003cem\u003eS100a8, S100a9, Mmp8\u003c/em\u003e, and \u003cem\u003eMmp9\u003c/em\u003e, consistent with a baseline pro-inflammatory state. By contrast, cluster 1 neutrophils were enriched for type I interferon\u0026ndash;stimulated genes (\u003cem\u003eIfit1, Ifit2, Rsad2, Gbp2/5\u003c/em\u003e) and metabolic regulators such as \u003cem\u003eAcod1\u003c/em\u003e, consistent with interferon-driven antimicrobial and oxidative effector programs. Cluster 2 neutrophils displayed a degranulation-associated signature, with high expression of \u003cem\u003eCamp, Ngp, Ltf, Chil3\u003c/em\u003e, and \u003cem\u003eCd177\u003c/em\u003e, together with redox enzymes (\u003cem\u003eG6pdx, Sqor\u003c/em\u003e), indicative of enhanced oxidative burst capacity consistent with tumor-suppressive effector functions. These findings suggest that P-VP treatment promotes the emergence of neutrophil subsets transcriptionally equipped for oxidative burst\u0026ndash;mediated effector activity (Supplementary Table\u0026nbsp;2). Given that neutrophils exert antitumor effects by inducing oxidative bursts that are toxic to cancer cells\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, we next performed flow cytometry analysis and confirmed the elevation of ROS- and iNOS-positive cells in the P-VP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef-g). In line with this observation, Gene Ontology (GO) analysis of the neutrophil subclusters showed upregulation of ROS metabolic processes in clusters 1 and 2, while these processes were downregulated in cluster 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). Taken together, these results indicated that the newly differentiated neutrophil subtypes induced by P-VP administration can suppress tumor growth through oxidative burst activity.\u003c/p\u003e\n\u003ch3\u003eP-VP Activates Neutrophils via TLR2 to Enhance Antitumor Cytotoxicity\u003c/h3\u003e\n\u003cp\u003eNext, to investigate how VP activates neutrophils, we examined variations in pattern recognition receptors (PRRs), which are critical mediators of host immune responses to microbial stimuli\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. To this end, we analyzed the expression of multiple PRR genes in tumors, including \u003cem\u003eTlr1\u003c/em\u003e, \u003cem\u003eTlr2\u003c/em\u003e, \u003cem\u003eTlr4\u003c/em\u003e, \u003cem\u003eTlr5\u003c/em\u003e, \u003cem\u003eTlr6\u003c/em\u003e, \u003cem\u003eTlr9\u003c/em\u003e, \u003cem\u003eNod1\u003c/em\u003e, and \u003cem\u003eNod2\u003c/em\u003e, after treating tumor-bearing mice with P-VP. The results showed that \u003cem\u003eTlr2\u003c/em\u003e was the only gene significantly upregulated by P-VP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Flow cytometry analysis further confirmed this finding by showing increased TLR2 expression on neutrophils, whereas TLR4 expression was not altered (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-c). Importantly, neutralization of TLR2 markedly diminished the antitumor activity of P-VP in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Consistently, neutrophils isolated from the mouse spleen treated with anti-TLR2 antibody lost their capacity to cause cytotoxicity when exposed to P-VP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Together, these results suggest that P-VP activates neutrophils through TLR2 to promote the production of ROS and NO, resulting in the inhibition of tumor growth.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eFerritin Dps Family Protein Vpar0143 Mediates VP-Induced Antitumor Neutrophil Activation\u003c/h3\u003e\n\u003cp\u003eNext, we tried to find the key bacterial antitumor substances. To this end, we purified the membrane fraction of VP (VP-MF) and further treated it with pronase, a mixture of nonspecific endo- and exoproteases, to eliminate the protein fraction (VP-MFP) from the membrane fraction, as it is known that bacterial proteins can affect immunity\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The antitumor effect was not observed in the VP-MFP-treated mice, while administration of VP-MF reduced tumor growth in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Additionally, the frequency of tumor-infiltrating neutrophils was increased only in VP-MF-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d), whereas VA-MF treatment failed to elicit such an effect (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-b). These results suggest that proteins within the membrane fraction are the main effector components of VP.