Secreted SULF1 Protein Modulates CD8+ T Cell Exhaustion by Promoting TAM Polarization in Gastric Cancer

Secreted SULF1 Protein Modulates CD8+ T Cell Exhaustion by Promoting TAM Polarization in Gastric Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Secreted SULF1 Protein Modulates CD8+ T Cell Exhaustion by Promoting TAM Polarization in Gastric Cancer Xiaodan Lu, Di Lu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8132447/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Apr, 2026 Read the published version in Genes & Immunity → Version 1 posted 9 You are reading this latest preprint version Abstract Gastric cancer (GC) progression is linked to immune escape in the tumor microenvironment, yet the molecules regulating tumor-associated macrophage polarization and CD8⁺ T-cell exhaustion are unclear. This study analyzed TCGA data to examine SULF1 expression and its prognostic role. It used CRISPR/Cas9 and lentiviral methods in GC cells to test proliferation, invasion, and apoptosis, plus co-culture and flow cytometry to assess SULF1’s impact on macrophages and CD8⁺ T-cells. STAT3 signaling was studied via immunoblotting and nuclear translocation assays, and a mouse model tested SULF1’s therapeutic relevance. Results showed SULF1 was up-regulated in GC, tied to advanced stages and poor survival. SULF1 knockdown inhibited GC cell traits, while overexpression boosted them. SULF1 activated macrophage STAT3, promoting M2 polarization and CD8⁺ T-cell dysfunction. In mice, SULF1 silencing reduced tumors and T-cell exhaustion, while supplementation reversed this. Conclusions: GC-secreted SULF1 creates an immunosuppressive microenvironment via STAT3-dependent pathways, and targeting SULF1–STAT3 may improve GC immunity. Biological sciences/Immunology/Tumour immunology/Immunosurveillance/Immunoediting Biological sciences/Immunology/Immune cell death Gastric cancer SULF1 Tumor-associated macrophages CD8⁺T cell exhaustion STAT3 signaling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Gastric cancer (GC), as one of the most common malignancies with high incidence and mortality rates worldwide, has always been a hot and difficult topic in oncology research due to its mechanisms of invasion, metastasis, and immune escape 1 . Although the comprehensive application of surgery, radiochemotherapy, targeted therapy, and immunotherapy in recent years has significantly improved the prognosis of some patients, the 5-year survival rate of patients with advanced GC is still less than 30% 2 . The tumor microenvironment (TME), as the "soil" in which tumor cells survive, is composed of immune cells, fibroblasts, extracellular matrix, and soluble factors. Its dynamic imbalance is the core force driving the progression of GC. Tumor-associated macrophages (TAMs), as the most abundant immune cell population in the TME, have interactions with CD8⁺ T cells that have become a key link in tumor immune escape 3 , 4 . M1 macrophages activate anti-tumor immunity by secreting pro-inflammatory cytokines such as IFN-γ and TNF-α. In contrast, M2 macrophages promote tumor progression by releasing immunosuppressive factors such as IL-10, TGF-β, and VEGF, which facilitate tumor angiogenesis, epithelial-mesenchymal transition (EMT), and CD8⁺ T cell exhaustion 5 , 6 . However, the specific mechanisms by which GC cells regulate TAM polarization and further induce CD8⁺ T cell exhaustion have not yet been fully elucidated. Sulfatase 1 (SULF1), an extracellular heparan sulfate (HS) endosulfatase, modulates the signaling of multiple growth factors, such as VEGF, FGF, and Wnt, by specifically removing the 6-O-sulfate groups from HS chains. This action influences cell proliferation, migration, and angiogenesis 7 . Previous studies have shown that SULF1 is dysregulated in various tumors, but its effects are highly tissue-specific. In hepatocellular carcinoma and pancreatic cancer, SULF1 exerts tumor-suppressive effects by inhibiting the FGF-2/VEGF signaling pathway. Low SULF1 expression is associated with increased tumor angiogenesis and poor patient prognosis 8 , 9 . In contrast, in breast and ovarian cancers, SULF1 promotes tumor cell proliferation, migration, and chemoresistance by enhancing oncogenic signaling pathways such as HB-EGF/EGFR and CXCL12/CXCR4 10, 11 . Notably, recent single-cell sequencing studies have revealed heterogeneous expression of SULF1 in tumor stromal cells, such as cancer-associated fibroblasts, and immune cells. This suggests that SULF1 may participate in shaping the immune microenvironment by regulating intercellular interactions 12 . The expression pattern of SULF1 and its correlation with immune cell infiltration in GC have not been previously reported. In this study, we integrated analysis of the TCGA database with in vitro and in vivo functional experiments to uncover for the first time a novel mechanism by which SULF1 secreted by GC cells promotes M2 polarization of TAMs via the STAT3 signaling pathway, thereby inducing CD8⁺ T cell exhaustion. This finding provides new evidence for immunotherapeutic strategies targeting TAM polarization and CD8⁺ T cell functional recovery. 2. Materials and Methods 2.1 Bioinformatics Analysis To comprehensively analyze the expression characteristics and clinical significance of SULF1 in GC, we downloaded RNA-seq data in STAR-counts format and corresponding clinical information from the TCGA database ( https://portal.gdc.cancer.gov ) and the GEO database ( https://www.ncbi.nlm.nih.gov/geo/ ). First, the raw count matrix was converted to TPM (Transcripts Per Million) format and normalized using log₂(TPM + 1). Subsequently, samples lacking complete clinical data or with duplicate sequencing were removed, resulting in a high-quality dataset for subsequent differential expression analysis, survival analysis, and functional enrichment. 2.2 Cell Culture Hs746T and MKN-74 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO₂. 2.3 Knockdown of SULF1 in Hs746T Cells Specific shRNA sequences targeting SULF1 were designed and cloned into the lentiviral shRNA expression vector (pLKO.1, Addgene, USA). The expression vector, along with packaging and envelope plasmids, was co-transfected into 293T cells using Lipofectamine 3 000 to produce lentiviral particles. After viral packaging, the supernatant was collected, filtered, and used to infect Hs746T cells. Infected cells were selected with puromycin (2 µg/mL), and the knockdown efficiency of SULF1 was confirmed by Western blot. 2.4 Overexpression of SULF1 in MKN-74 Cells A plasmid containing the SULF1 gene was constructed and transfected into MKN-74 cells. Stable overexpression cell lines were obtained through antibiotic selection, and the overexpression of SULF1 was confirmed by Western blot. 2.5 Western Blot (WB) Cells were lysed in RIPA buffer with protease/phosphatase inhibitors. 30 µg proteins were resolved by 12% SDS-PAGE, transferred to NC membranes, blocked with 5% non-fat milk, and incubated overnight (4℃) with primary antibodies: SULF1 (27438-1-AP, Proteintech), STAT3 (10253-2-AP, Proteintech), P-STAT3-Tyr705 (80199-2-RR, Proteintech), and β-actin (20536-1-AP, Proteintech). After HRP-conjugated secondary antibody (SA00001-2, Proteintech) incubation, bands were visualized by ECL and quantified via ImageJ. 2.6 Transwell Assay Starved cells were prepared as a cell suspension and added to the upper chamber of a Transwell insert. The insert was placed in a 24-well plate with serum-containing medium in the lower chamber. After 12 hours of incubation, the Transwell insert was removed, fixed with formaldehyde for 30 minutes, air-dried, and stained with crystal violet for 40 minutes. The cells were observed under a microscope. 2.7 Flow Cytometry Cell suspensions were prepared and stained with CoraLite® Plus 488-Annexin V and PI apoptosis detection kit (PF00005, Proteintech) for apoptosis analysis. Cells were also stained with the following antibodies: CoraLite® Plus 647-conjugated iNOS Polyclonal antibody (CL647-18985, Proteintech), CoraLite® Plus 488 Anti-Human CD86 (CL488-65165, Proteintech), CoraLite® Plus 488 Anti-Mouse CD86 (CL488-65068, Proteintech), CoraLite® Plus 488 Anti-Mouse CD206 Rabbit Recombinant Antibody (CL488-98031, Proteintech), and CoraLite® Plus 647 Anti-Human CD163 (GHI/61) Mouse IgG2a Recombinant Antibody (CL647-65561, Proteintech). Additional antibodies used for macrophage surface labeling included Alexa Fluor® 488 anti-mouse CD45 Antibody (160306, Biolegend), APC/Cyanine7 anti-mouse CD3 Antibody (100221, Biolegend), APC/Fire™ 810 anti-mouse CD8a Antibody (104007, Biolegend), Spark YG™ 570 anti-mouse/human CD44 Antibody (103073, Biolegend), Alexa Fluor® 700 anti-mouse CD62L Antibody (104426, Biolegend), Spark Blue™ 574 anti-mouse CD279 (PD-1) (Flexi-Fluor™) Antibody (285153, Biolegend), and Alexa Fluor® 647 anti-mouse CD366 (Tim-3) Antibody (119743, Biolegend). After incubation, cells were washed with PBS and analyzed by flow cytometry to determine the rates of apoptosis and the proportions of M1 and M2 macrophages, as well as activated and exhausted CD8⁺ T cells. 2.8 Confocal Microscopy Subcutaneous tumor tissues from mice were sectioned and deparaffinized. Cells grown to an appropriate density in 24-well plates were fixed with 4% paraformaldehyde. The tumor sections and cells were permeabilized with 0.1% Triton X-100, blocked with skim milk, and incubated with a fluorescently labeled primary antibody specific to STAT3 (10253-2-AP, Proteintech). The corresponding secondary antibody, Fluorescein (FITC)–conjugated Goat Anti-Rabbit IgG(H + L) (SA00003-2, Proteintech), and a blue fluorescent nuclear marker, DAPI (PR30021, Proteintech), were added. The tumor sections and cell slides were incubated overnight at 4°C in a humidified chamber to allow antibodies to bind to the target proteins. After incubation, the sections and slides were washed with PBS to remove unbound antibodies and markers, and mounted with an anti-fade mounting medium. The sections and slides were observed and imaged under a confocal microscope, with adjustments made to the microscope parameters to capture blue and green fluorescence signals to clearly visualize the distribution of fluorescence. 2.9 ELISA Assay Human/mouse IL-10 (HM10203 / MU30055, Bioswamp), TGF-β (HM10058 / MU30071, Bioswamp), and INF-γ (HM10115 / MU30038, Bioswamp) ELISA kits were used for quantitative detection. Samples were centrifuged at 3000 × g for 10 minutes at 4°C, and the supernatant was collected. The assays were performed strictly according to the manufacturer's instructions, including sample addition, incubation, and color development. The absorbance was measured at 450 nm using a microplate reader, and the absolute concentrations of IL-10 and TGF-β were calculated based on the standard curve. 2.10 Animal Experiment Balb/c mice were used to establish subcutaneous xenograft models. Mice were randomly divided into groups and subcutaneously implanted with Hs746T, Hs746T-SULF1-KD, and MKN-74, MKN-74-SULF1-OE cells. These models were treated with SULF1-His protein and anti-SULF1 antibody, respectively. Tumor volumes were measured every 5 days. On day 30, mice were euthanized, tumors were excised, weighed, and tumor tissue proteins were extracted for subsequent analysis. All animal experiments were conducted in accordance with the approval and guidelines of the ethics review committee. 2.11 Statistical Analysis All experiments were repeated at least three times, and data were presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using GraphPad Prism 8.0: Student's t-test for two-group comparisons and one-way analysis of variance (ANOVA) for multiple-group comparisons. A p-value < 0.05 was considered statistically significant. 3. Results 3.1 Expression and Function of SULF1 in GC To explore SULF1’s expression and role in gastric cancer (GC), TCGA database analysis showed significantly higher SULF1 expression in GC tissues than normal tissues, with upregulation during disease progression (Fig. 1 A). Survival analysis linked high SULF1 expression to shorter patient survival, suggesting its pro-tumor effect (Fig. 1 B). Among five GC cell lines, Hs746T had the highest and MKN-74 the lowest SULF1 levels (Fig. 1 C), so these two lines were selected for subsequent experiments. Lentiviral vectors mediated SULF1 knockdown (KD) in Hs746T (Fig. 1 D) and overexpression (OE) in MKN-74 (Fig. 1 E). Flow cytometry demonstrated SULF1-KD reduced GC cell viability (Fig. 1 F) while SULF1-OE promoted proliferation (Fig. 1 G). Transwell assays showed SULF1-KD inhibited metastasis and invasion, whereas SULF1-OE enhanced these abilities (Fig. 1 H & I). Additionally, SULF1-KD increased GC cell apoptosis, while SULF1-OE exerted the opposite effect (Fig. 1 J & K). These data confirm SULF1’s regulatory role in GC progression. 3.2 Correlation of SULF1 with Immune Responses To elucidate SULF1’s mechanism, TCGA data immune scoring revealed a positive correlation between SULF1 and macrophages (Fig. 2 A). Co-culture of SULF1-KD/OE GC cells with macrophages showed SULF1-KD increased M1 and decreased M2 polarization, while SULF1-OE reversed this trend (Fig. 2 B & C). Exogenous SULF1-His treatment similarly inhibited M1 and promoted M2 polarization (Fig. 2 D). SULF1-His-activated tumor-associated macrophages (TAMs) enhanced GC cell proliferation (Fig. 2 E & F), metastasis/invasion (Fig. 2 G & H), and reduced apoptosis (Fig. 2 I & J). Rescue experiments showed SULF1-His restored the pro-tumor phenotype in SULF1-KD cells (enhanced proliferation, reduced apoptosis; Fig. 2 K, M, O), while anti-SULF1 antibody abrogated the pro-tumor effect of SULF1-OE cells (Fig. 2 L, N, P), confirming SULF1’s direct role. 3.3 Mechanism of SULF1 Activation of TAMs The STAT3 signaling pathway is believed to be closely related to TAM polarization 13 . Therefore, in this study, macrophages were treated with SULF1-His to detect the activation of STAT3 in macrophages. The results showed that after treatment with SULF1-His, the level of STAT3 protein remained unchanged, while the level of P-STAT3-Tyr705 significantly increased (Fig. 3 A). Confocal imaging results also showed an increased nuclear localization of P-STAT3-Tyr705 (Fig. 3 B). Meanwhile, the levels of cytokines IL-10 and TGF-β, which are closely related to M2 polarization, were detected. ELISA results indicated that the addition of SULF1-His elevated the levels of these cytokines (Fig. 3 C & D). To further investigate the activation of STAT3 and the levels of IL-10 and TGF-β, macrophages were co-cultured with GC cells after SULF1-KD/OE. The results showed that regardless of KD or OE, the expression level of STAT3 remained unchanged. However, after KD, the expression level of P-STAT3-Tyr705 decreased (Fig. 3 E), and the levels of IL-10 and TGF-β cytokines were reduced (Fig. 3 F & G). Conversely, after OE, the expression level of P-STAT3-Tyr705 increased (Fig. 3 H), and the levels of IL-10 and TGF-β cytokines were elevated (Fig. 3 I & J). These findings indicate that SULF1 promotes TAM polarization by increasing the level of p-STAT3-Tyr705. 