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo identify a specific antitumor protein, we performed a proteome analysis and compared the proteins from the membrane fraction of VP to those of VA based on the iBAQ intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and Supplementary Table\u0026nbsp;3a-b). We initially evaluated one of the most abundant proteins, an S-layer domain-related protein (Vpar0556), from the VP membrane fraction (Supplementary Table\u0026nbsp;3a). This protein was also abundant in VA and did not inhibit tumor growth or induce an increase in neutrophils infiltrating the tumor (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). Next, we searched for a highly abundant protein found only in the VP and selected Vpar0143, a ferritin Dps family protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed and Supplementary Table\u0026nbsp;3b). Administration of Vpar0143 to mice bearing MC38 tumors inhibited tumor growth in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee) and increased the frequency of tumor-infiltrating neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Moreover, a cytotoxicity assay showed that neutrophils isolated from mice administered Vpar0143 had enhanced cytotoxicity compared to neutrophils isolated from PBS-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). This protein also promoted neutrophil-mediated antitumor activity through induction of ROS and NO production (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh-i). Consistent with the results of P-VP itself, Vpar0143 specifically upregulated TLR2 expression in neutrophils (Extended Data Fig.\u0026nbsp;6a-b). All of these findings point to important roles for the ferritin Dps family protein Vpar0143 in the antitumor activity of VP via neutrophil stimulation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious reports have shown that commensal bacteria are closely related to the immunotherapy response; however, the bacteria identified vary between studies. For example, \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eAkkermansia\u003c/em\u003e have previously been shown to have anticancer efficacy\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and we discovered that VP had the most robust antitumor effects out of the 16 selected bacteria under the same conditions. Beyond confirming the efficacy of VP, our study elucidates its underlying antitumor mechanisms. Previous studies on the antitumor mechanisms of the human microbiota have centered on improving adaptive immune responses, especially CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T-cells, although microbiota also have important roles in the establishment of the innate immune system\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In the present study, single-cell transcriptome analysis showed that VP induced neutrophil subtypes with antitumor effects by upregulating ROS and NO production.\u003c/p\u003e\u003cp\u003eThe role of neutrophils in cancer remains in debate. Neutrophils have potent antitumor effects through ROS or NO production, but in different settings, these oxygen free radicals can induce host immunosuppression and tumor progression because they are also toxic to normal immune cells including cytotoxic T-cells\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Peishan et al. reported that these dual roles of neutrophils depend on the immune status of the host. The study found that neutrophils had a \u0026lsquo;net tumoricidal\u0026rsquo; effect when NK cells were absent in the host, but they had a \u0026lsquo;net tumor-promoting\u0026rsquo; effect when NK cells were present because the NK cells were able to block the increased tumoricidal activity of neutrophils. This is because neutrophils are cytotoxic not only to tumors but also to effector immune cells such as NK cells. It would therefore potentially be necessary to check the patient's immune status prior to the clinical application of neutrophil-based cancer therapy.\u003c/p\u003e\u003cp\u003eNeutrophils and T-cells may have a complementary relationship in terms of cancer treatment. The maturation of neutrophils to antigen-presenting cells (APCs) is promoted by IFN-γ and granulocyte\u0026ndash;macrophage colony-stimulating factor (GM-CSF), which also activate adaptive T-cells\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Additionally, neutrophils polarize CD4\u003csup\u003e\u0026ndash;\u003c/sup\u003eCD8\u003csup\u003e\u0026ndash;\u003c/sup\u003eTCRαβ\u003csup\u003e+\u003c/sup\u003e double-negative unconventional T-cells (UTC\u003csub\u003eαβ\u003c/sub\u003e) toward the type 1 immune response and produce IFN-γ by interacting with macrophages\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In addition, a recent study uncovered an interaction between T-cells and neutrophils in T-cell immunotherapies. T-cells mediate an initial antitumor immune response, while neutrophils are responsible for removing tumor antigen-loss