3.4 SULF1 Activation of TAMs Promotes CD8⁺ T Cell Exhaustion Immune scoring analysis of clinical data from GC patients in the TCGA database also revealed a negative correlation between SULF1 and CD8⁺ T cells (Fig. 4 A). To verify the relationship between SULF1 and CD8⁺ T cells, CD8⁺ T cells were co-cultured with GC cells after SULF1-KD/OE for 24 hours. It was found that CD8⁺ T cells were activated during the co-culture process. However, neither SULF1-KD nor OE significantly affected the degree of CD8⁺ T cell activation (Fig. 4 B & C), and similar results were obtained for cell exhaustion (Fig. 4 D & E). Recent studies have indicated that an increase in TAMs within the tumor microenvironment can promote CD8⁺ T cell exhaustion 14 . Therefore, macrophages were co-cultured with GC cells after SULF1-KD/OE, followed by co-culture with CD8⁺ T cells for 24 hours to detect the proliferation and apoptosis levels of CD8⁺ T cells. Flow cytometry results showed that after SULF1-KD, proliferation was enhanced (Fig. 4 F) and apoptosis levels were reduced (Fig. 4 H). Conversely, after SULF1-OE, proliferative capacity was weakened (Fig. 4 G) and apoptosis levels increased (Fig. 4 I). CD8⁺ T cell exhaustion was also examined, and it was found that after SULF1-KD, the level of CD8⁺ T cell exhaustion was inhibited (Fig. 4 J), while after SULF1-OE, the level of CD8⁺ T cell exhaustion was enhanced (Fig. 4 K). Subsequently, CD8⁺ T cells were directly treated with SULF1-His, and their proliferation, apoptosis, and T cell exhaustion levels were detected. Compared with the untreated group, the addition of SULF1-His did not significantly change the cell proliferation level (Fig. 4 L), nor did it significantly affect apoptosis and T cell exhaustion levels (Fig. 4 M & N). These findings clearly demonstrate that SULF1-induced CD8⁺ T cell exhaustion is mediated by TAM polarization. 3.5 Animal Experiments To investigate the impact of SULF1 on gastric cancer, we established Balb/c mouse tumor models using Hs746T, Hs746T-SULF1-KD, and MKN-74, MKN-74-SULF1-OE cells. These models were treated with exogenous SULF1-His protein and anti-SULF1 antibody, respectively. The results showed that SULF1-KD in Hs746T cells significantly inhibited tumor formation, but this inhibition was reversed by the addition of SULF1-His (Fig. 5 A & B). Conversely, SULF1-OE in MKN-74 cells promoted tumor formation, an effect that was effectively inhibited by anti-SULF1 antibody treatment (Fig. 5 C & D). Flow cytometry analysis revealed that the proportion of M2 macrophages in tumor tissues from Hs746T-SULF1-KD group was lower than that from Hs746T group, but this proportion was restored upon SULF1-His treatment (Fig. 5 E). In contrast, MKN-74-SULF1-OE group exhibited a higher proportion of M2 macrophages compared to MKN-74 group, which decreased after anti-SULF1 treatment (Fig. 5 F). Exhaustion levels of CD8⁺ T cells were also examined. Mice with Hs746T-SULF1-KD exhibited reduced CD8⁺ T cell exhaustion, whereas those with Hs746T-SULF1-KD + SULF1-His showed increased exhaustion levels (Fig. 5 G & I). Similarly, MKN-74-SULF1-OE group had higher CD8⁺ T cell exhaustion levels, which decreased following anti-SULF1 antibody treatment (Fig. 5 H & L). ELISA assays indicated that SULF1 KD led to decreased levels of IL-10 and TGF-β, while SULF1-His treatment increased these cytokine levels. IFN-γ levels were elevated in SULF1 KD mice but decreased upon SULF1-His treatment (Fig. 5 J). In MKN-74-SULF1-OE group, IFN-γ levels were reduced, but increased after anti-SULF1 antibody treatment. WB analysis showed that in Hs746T cells, SULF1 KD inhibited P-STAT3-Tyr705 and reduced its nuclear localization. SULF1-His treatment activated P-STAT3-Tyr705 and increased its nuclear presence (Fig. 5 K & O). In contrast, SULF1-OE in MKN-74 cells led to opposite changes in IL-10, TGF-β, IFN-γ, and P-STAT3-Tyr705 levels (Fig. 5 M & K & P). In summary, these in vivo experimental results further confirmed the observations from our in vitro studies, demonstrating that SULF1 significantly affects tumor formation and the immune microenvironment in gastric cancer by modulating various cytokines and signaling pathways. 4. Discussion The progression of gastric cancer is contingent upon the dynamic interplay between tumor cells and immune cells within the microenvironment, with the polarization state of TAMs being a pivotal factor in regulating immune evasion 15 . This study, for the first time, elucidates that SULF1 secreted by gastric cancer cells promotes the polarization of TAMs towards the immunosuppressive M2 phenotype by activating the STAT3 signaling pathway, thereby inducing CD8⁺ T cell exhaustion. This finding offers a novel perspective for understanding the regulatory mechanisms of the gastric cancer immune microenvironment. By analyzing data from the TCGA database and conducting cellular functional experiments, we discovered that SULF1 is highly expressed in gastric cancer tissues, with its levels positively correlated with tumor progression and patient prognosis. Additionally, our study found that SULF1-KD significantly inhibits the viability of gastric cancer cells and induces apoptosis, whereas overexpression enhances their invasive capacity. These findings further confirm the central role of SULF1 in tumor evolution. This is consistent with previous studies showing SULF1's oncogenic role in breast and ovarian cancers but contrasts sharply with its tumor-suppressive effects in liver and pancreatic cancers 16 – 18 . This tissue specificity likely arises from differences in the baseline sulfation levels of HS and the growth factor networks in different tumor microenvironments. For instance, in liver cancer, SULF1 inhibits angiogenesis by suppressing FGF signaling, whereas in gastric cancer, SULF1 indirectly activates TAM polarization by relieving HS-mediated inhibition of the JAK2/STAT3 pathway. This suggests that the functional plasticity of SULF1 is closely related to the "sulfation background" in its microenvironment, and future research should employ single-cell sulfation genomics to