variants. The complete eradication of the tumor relied on the presence of neutrophils and was partially dependent on inducible NOS\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRecent efforts have attempted to utilize neutrophil-activated oxidative damage for cancer treatment. A previous study demonstrated that the combined actions of tumor necrosis factor (TNF), CD40 agonist, and tumor-binding antibody activated neutrophil infiltration and induced an inflammatory cascade driving ROS production, which drove T-cell\u0026ndash;independent tumor clearance\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Given that neutrophils are the first antimicrobial responders, microbes can be an promising adjuvant for neutrophil-activation therapy. Another recent study highlighted the potential applications of microbial immunotherapy in the treatment of solid tumors\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The therapeutic impact of checkpoint inhibitor therapy was found to be improved by intratumoral injection of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, which increased CD8 T-cell function as well as secretion of ROS by neutrophil activation\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Our study also showed the potential of commensal bacteria as an adjuvant for microbe-based neutrophil-activation therapy.\u003c/p\u003e\u003cp\u003eWe further discovered a bacteria-driven antitumor protein with the potential for safe, stable and controllable therapeutic development. Ferritin Dps family protein is a molecule with iron-storage activity and is known to affect host immunity in addition to protecting bacterial DNA from oxidizing free radicals\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. One of the most-studied bacterial ferritins is \u003cem\u003eHelicobacter pylori\u003c/em\u003e neutrophil-activating protein (HP-NAP), a TLR2 agonist that activates neutrophils to generate oxygen free radicals and that induces other innate immune cells to secrete proinflammatory cytokines such as TNF-α, IL-6, IL-12 and IL-23\u003csup\u003e33\u003c/sup\u003e. Another study showed that this protein also has antitumor activity and enhances the immunotherapy response\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. We observed a similar mechanistic link between Vpar0143 and MC38 tumors, yet the complexity of bacterial components opens the possibility of other effective bacterial antitumor compounds. Therefore, further studies are warranted on the antitumor efficacy of other constitutive components of VP.\u003c/p\u003e\u003cp\u003eFurther investigations into the antitumor mechanisms of VP or Vpar0143 using various preclinical models including metastasis tumor models and spontaneous cancer models may be essential for exploring clinical application, as we have only confirmed the antitumor efficacy of VP and Vpar0143 in subcutaneous tumor models.\u003c/p\u003e\u003cp\u003eIn conclusion, we have demonstrated that VP and its membrane protein, Vpar0143, can be effective antitumor treatments, as they were able to significantly inhibit tumor growth and promote neutrophil-mediated antitumor immunity by inducing ROS and NO production. This study provides a new perspective on the clinical potential of VP in cancer treatment and the development of a novel neutrophil-targeting cancer therapeutic.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.Y., H.J.Y., and G.P.K. conceived and designed the study. S.Y. and H.J.Y. drafted the manuscript with input from all authors. J.S. performed the single-cell RNA-seq data analysis. S.J., J.P., and M.J. assisted with \u003cem\u003ein vivo\u003c/em\u003e experiments and sample processing. D.H. and K.L. performed the proteomic analysis. G.P.K. supervised the project and acquired funding. H.K.L. provided critical advice and reviewed the manuscript. L.S., G.L., and J.Y. contributed to data acquisition and analysis. J.S.C. performed cloning and protein production. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Bio\u0026amp;Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (RS-2022-NR067344). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (RS-2024-00336212).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConflict of interest statement\u003c/p\u003e\n\u003cp\u003eG.K. is a founder of KoBioLabs, Inc., a company characterizing the role of host-microbiome interaction. The other authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHelmink, B. A., Khan, M. A. 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HP-NAP of Helicobacter pylori : The Power of the Immunomodulation. \u003cstrong\u003e13\u003c/strong\u003e, 1\u0026ndash;7 (2022).\u003c/li\u003e\n\u003cli\u003eAmedei, A. \u003cem\u003eet al.\u003c/em\u003e The neutrophil-activating protein of Helicobacter pylori promotes Th1 immune responses. \u003cstrong\u003e116\u003c/strong\u003e, 1092\u0026ndash;1101 (2006).\u003c/li\u003e\n\u003cli\u003eLim, M. Y. \u003cem\u003eet al.\u003c/em\u003e The effect of heritability and host genetics on the gut microbiota and metabolic syndrome. \u003cem\u003eGut\u003c/em\u003e\u003cstrong\u003e66\u003c/strong\u003e, 1031\u0026ndash;1038 (2017).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eBacteria culture and pasteurization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrief information on the strains used in this study is listed in Supplementary Table 1. \u003cem\u003eBifidobacterium adolescentis\u003c/em\u003e SNUG30339, \u003cem\u003eBifidobacterium longum\u003c/em\u003e SNUG30192, \u003cem\u003eCollinsella aerofaciens\u003c/em\u003e SNUG30018, \u003cem\u003eParabacteroides merdae\u003c/em\u003e SNUG30168, \u003cem\u003eBacteroides caccae\u003c/em\u003e SNUG30273, \u003cem\u003eBacteroides thetaiotamicron\u003c/em\u003e SNUG30057, \u003cem\u003eAlistipes indistinctus\u003c/em\u003e SNUG30286, \u003cem\u003eEnterococcus faecium\u003c/em\u003e SNUG10198, \u003cem\u003eEnterococcus hirae\u003c/em\u003e SNUG10078, \u003cem\u003eEubacterium callanderi\u003c/em\u003e SNUG40125, \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e SNUG30106, and \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e SNUG61027 were isolated from freshly collected fecal samples, or \u003cem\u003eLactobacillus crispatus\u003c/em\u003e SNUV206 and \u003cem\u003eVeillonella atypica\u003c/em\u003e SNUV638 were isolated from fresh vaginal fluid samples that were obtained from healthy individuals who had not been treated with antibiotics during the preceding year\u003csup\u003e35\u003c/sup\u003e. \u003cem\u003eRuminococcus bromii\u003c/em\u003e ATCC27255 was purchased from American Type Culture Collection (ATCC), and \u003cem\u003eRuminococcus faecis\u003c/em\u003e KCTC5757 and \u003cem\u003eVeillonella parvula\u003c/em\u003e KCTC5019 were purchased from the Korean Collection for Type Cultures (KCTC Seoul, Korea). \u003cem\u003eBifidobacterium adolescentis\u003c/em\u003e SNUG30339, \u003cem\u003eBifidobacterium longum\u003c/em\u003e SNUG30192, and \u003cem\u003eLactobacillus crispatus\u003c/em\u003e SNUV206 were cultured in MRS media (BD Difco). \u003cem\u003eVeillonella parvula\u003c/em\u003e KCTC5019 and \u003cem\u003eVeillonella atypica\u003c/em\u003e SNUV638 were cultured in GAM Broth supplemented with 0.75% Sodium DL-lactate, 2 mg/L putrescine. Other bacteria were cultured in BHI broth. \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e SNUG61027 was cultured in BHI broth with 16 g/l soy-peptone, 4 g/l threonine, and a mix of glucose and N-acetylglucosamine (25 mM each). All media were supplemented with 0.05% L-cysteine and all bacteria were cultured anaerobically at 37 °C. Cultures were washed and concentrated in anaerobic PBS with 25% (vol/vol) glycerol under strict anaerobic conditions. Additionally, an identical quantity of \u003cem\u003eA. muciniphila\u003c/em\u003e grown on the synthetic medium was inactivated by pasteurization for 30 min at 70 °C. Cultures were then immediately frozen and stored at −80 °C. Bacterial viable count was performed using LIVE/DEAD™ BacLight™ Bacterial Viability and Counting Kit according to the manufacturer’s instructions (Thermo Fisher Scientific).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale C57BL/6 and NOD/ShiLtJ-Prkdcem1AMCIl2rgem1AMC (NSGA) mice were purchased from JA bio. Male athymic nude mice (Koat:Athymic NCr-nu/nu) were purchased from Koatech. All mice used in this study were 8 weeks old. All animal procedures were approved by Institutional Animal Care and Use Committee (IACUC) of Seoul National University, and they were performed in accordance with institutional guidelines and international law and policies (IACUC No. SNU-200820-3). To generate the mouse tumor model, MC38, B16F10, and CT26 (2.5 × 10\u003csup\u003e5\u003c/sup\u003e) cells were subcutaneously (s.c.) implanted into the right flank. Tumors were measured with a caliper every 2-4 days beginning on day 7 after tumor inoculation. Tumor volume was calculated by the following equation, where L is the tumor length (larger dimension) and W is the width (smaller dimension): 𝑇𝑢𝑚𝑜𝑟 𝑣𝑜𝑙𝑢𝑚𝑒 = (𝐿 × 𝑊2)/2. Bacteria (1.6 × 10\u003csup\u003e8\u003c/sup\u003e cells/mouse for intratumoral injection; 5 × 10\u003csup\u003e9\u003c/sup\u003e cells/mouse for intraperitoneal injection) were administered intratumorally (i.t.) or intraperitoneally (i.p.) once every 3 days, starting on day 7 after tumor inoculation. For oral treatment, \u003cem\u003eVeillonella parvula\u003c/em\u003e (1 × 10\u003csup\u003e12\u003c/sup\u003e cells/mouse) was administered