further elucidate this relationship. Tumor cells reshape macrophage polarization through secreted factors as an important strategy for immune evasion. Our study found that the SULF1-STAT3 axis complements previously reported pathways such as HIF-1α and NF-κB. Notably, SULF1-His can directly induce the nuclear translocation of macrophage p-STAT3-Tyr705, while SULF1-KD in tumor cells indirectly inhibits macrophage STAT3 activation through a secretion defect. This indicates a "secretion-response" positive feedback loop between tumor cells and macrophages. This mechanism, together with the finding that SPP1⁺ TAMs induce T cell exhaustion through the GDF15-TGFβ axis 19 , reveals the multi-dimensional characteristics of TAM polarization regulation. However, the extracellular enzymatic nature of SULF1 endows it with the potential for "remote regulation" of the microenvironment, making it a potentially more universal intervention target. Regarding the mechanism of CD8⁺ T cell exhaustion, our study confirmed that the effects of SULF1 are entirely mediated by TAMs, which is different from the previously reported mode of tumor cells directly secreting PD-L1 to inhibit T cells 20 . Importantly, M2 macrophages not only inhibit T cell proliferation by secreting IL-10 and TGF-β but also enhance exhaustion signals by upregulating PD-L1 expression, forming a dual-inhibition network. Animal experiments in our study showed that SULF1-KD in tumor tissues increased IFN-γ levels and reduced exhaustion markers, further confirming the central role of TAM polarization in T cell functional remodeling. This finding provides a theoretical basis for the combined treatment strategy of "reprogramming TAM polarization + restoring T cell function," such as the potential synergistic effect of using SULF1 neutralizing antibodies with PD-1 inhibitors. Compared with similar studies, our research has completely constructed the regulatory axis of "tumor cell secreted factors - macrophage polarization - effector T cell function" through in vivo and in vitro experiments and has verified its clinical transformation potential in animal models. However, several scientific questions remain to be addressed: First, as a secreted protein, the specific binding receptors of SULF1 on macrophages in the microenvironment (LRP1, CD44) have not been identified, which may affect the development of targeted drugs. Second, the spatial co-localization evidence of SULF1 expression with TAM polarization and CD8⁺ T cell exhaustion in clinical samples still needs to be supplemented by double immunofluorescence experiments. Moreover, whether SULF1 indirectly affects the microenvironment by regulating other immune cell subsets (NK cells, Treg cells) also needs further exploration. From the perspective of translational medicine, the SULF1-STAT3 axis revealed in this study provides a dual intervention target for gastric cancer immunotherapy: inhibiting SULF1 can block the upstream signal of TAM polarization, while inhibiting STAT3 can directly reverse the M2 phenotype. Existing research has shown that STAT3 inhibitors (OPB-31121) can enhance the efficacy of PD-1 inhibitors in preclinical models, and our study further suggests that combined blockade of SULF1 may produce a synergistic effect. Additionally, designing small-molecule inhibitors or monoclonal antibodies based on the enzymatic activity of SULF1 holds promise as a new strategy for "de-immunosuppressive" therapy. In summary, by integrating multi-omics data with functional experiments, our study systematically elucidates the molecular mechanisms by which SULF1 regulates the immune microenvironment in gastric cancer. This not only expands our understanding of the tumor-immune interaction network but also provides a new direction for overcoming current immunotherapy resistance. Future work should validate this mechanism in larger clinical cohorts and explore personalized targeting strategies using organoid models to promote the translation of basic research into clinical applications. 5. Conclusion This study has uncovered the complete molecular pathway of the "GC-SULF1-TAM-CD8⁺ T cell" immunosuppressive axis, offering new insights for the precision immunotherapy of GC. Future targeting of SULF1 or combination with STAT3 inhibitors may help overcome current immunotherapy resistance. Declarations Disclosure funding statement The authors did not receive support from any organization for the submitted work. Conflict of interest disclosure The authors have no conflict of interest. Ethical Statement Informed Consent: All authors have read and approved the final manuscript and consent to its submission for publication. Registry and the Registration No. of the study/trial: N/A. Animal Studies: All animal experiments conformed to the internationally accepted principles for the care and use of laboratory animals (Medical Ethics Committee of Henan Provincial People's Hospital). Author Contributions: Conceptualization, Xiaodan Lu and Di Lu; Methodology, Xiaodan Lu; Validation, Xiaodan Lu and Di Lu; Formal Analysis, Xiaodan Lu; Investigation, Xiaodan Lu and Di Lu; Data Curation, Xiaodan Lu; Writing – Original Draft Preparation, Di Lu; Writing – Review & Editing, Xiaodan Lu; Visualization, Di Lu; Supervision, Xiaodan Lu; Project Administration, Xiaodan Lu; Funding Acquisition, Xiaodan Lu. All authors have read and agreed to the published version of the manuscript. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021; 71(3): 209–249. El Halabi M, Horanieh R, Tamim H, Mukherji D, Jdiaa S, Temraz S et al. The impact of age on prognosis in patients with gastric cancer: experience in a tertiary care centre. Journal of gastrointestinal oncology 2020; 11(6): 1233–1241. 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Additional Declarations There is NO conflict of interest to disclose. there is NO conflict of interest to disclose Cite Share Download PDF Status: Published Journal Publication published 15 Apr, 2026 Read the published version in Genes & Immunity → Version 1 posted Editorial decision: revise 19 Jan, 2026 Review # 2 received at journal 06 Jan, 2026 Reviewer # 2 agreed at journal 06 Jan, 2026 Review # 1 received at journal 15 Dec, 2025 Reviewer # 1 agreed at journal 03 Dec, 2025 Reviewers invited by journal 03 Dec, 2025 Submission checks completed at journal 18 Nov, 2025 Editor assigned by journal 17 Nov, 2025 First submitted to journal 17 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8132447","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":554814691,"identity":"d4f767c7-9967-474c-9d72-d7d68df7eaa7","order_by":0,"name":"Xiaodan Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYBACAzBZISHHxszYcCAByJAnTssZG2N+9ubGBw/OWBgbNhCjhbEtLXFmz/Fmw4dtFYkMBwhoMWfvPfi5gO0w44YbiW0SifMkEhgbmB8+uoFHi2XPuWTpGTyHmQ3AWrZJ5LEzsBkb5+Bz2I0cA2keicNsMC3FjA08bNJ4tdx/Y/ybx+AwD0TLHInEhgOEtNzgMZPmSUiTkOw52GyQ2ECMljM5ZtY8B2wM+NkbGx8kHJMwNmwm5JfjZ4xv8/6TqG9jZn9w8EdNnZw8e/PDx/i0YAHMpCkfBaNgFIyCUYAFAAAPg0+f6vAv9AAAAABJRU5ErkJggg==","orcid":"","institution":"Henan Provincial