every other day, starting from 7 days after tumor inoculation. Membrane fractions of \u003cem\u003eVeillonella\u003c/em\u003e species were administered every 3 days i.p. at a dose of 50 μg/mouse. For the NK cell-depletion study, the mice were i.p. administered with control IgG (Clone 2A3; Bio X Cell) or anti-NK1.1 (Clone PK136; Bio X Cell) starting on day 6 after tumor inoculation (one day before \u003cem\u003eVeillonella parvula\u003c/em\u003e administration) at a dose of 200 µg per mouse every 3 days until the end of the experiment. To neutralize TLR2, control IgG or TLR2 neutralizing antibody (Clone C9A12; Invivogen) was administered i.p. starting one day before \u003cem\u003eVeillonella parvula\u003c/em\u003e administration at a dose of 50 μg/mouse every other day until the end of the experiment. For checkpoint blockade immunotherapy study, a tumor was implanted into the right flank as described above. Control IgG or PD1 blocking antibody (clone RMP1-14, InVivoPlus grade, BioXCell) was administered i.p. starting on day 6 after tumor inoculation (one day before \u003cem\u003eVeillonella parvula\u003c/em\u003e administration) at a dose of 100 µg per mouse every 3 days until the end of the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-cell transcriptome cell preparation and sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMC38 cells (2.5 × 10\u003csup\u003e5\u003c/sup\u003e) were s.c. implanted into the right flank of 8-week-old male C57BL/6 mice. PBS or P-VP (5 × 10\u003csup\u003e9\u003c/sup\u003e cells/mouse) was administered twice i.p. on days 7 and 10. On day 11, tumor tissues were harvested and dissociated into single-cell suspensions using a Mouse Tumor Dissociation Kit according to the manufacturer’s instructions (130-096-730; Miltenyi Biotec). After dissociation, cells from each group were pooled for further analysis. Next, the dissociated cells were stained with Trypan blue, and their number and viability were assessed using a Countess II Automated Cell Counter (Thermo Fisher). Following QC, the single-cell suspension was loaded onto Chromium Chip A (10X Genomics PN 230027), and GEM generation, cDNA synthesis, cDNA amplification, and library preparation of 1,700-6,400 cells were processed using the Chromium Single Cell 5′ Reagent Kit (10X Genomics PN 1000006) according to the manufacturer’s protocol. cDNA amplification included 13-16 cycles, and 11-50 ng of the material was used to prepare sequencing libraries with 14-16 cycles of PCR. Indexed libraries were pooled equimolar and sequenced on a NovaSeq 6000 in a PE28/91 run using the NovaSeq 6000 S1 Reagent Kit (100 cycles) (Illumina). An average of 160 million paired reads were generated per sample.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle\u003c/strong\u003e\u003cstrong\u003e-cell RNA-Seq analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e10X datasets were further processed using Cell Ranger v2.2.0 software using default parameters (10X Genomics) to generate fastq files. The Cell Ranger pipeline took the fastq files and generated expression matrices. A feature-barcode matrix was generated after preprocessing, aligning, counting UMI, and filtering cells with default options. We used R software version 4.3.0 to perform all further analyses using the Seurat package version 4.3.0. We removed low-quality cells with \u0026lt;200 and \u0026gt;9000 detected genes per cell and a mitochondrial RNA content of \u0026gt;10%. Normalization of the single-cell RNA-seq data was performed with the number of variable genes set at 10,000. Low-dimensional projection was performed using UMAP based on the top 15 principal components and for preliminary clustering of cells (resolution = 0.5). Differential gene expression analysis was performed using the FindMarkers function. Automated cell-type labeling was performed on the single-cell RNA-seq counts using the R package SingleR with reference purified microarray expression profiles from the Immunological Genome Project dataset.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene ontology biological processes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo perform the GO biological process enrichment analysis, we used the gost function from the gprofiler2 package version 0.2.2 to compare the genes identified in the neutrophil subclusters against genes that were present in the whole neutrophil cluster. Upregulated and downregulated genes were analyzed separately. Adjusted p values were calculated using the Benjamin \u0026amp; Hochberg (BH) false discovery rate method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell analysis was performed on a BD FACSCanto™ II (BD biosciences). For cytometry analysis, tissues including tumor, spleen, and bone marrow were harvested after intraperitoneal administration of pasteurized \u003cem\u003eV. parvula\u003c/em\u003e to MC38-bearing mice, once every 3 days for a total of 4 times. Single cell suspensions from tumors were prepared after incubation of the dissected tissue for 30 min at 37 °C in RPMI culture medium containing 40 μg/mL DNase I (Sigma) and 2 mg/mL collagenase IV (Sigma). Mouse spleens were homogenized and splenic single cell suspensions were prepared after erythrocyte lysis with red blood cell lysis buffer (Sigma). The bone marrow cells were collected by flushing the femurs and treated with red blood cell lysis buffer (Sigma). Staining for flow cytometry was conducted in round bottom 96-well plates in the dark. Viability staining was conducted with Zombie Aqua™ Fixable Viability Kit (Biolegend) for 20 min at RT, followed by a surface staining cocktail for 30 min on ice. For intracellular cytokine staining, isolated cells were stimulated with 50 ng/mL phorbol-12-myristate-13-acetate (Sigma), 1 μg/mL ionomycin (Sigma) and 1 μL/mL GolgiPlug (BD biosciences) for 3 h at 37 °C prior to the addition of antibodies. After surface staining, cells were fixed and permeabilized for 20 min on ice with Cytofix/Cytoperm and washed with Perm/Wash buffer (BD Biosciences). Intracellular staining was then conducted using antibodies diluted in Perm/Wash buffer for 30 min on ice, followed by washing in Perm/Wash buffer. To determine intracellular levels of ROS, single cell suspensions from tumors were stained with antibodies against surface markers and then were stained with CellROX™ Green Flow Cytometry Assay Kit (Thermo Fisher). Data analysis was performed using FlowJo (Tree Star) software.\u003c/p\u003e\n\u003cp\u003eThe following antibodies were used for flow cytometry analysis: anti-mouse CD45-BV421 (clone 30-F11, Biolegend), anti-mouse CD45-FITC (clone 30-F11, Biolegend), anti-mouse CD3-BV421 (clone 17A2, Biolegend), anti-mouse CD3-FITC (clone 17A2, Biolegend), anti-mouse CD3-PerCP/Cyanine5.5 (clone 17A2, Biolegend), anti-mouse CD4-BV421 (clone GK1.5, Biolegend), anti-mouse CD4-FITC (clone GK1.5, Biolegend), anti-mouse CD8a-PE/Cyanine7 (clone 53-6.7, Biolegend), anti-mouse NK1.1-PE (clone PK136, Biolegend), anti-mouse/human CD11b-PE/Cyanine7 (clone M1/70, Biolegend), anti-mouse F4/80-FITC (clone BM8, Biolegend), anti-mouse F4/80-PerCP/Cyanine5.5 (clone BM8, Biolegend), anti-mouse F4/80-PE (clone BM8, Biolegend), anti-mouse Ly6C-PE (clone HK1.4, Biolegend), anti-mouse Ly6G-APC (clone 1A8, Biolegend), anti-mouse CD54 (clone YN1/1.7.4, Biolegend), anti-mouse CD16-PerCP/Cyanine5.5 (clone S17014E, Biolegend), anti-mouse CD170 (Siglec-F)-PE (clone S17007L, Biolegend), anti-mouse Nos2-PE (iNOS; clone W16030C, Biolegend), anti-mouse IFN-γ-APC (clone XMG1.2, Biolegend), anti-mouse/human Granzyme B-FITC (clone GB11, Biolegend), anti-mouse Perforin-APC (clone S16009B, Biolegend), anti-mouse CD282 (TLR2)-PE (clone CB225, Biolegend), anti-mouse CD284 (TLR4)-PE (clone SA15-21, Biolegend).\u003c/p\u003e\n\u003cp\u003eThe following markers were used for identifying different immune cell subsets: CD45+CD3+ for T-cells, CD45+CD3+CD4+ for CD4+ T-cells, CD45+CD3+CD8+ for CD8+ T-cells, CD45+CD3-NK1.1+ for NK cells, CD45+CD11b+ for myeloid cells, CD45+CD11b+F4/80+ for macrophages, CD45+CD11b+Ly6CloLy6G+ for neutrophils, and CD45+CD11b+Ly6ChiLy6G- for monocytes. The flow cytometry gating strategies are shown in Extended Data Fig. 7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse cancer cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMC38 (murine colon adenocarcinoma cell line) was purchased from Kerafast. B16F10 (murine melanoma cell line) and CT26 (murine colorectal carcinoma cell line) were obtained from KCLB (Korean Cell Line Bank). Cancer cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 IU/mL penicillin/streptomycin (Sigma) in a 5% CO2 humidified incubator at 37 °C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of neutrophils\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo isolate neutrophils for ex vivo coculture experiments, splenocytes were isolated as described under ‘Flow cytometry analysis’. Neutrophils were purified using anti-Ly6G magnetic beads (Miltenyi Biotech) according to the manufacturer’s instructions. Purity of the magnetically isolated populations was \u0026gt; 90%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of cytokines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIFN-γ were measured by ELISA MAX™ Deluxe Set (Biolegend) following the manufacturer’s instructions. Splenocytes were isolated from MC38-bearing mice as described under ‘Flow cytometry analysis’. Splenocytes were treated with the pasteurized mix of 16 bacteria described under ‘Bacteria culture and pasteurization’ at 1:100 = splenocyte:bacteria cells for 16 h, and then cultured supernatant was harvested and stored at -80 °C until measurement by ELISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxicity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMC38 colon carcinoma cells were incubated in 96-well plate at 5 × 10\u003csup\u003e3\u003c/sup\u003e cells per plate one day before the cytotoxicity assay. Cytotoxicity was measured using CyQUANT LDH Cytotoxicity Assay (Invitrogen) according to the manufacturer’s instructions. For ex vivo experiments, splenic neutrophils isolated from mice treated with PBS or pasteurized \u003cem\u003eV. parvula\u003c/em\u003e/Vpar0143 were cocultured with MC38 cells at 1:10, 1:50, and 1:100 (MC38 cells:neutrophils) for 16 h. For in vitro experiments, MC38 cells were cocultured with pasteurized \u003cem\u003eV. parvula\u003c/em\u003e at 1:10000 (MC38 cells:bacteria cells) with splenocytes at 1:100 (MC38 cells:splenocytes) or without splenocytes. For TLR2 blocking experiment, splenic neutrophils were treated with 20μg/mL of anti-mouse TLR2 antibody (Clone C9A12; Invivogen) for 4 h before pasteurized \u003cem\u003eV. parvula\u003c/em\u003e treatment, and then cocultured with MC38 cells (1:100 = MC38 cells:neutrophils) and pasteurized \u003cem\u003eV. parvula\u003c/em\u003e (1:100 = neutrophils:bacteria cells) in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator at 37 °C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA isolation and quantitative real time PCR (RT‒qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was extracted from tumor tissues using the Easy-spin Total RNA extraction kit (iNtRON Biotechnology). Complementary DNA was synthesized using the High-Capacity RNA-to-cDNA Kit according to the manufacturer’s instructions (Applied Biosystems). RT‒qPCR was performed by using THUNDERBIRD™ SYBR® qPCR Master Mix (TOYOBO) and QuantStudio™ 6 Flex Real-Time PCR System (Thermo Fisher Scientific). A list of the sequences of the primers used for RT–qPCR is provided in Supplementary Table 4. Relative mRNA expression was calculated using the comparative (2\u003csup\u003e−ΔΔCT\u003c/sup\u003e) method and normalized to \u003cem\u003eβ-actin\u003c/em\u003e housekeeping gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMembrane fraction extraction of \u003cem\u003eVeillonella\u003c/em\u003e species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eVeillonella \u003c/em\u003especies grown anaerobically were harvested by centrifugation at 5,000 × g for 20 min at 4 °C and resuspended in 0.1 M pH 7.3 Tris-HCl, and then the culture suspension was frozen at -80 °C overnight. Cells were thawed and lysed by sonication in an ice water bath in cycles of 15 sec sonication followed by 45 sec cooling until the lysate changes from an opaque solution into a less turbid solution. Cellular debris were pelleted by centrifugation at 6,500 × g for 20 min at 4 °C. The supernatant was collected and the pellet was discarded. This step was repeated an additional time. The supernatant was ultracentrifuged at 120,000 × g for 1 h at 4 °C and the pellet washed in Tris-HCl (0.1 M pH 7.3) and spun at 85,000 × g for 20 min at 4 °C. The wash was repeated twice. The pellet was resuspended in PBS pH 7 for in vivo experiments or ddH2O for LC‒MS/MS analysis. Pronase (Sigma) was also added to suspended membrane pellet at 200 µg/mL, which was incubated at 37 °C overnight, and then the enzyme activity was heat-inactivated at 95 °C for 10 min. The BCA Protein Assay kit (Thermo Fisher Scientific) was used to determine the protein concentration according to the manufacturer’s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC–MS/MS analysis of membrane fraction proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cellular proteins of \u003cem\u003eV. parvula\u003c/em\u003e were isolated by carbonate extraction method. The extracted membrane proteins were sent to the Proteomics Core Facility at Seoul National University Hospital in Seoul, Korea for protein identification and analysis. The samples were analyzed using an LC–MS instrument consisting of an Ultimate 3000 RSLC system (Dionex), coupled via a nanoelectrospray ion source (Thermo Fisher Scientific) to a Q Exactive Plus Orbitrap (Thermo Fisher Scientific), according to previously described procedures. MS raw files were searched using MaxQuant (version 1.6.1.0) and the Andromeda search engine against the UniProt reference proteome database (\u003cem\u003eV. parvula\u003c/em\u003e; UP000007968 and \u003cem\u003eV. atypica\u003c/em\u003e; UP000005942). The false-discovery rate was set to 0.01 for both proteins and peptides with a minimum length of six amino acids, and was determined by searching a reverse database.