People's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Xiaodan","middleName":"","lastName":"Lu","suffix":""},{"id":554814692,"identity":"9738ac00-2154-4105-af23-45b1bde07b7b","order_by":1,"name":"Di Lu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Di","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2025-11-17 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09:39:26","extension":"xml","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":79792,"visible":true,"origin":"","legend":"","description":"","filename":"2025GENE6240structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8132447/v1/f735728975119ce5bf6c628e.xml"},{"id":97671317,"identity":"1f410600-8c14-41f8-ae7f-4a902794144f","added_by":"auto","created_at":"2025-12-08 09:32:26","extension":"html","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":88000,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8132447/v1/cff9c664b80d636072b8acc7.html"},{"id":97551386,"identity":"746200bb-b470-4269-9a31-b84aa4709fc6","added_by":"auto","created_at":"2025-12-05 17:18:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":452233,"visible":true,"origin":"","legend":"\u003cp\u003eExpression and Function of SULF1 in GC. A: Analysis of SULF1 expression differences between GC and normal tissues using the TCGA database. B: Survival analysis based on TCGA data to evaluate the relationship between SULF1 expression and survival in GC patients. C: WB screening of SULF1 expression in different gastric cancer cell lines. D: WB validation of SULF1-KD efficiency in Hs746T cells using lentivirus. E: WB validation of SULF1-OE efficiency in MKN-74 cells using lentivirus. F: Flow cytometry analysis of changes in cell viability in Hs746T cells after SULF1-KD. G: Flow cytometry analysis of cell growth in NCI-N87 cells after SULF-OE. H \u0026amp; I: Transwell analysis of the impact of SULF1-KD/OE on the metastatic and invasive capabilities of GC cells. J \u0026amp; K: Flow cytometry analysis of apoptosis changes in GC cells after SULF1-KD/OE. Data are presented as the mean ± SD; n = 3 per group. The differences between the two groups were compared using a one-way ANOVA followed by Dunnett's multiple comparisons test. Asterisks (*) indicate significance levels, * represents p \u0026lt; 0.05, ** represents p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8132447/v1/11e20ad9af6067412048595d.png"},{"id":97672590,"identity":"e57a3d86-534a-4ed2-a0e1-72f6dd5d9ada","added_by":"auto","created_at":"2025-12-08 09:38:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":912431,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of SULF1 with Immune Responses. A: Analysis of the correlation between SULF1 and macrophages using the TCGA database. B \u0026amp; C: Flow cytometry analysis of the effects of SULF1 on the polarization of M1 and M2 macrophages after co-culture with GC-SULF1-KD/OE cells. D: Flow cytometry analysis of the regulatory effects of exogenous SULF1 treatment on the polarization levels of M1 and M2 macrophages. E \u0026amp; F: Flow cytometry validation of the impact of SULF1-His addition on the proliferation of GC cells. G \u0026amp; H: Transwell analysis of the effects of SULF1-His addition on the metastatic and invasive capabilities of GC cells. I \u0026amp; J: Flow cytometry validation of the impact of SULF1-His addition on the apoptosis levels of GC cells. K \u0026amp; M \u0026amp; O: Flow cytometry analysis of the regulatory effects of SULF1-His on the proliferation, metastasis, invasion, and apoptosis levels of GC-SULF1-KD cells. L \u0026amp; N \u0026amp; P: Flow cytometry analysis of the regulatory effects of anti-SULF1 on the proliferation, metastasis, invasion, and apoptosis levels of GC-SULF1-OE cells.Data are presented as the mean ± SD; n = 3 per group. The differences between the two groups were compared using a one-way ANOVA followed by Dunnett's multiple comparisons test. Asterisks (*) indicate significance levels, * represents p \u0026lt; 0.05, ** represents p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8132447/v1/c87aeb23ab2eb03afa57835d.png"},{"id":97551389,"identity":"37ae09f3-18de-4fef-95d2-f8cbc2391477","added_by":"auto","created_at":"2025-12-05 17:18:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":212719,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of SULF1 Activation of TAMs. A: WB analysis of changes in STAT3 and P-STAT3-Tyr705 protein expression in macrophages treated with SULF1-His. B: Confocal imaging analysis of changes in P-STAT3-Tyr705 cellular localization in macrophages treated with SULF1-His. C \u0026amp; D: ELISA detection of changes in IL-10 and TGF-β cytokine levels in macrophages treated with SULF1-His. E \u0026amp; H: WB analysis of the effects of co-culture of GC-SULF1-KD/OE cells with macrophages on the expression levels of STAT3 and P-STAT3-Tyr705. F \u0026amp; G: ELISA detection of changes in IL-10 and TGF-β levels after co-culture of M0 macrophages with GC-SULF1-KD cells. I \u0026amp; J: ELISA detection of changes in IL-10 and TGF-β levels after co-culture of M0 macrophages with GC-SULF1-OE cells.Data are presented as the mean ± SD; n = 3 per group. The differences between the two groups were compared using a one-way ANOVA followed by Dunnett's multiple comparisons test. Asterisks (*) indicate significance levels, * represents p \u0026lt; 0.05, ** represents p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8132447/v1/527729bc635324504463d6dd.png"},{"id":97551392,"identity":"a81ed097-a047-47ed-939d-352509173128","added_by":"auto","created_at":"2025-12-05 17:18:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":585134,"visible":true,"origin":"","legend":"\u003cp\u003eSULF1 Activation of TAMs Promotes CD8⁺ T Cell Exhaustion. A: Analysis of the correlation between SULF1 and CD8⁺ T cells based on TCGA data. B \u0026amp; C: Flow cytometry analysis of the effects of SULF1-KD/OE on the activation levels of CD8⁺ T cells. D \u0026amp; E: Flow cytometry analysis of the effects of SULF1-KD/OE on the exhaustion levels of CD8⁺ T cells. F \u0026amp; H \u0026amp; J: Flow cytometry analysis of the regulatory effects of SULF1-KD on the proliferation, apoptosis, and exhaustion levels of CD8⁺ T cells co-cultured with M0 macrophages. G \u0026amp; I \u0026amp; K: Flow cytometry analysis of the regulatory effects of SULF1-OE on the proliferation, apoptosis, and exhaustion levels of CD8⁺ T cells co-cultured with M0 macrophages. L \u0026amp; M \u0026amp; N: Flow cytometry analysis of the regulatory effects of SULF1-His on the proliferation, apoptosis, and exhaustion levels of CD8⁺ T cells.Data are presented as the mean ± SD; n = 3 per group. The differences between the two groups were compared using a one-way ANOVA followed by Dunnett's multiple comparisons test. Asterisks (*) indicate significance levels, * represents p \u0026lt; 0.05, ** represents p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8132447/v1/9e4248addd4dc71ad7a1619f.png"},{"id":97551398,"identity":"3ba87c0b-6d68-473e-b005-c7ea68bdbf14","added_by":"auto","created_at":"2025-12-05 