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid constructs and protein expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe homologous protein to Vpar0143 or Vpar0556 was identified in \u003cem\u003eE. coli\u003c/em\u003e DH5α (EcPrc) in the UniProt database (https://www.uniprot.org/). Each of the corresponding cDNAs was cloned into the pET-28a plasmid (Novagen) digested with \u003cem\u003eNde\u003c/em\u003e\u003cem\u003eⅠ\u003c/em\u003e\u003cem\u003e/Xho\u003c/em\u003e\u003cem\u003eⅠ\u003c/em\u003e via Gibson assembly (NEB), which contains an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter. A list of the primer sequences used to generate the construct is provided in Supplementary Table 5. A His tag was added to the C-terminus of each protein for subsequent purification. The correct sequences of the resulting plasmids were then confirmed. These vectors were transformed into BL21 \u003cem\u003eEscherichia coli\u003c/em\u003e (RRID: WBHT115(DE3)) and grown in LB broth containing kanamycin (100 μg/mL), into which 1.0 mM IPTG was added during the mid-exponential growth stage, after which a further 4 h was provided for protein expression. The cells were pelleted by centrifuging for 10 min at 5,000 × g and the cell pellets were stored at −80 °C until lysis. The cell pellets of BL21 (DE3) \u003cem\u003eE. coli\u003c/em\u003e expressing Vpar0143 were resuspended and lysed by sonication Vibra-Cell™ VCX500 (Sonics \u0026amp; Materials). Vpar0556 expressing BL21 (DE3) \u003cem\u003eE. coli\u003c/em\u003e pellets were resuspended with 8 M urea buffer and incubated for 1 h at 37 °C. After incubation, the resuspended BL21 (DE3) was centrifuged 10 min at 20,000 × g and the supernatant was used for protein purification. For protein purification, Ni-NTA agarose resin (QIAGEN) and the HisTALON buffer kit (Takara Bio) were used according to the manufacturer’s instructions, with minor modifications. The purified proteins were then dialyzed against endotoxin-free dialysis buffer. The purification of each protein was confirmed using SDS–polyacrylamide gel electrophoresis and Coomassie blue staining by the presence of a single band at the expected size.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults are presented as the means ± SEM. Data were analyzed by two-tailed Student’s t test or Mann-Whitney U test as appropriate. Multiple-group comparisons were performed using two-way ANOVA with Tukey’s or Sidak’s multiple comparisons test. Statistical analyses were performed with GraphPad Prism software (GraphPad Inc.) and statistical significance was set at p \u0026lt; 0.05.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7615171/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7615171/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRecent scientific efforts have focused on identifying commensal microbiota with antitumor activity, yet most cancer studies remain limited to taxonomic associations or treatment-induced microbial changes, with only a few addressing underlying mechanisms. These mechanistic studies have focused mainly on lymphoid cells such as CD8⁺ T cells, while neutrophil-mediated mechanisms have received little attention despite their abundance and functions. Here, we screened 16 taxa linked to positive responses to cancer immunotherapy and identified \u003cem\u003eVeillonella parvula\u003c/em\u003e as effective in reducing tumor growth in a murine model. Using single-cell transcriptomics and flow cytometry, we show that \u003cem\u003eV. parvula\u003c/em\u003e reshapes the tumor microenvironment by promoting infiltration of neutrophil subsets that secrete reactive oxygen and nitrogen species, driving tumor cell death. We further identify Vpar0143, a ferritin Dps family protein, as the bacterial effector responsible for neutrophil activation and the antitumor effect. Together, these findings reveal a commensal\u0026ndash;neutrophil axis harnessable for microbe-based cancer therapy.\u003c/p\u003e","manuscriptTitle":"The Gut Commensal Bacteria Veillonella parvula-Induced Neutrophil Activation Mediates Antitumor Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-16 05:48:12","doi":"10.21203/rs.3.rs-7615171/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ca46e98d-3f18-404b-b384-a8462d7710e3","owner":[],"postedDate":"October 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56296626,"name":"Biological sciences/Microbiology"},{"id":56296627,"name":"Biological sciences/Immunology"}],"tags":[],"updatedAt":"2026-03-04T09:06:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-16 05:48:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7615171","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7615171","identity":"rs-7615171","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}