17:18:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1449653,"visible":true,"origin":"","legend":"\u003cp\u003eAnimal Experiments. A \u0026amp; B: Tumor formation experiments observing the tumorigenesis of SULF1-KD GC cells treated with SULF1-His in Balb/c mice. C \u0026amp; D: Tumor formation experiments observing the tumorigenesis of SULF1-OE GC cells treated with anti-SULF1 in Balb/c mice. E \u0026amp; F: Flow cytometry analysis of macrophage polarization states in tumor tissues. G \u0026amp; H \u0026amp; I \u0026amp; L: Flow cytometry detection of CD8⁺ T cell content and exhaustion levels in tumor tissues. J \u0026amp; M: ELISA detection of cytokine levels of IL-10, TGF-β, and IFN-γ in tumor tissues. K \u0026amp; N: WB detection of the effects of SULF1 on the protein levels of STAT3 and p-STAT3-Tyr705 in tumor tissues. O \u0026amp; P: Immunofluorescence observation of the nuclear localization of p-STAT3-Tyr705 in tumor tissue cells affected by SULF1. Data are presented as the mean ± SD; n = 3 per group. The differences between the two groups were compared using a one-way ANOVA followed by Dunnett's multiple comparisons test. Asterisks (*) indicate significance levels, * represents p \u0026lt; 0.05, ** represents p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8132447/v1/8ce8bbb09036c81c5bba4979.png"},{"id":107045268,"identity":"436ea70b-72c3-4aa1-ad84-bf8e1069fa05","added_by":"auto","created_at":"2026-04-16 07:13:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5485981,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8132447/v1/87ac01ac-5adc-4b84-98d2-528f95c48a2c.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.\nthere is NO conflict of interest to disclose","formattedTitle":"Secreted SULF1 Protein Modulates CD8+ T Cell Exhaustion by Promoting TAM Polarization in Gastric Cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGastric cancer (GC), as one of the most common malignancies with high incidence and mortality rates worldwide, has always been a hot and difficult topic in oncology research due to its mechanisms of invasion, metastasis, and immune escape\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Although the comprehensive application of surgery, radiochemotherapy, targeted therapy, and immunotherapy in recent years has significantly improved the prognosis of some patients, the 5-year survival rate of patients with advanced GC is still less than 30%\u003csup\u003e2\u003c/sup\u003e. The tumor microenvironment (TME), as the \"soil\" in which tumor cells survive, is composed of immune cells, fibroblasts, extracellular matrix, and soluble factors. Its dynamic imbalance is the core force driving the progression of GC. Tumor-associated macrophages (TAMs), as the most abundant immune cell population in the TME, have interactions with CD8⁺ T cells that have become a key link in tumor immune escape\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. M1 macrophages activate anti-tumor immunity by secreting pro-inflammatory cytokines such as IFN-γ and TNF-α. In contrast, M2 macrophages promote tumor progression by releasing immunosuppressive factors such as IL-10, TGF-β, and VEGF, which facilitate tumor angiogenesis, epithelial-mesenchymal transition (EMT), and CD8⁺ T cell exhaustion\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, the specific mechanisms by which GC cells regulate TAM polarization and further induce CD8⁺ T cell exhaustion have not yet been fully elucidated.\u003c/p\u003e\u003cp\u003eSulfatase 1 (SULF1), an extracellular heparan sulfate (HS) endosulfatase, modulates the signaling of multiple growth factors, such as VEGF, FGF, and Wnt, by specifically removing the 6-O-sulfate groups from HS chains. This action influences cell proliferation, migration, and angiogenesis\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that SULF1 is dysregulated in various tumors, but its effects are highly tissue-specific. In hepatocellular carcinoma and pancreatic cancer, SULF1 exerts tumor-suppressive effects by inhibiting the FGF-2/VEGF signaling pathway. Low SULF1 expression is associated with increased tumor angiogenesis and poor patient prognosis\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In contrast, in breast and ovarian cancers, SULF1 promotes tumor cell proliferation, migration, and chemoresistance by enhancing oncogenic signaling pathways such as HB-EGF/EGFR and CXCL12/CXCR4\u003csup\u003e10, 11\u003c/sup\u003e. Notably, recent single-cell sequencing studies have revealed heterogeneous expression of SULF1 in tumor stromal cells, such as cancer-associated fibroblasts, and immune cells. This suggests that SULF1 may participate in shaping the immune microenvironment by regulating intercellular interactions\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The expression pattern of SULF1 and its correlation with immune cell infiltration in GC have not been previously reported.\u003c/p\u003e\u003cp\u003eIn this study, we integrated analysis of the TCGA database with in vitro and in vivo functional experiments to uncover for the first time a novel mechanism by which SULF1 secreted by GC cells promotes M2 polarization of TAMs via the STAT3 signaling pathway, thereby inducing CD8⁺ T cell exhaustion. This finding provides new evidence for immunotherapeutic strategies targeting TAM polarization and CD8⁺ T cell functional recovery.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Bioinformatics Analysis\u003c/h2\u003e\u003cp\u003eTo comprehensively analyze the expression characteristics and clinical significance of SULF1 in GC, we downloaded RNA-seq data in STAR-counts format and corresponding clinical information from the TCGA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov\u003c/span\u003e\u003cspan address=\"https://portal.gdc.cancer.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the GEO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). First, the raw count matrix was converted to TPM (Transcripts Per Million) format and normalized using log₂(TPM\u0026thinsp;+\u0026thinsp;1). Subsequently, samples lacking complete clinical data or with duplicate sequencing were removed, resulting in a high-quality dataset for subsequent differential expression analysis, survival analysis, and functional enrichment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Cell Culture\u003c/h2\u003e\u003cp\u003eHs746T and MKN-74 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37\u0026deg;C with 5% CO₂.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Knockdown of SULF1 in Hs746T Cells\u003c/h2\u003e\u003cp\u003eSpecific shRNA sequences targeting SULF1 were designed and cloned into the lentiviral shRNA expression vector (pLKO.1, Addgene, USA). The expression vector, along with packaging and envelope plasmids, was co-transfected into 293T cells using Lipofectamine 3 000 to produce lentiviral particles. After viral packaging, the supernatant was collected, filtered, and used to infect Hs746T cells. Infected cells were selected with puromycin (2 \u0026micro;g/mL), and the knockdown efficiency of SULF1 was confirmed by Western blot.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Overexpression of SULF1 in MKN-74 Cells\u003c/h2\u003e\u003cp\u003eA plasmid containing the SULF1 gene was constructed and transfected into MKN-74 cells. Stable overexpression cell lines were obtained through antibiotic selection, and the overexpression of SULF1 was confirmed by Western blot.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Western Blot (WB)\u003c/h2\u003e\u003cp\u003eCells were lysed in RIPA buffer with protease/phosphatase inhibitors. 30 \u0026micro;g proteins were resolved by 12% SDS-PAGE, transferred to NC membranes, blocked with 5% non-fat milk, and incubated overnight (4℃) with primary antibodies: SULF1 (27438-1-AP, Proteintech), STAT3 (10253-2-AP, Proteintech), P-STAT3-Tyr705 (80199-2-RR, Proteintech), and β-actin (20536-1-AP, Proteintech). After HRP-conjugated secondary antibody (SA00001-2, Proteintech) incubation, bands were visualized by ECL and quantified via ImageJ.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Transwell Assay\u003c/h2\u003e\u003cp\u003eStarved cells were prepared as a cell suspension and added to the upper chamber of a Transwell insert. The insert was placed in a 24-well plate with serum-containing medium in the lower chamber. After 12 hours of incubation, the Transwell insert was removed, fixed with formaldehyde for 30 minutes, air-dried, and stained with crystal violet for 40 minutes. The cells were observed under a microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Flow Cytometry\u003c/h2\u003e\u003cp\u003eCell suspensions were prepared and stained with CoraLite\u0026reg; Plus 488-Annexin V and PI apoptosis detection kit (PF00005, Proteintech) for apoptosis analysis. Cells were also stained with the following antibodies: CoraLite\u0026reg; Plus 647-conjugated iNOS Polyclonal antibody (CL647-18985, Proteintech), CoraLite\u0026reg; Plus 488 Anti-Human CD86 (CL488-65165, Proteintech), CoraLite\u0026reg; Plus 488 Anti-Mouse CD86 (CL488-65068, Proteintech), CoraLite\u0026reg; Plus 488 Anti-Mouse CD206 Rabbit Recombinant Antibody (CL488-98031, Proteintech), and CoraLite\u0026reg; Plus 647 Anti-Human CD163 (GHI/61) Mouse IgG2a Recombinant Antibody (CL647-65561, Proteintech). Additional antibodies used for macrophage surface labeling included Alexa Fluor\u0026reg; 488 anti-mouse CD45 Antibody (160306, Biolegend), APC/Cyanine7 anti-mouse CD3 Antibody (100221, Biolegend), APC/Fire\u0026trade; 810 anti-mouse CD8a Antibody (104007, Biolegend), Spark YG\u0026trade; 570 anti-mouse/human CD44 Antibody (103073, Biolegend), Alexa Fluor\u0026reg; 700 anti-mouse CD62L Antibody (104426, Biolegend), Spark Blue\u0026trade; 574 anti-mouse CD279 (PD-1) (Flexi-Fluor\u0026trade;) Antibody (285153, Biolegend), and Alexa Fluor\u0026reg; 647 anti-mouse CD366 (Tim-3) Antibody (119743, Biolegend). After incubation, cells were washed with PBS and analyzed by flow cytometry to determine the rates of apoptosis and the proportions of M1 and M2 macrophages, as well as activated and exhausted CD8⁺ T cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Confocal Microscopy\u003c/h2\u003e\u003cp\u003eSubcutaneous tumor tissues from mice were sectioned and deparaffinized. Cells grown to an appropriate density in 24-well plates were fixed with 4% paraformaldehyde. The tumor sections and cells were permeabilized with 0.1% Triton X-100, blocked with skim milk, and incubated with a fluorescently labeled primary antibody specific to STAT3 (10253-2-AP, Proteintech). The corresponding secondary antibody, Fluorescein (FITC)\u0026ndash;conjugated Goat Anti-Rabbit IgG(H\u0026thinsp;+\u0026thinsp;L) (SA00003-2, Proteintech), and a blue fluorescent nuclear marker, DAPI (PR30021, Proteintech), were added. The tumor sections and cell slides were incubated overnight at 4\u0026deg;C in a humidified chamber to allow antibodies to bind to the target proteins. After incubation, the sections and slides were washed with PBS to remove unbound antibodies and markers, and mounted with an anti-fade mounting medium. The sections and slides were observed and imaged under a confocal microscope, with adjustments made to the microscope parameters to capture blue and green fluorescence signals to clearly visualize the distribution of fluorescence.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 ELISA Assay\u003c/h2\u003e\u003cp\u003eHuman/mouse IL-10 (HM10203 / MU30055, Bioswamp), TGF-β (HM10058 / MU30071, Bioswamp), and INF-γ (HM10115 / MU30038, Bioswamp) ELISA kits were used for quantitative detection. Samples were centrifuged at 3000 \u0026times; g for 10 minutes at 4\u0026deg;C, and the supernatant was collected. The assays were performed strictly according to the manufacturer's instructions, including sample addition, incubation, and color development. The absorbance was measured at 450 nm using a microplate reader, and the absolute concentrations of IL-10 and TGF-β were calculated based on the standard curve.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Animal Experiment\u003c/h2\u003e\u003cp\u003eBalb/c mice were used to establish subcutaneous xenograft models. Mice were randomly divided into groups and subcutaneously implanted with Hs746T, Hs746T-SULF1-KD, and MKN-74, MKN-74-SULF1-OE cells. These models were treated with SULF1-His protein and anti-SULF1 antibody, respectively. Tumor volumes were measured every 5 days. On day 30, mice were euthanized, tumors were excised, weighed, and tumor tissue proteins were extracted for subsequent analysis. All animal experiments were conducted in accordance with the approval and guidelines of the ethics review committee.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll experiments were repeated at least three times, and data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). Statistical analysis was performed using GraphPad Prism 8.0: Student's t-test for two-group comparisons and one-way analysis of variance (ANOVA) for multiple-group comparisons. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Expression and Function of SULF1 in GC\u003c/h2\u003e\u003cp\u003eTo explore SULF1\u0026rsquo;s expression and role in gastric cancer (GC), TCGA database analysis showed significantly higher SULF1 expression in GC tissues than normal tissues, with upregulation during disease progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Survival analysis linked high SULF1 expression to shorter patient survival, suggesting its pro-tumor effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Among five GC cell lines, Hs746T had the highest and MKN-74 the lowest SULF1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), so these two lines were selected for subsequent experiments. Lentiviral vectors mediated SULF1 knockdown (KD) in Hs746T (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) and overexpression (OE) in MKN-74 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Flow cytometry demonstrated SULF1-KD reduced GC cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) while SULF1-OE promoted proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Transwell assays showed SULF1-KD inhibited metastasis and invasion, whereas SULF1-OE enhanced these abilities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH \u0026amp; I). Additionally, SULF1-KD increased GC cell apoptosis, while SULF1-OE exerted the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ \u0026amp; K). These data confirm SULF1\u0026rsquo;s regulatory role in GC progression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Correlation of SULF1 with Immune Responses\u003c/h2\u003e\u003cp\u003eTo elucidate SULF1\u0026rsquo;s mechanism, TCGA data immune scoring revealed a positive correlation between SULF1 and macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Co-culture of SULF1-KD/OE GC cells with macrophages showed SULF1-KD increased M1 and decreased M2 polarization, while SULF1-OE reversed this trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB \u0026amp; C). Exogenous SULF1-His treatment similarly inhibited M1 and promoted M2 polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). SULF1-His-activated tumor-associated macrophages (TAMs) enhanced GC cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE \u0026amp; F), metastasis/invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG \u0026amp; H), and reduced apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI \u0026amp; J). Rescue experiments showed SULF1-His restored the pro-tumor phenotype in SULF1-KD cells (enhanced proliferation, reduced apoptosis; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK, M, O), while anti-SULF1 antibody abrogated the pro-tumor effect of SULF1-OE cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL, N, P), confirming SULF1\u0026rsquo;s direct role.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Mechanism of SULF1 Activation of TAMs\u003c/h2\u003e\u003cp\u003eThe STAT3 signaling pathway is believed to be closely related to TAM polarization \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Therefore, in this study, macrophages were treated with SULF1-His to detect the activation of STAT3 in macrophages. The results showed that after treatment with SULF1-His, the level of STAT3 protein remained unchanged, while the level of P-STAT3-Tyr705 significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Confocal imaging results also showed an increased nuclear localization of P-STAT3-Tyr705 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Meanwhile, the levels of cytokines IL-10 and TGF-β, which are closely related to M2 polarization, were detected. ELISA results indicated that the addition of SULF1-His elevated the levels of these cytokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC \u0026amp; D). To further investigate the activation of STAT3 and the levels of IL-10 and TGF-β, macrophages were co-cultured with GC cells after SULF1-KD/OE. The results showed that regardless of KD or OE, the expression level of STAT3 remained unchanged. However, after KD, the expression level of P-STAT3-Tyr705 decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), and the levels of IL-10 and TGF-β cytokines were reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF \u0026amp; G). Conversely, after OE, the expression level of P-STAT3-Tyr705 increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), and the levels of IL-10 and TGF-β cytokines were elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI \u0026amp; J). These findings indicate that SULF1 promotes TAM polarization by increasing the level of p-STAT3-Tyr705.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4 SULF1 Activation of TAMs Promotes CD8⁺ T Cell Exhaustion\u003c/h2\u003e\u003cp\u003eImmune scoring analysis of clinical data from GC patients in the TCGA database also revealed a negative correlation between SULF1 and CD8⁺ T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To verify the relationship between SULF1 and CD8⁺ T cells, CD8⁺ T cells were co-cultured with GC cells after SULF1-KD/OE for 24 hours. It was found that CD8⁺ T cells were activated during the co-culture process. However, neither SULF1-KD nor OE significantly affected the degree of CD8⁺ T cell activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB \u0026amp; C), and similar results were obtained for cell exhaustion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD \u0026amp; E). Recent studies have indicated that an increase in TAMs within the tumor microenvironment can promote CD8⁺ T cell exhaustion\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Therefore, macrophages were co-cultured with GC cells after SULF1-KD/OE, followed by co-culture with CD8⁺ T cells for 24 hours to detect the proliferation and apoptosis levels of CD8⁺ T cells. Flow cytometry results showed that after SULF1-KD, proliferation was enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) and apoptosis levels were reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Conversely, after SULF1-OE, proliferative capacity was weakened (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) and apoptosis levels increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). CD8⁺ T cell exhaustion was also examined, and it was found that after SULF1-KD, the level of CD8⁺ T cell exhaustion was inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ), while after SULF1-OE, the level of CD8⁺ T cell exhaustion was enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). Subsequently, CD8⁺ T cells were directly treated with SULF1-His, and their proliferation, apoptosis, and T cell exhaustion levels were detected. Compared with the untreated group, the addition of SULF1-His did not significantly change the cell proliferation level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL), nor did it significantly affect apoptosis and T cell exhaustion levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM \u0026amp; N). These findings clearly demonstrate that SULF1-induced CD8⁺ T cell exhaustion is mediated by TAM polarization.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Animal Experiments\u003c/h2\u003e\u003cp\u003eTo investigate the impact of SULF1 on gastric cancer, we established Balb/c mouse tumor models using Hs746T, Hs746T-SULF1-KD, and MKN-74, MKN-74-SULF1-OE cells. These models were treated with exogenous SULF1-His protein and anti-SULF1 antibody, respectively. The results showed that SULF1-KD in Hs746T cells significantly inhibited tumor formation, but this inhibition was reversed by the addition of SULF1-His (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u0026amp; B). Conversely, SULF1-OE in MKN-74 cells promoted tumor formation, an effect that was effectively inhibited by anti-SULF1 antibody treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC \u0026amp; D). Flow cytometry analysis revealed that the proportion of M2 macrophages in tumor tissues from Hs746T-SULF1-KD group was lower than that from Hs746T group, but this proportion was restored upon SULF1-His treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). In contrast, MKN-74-SULF1-OE group exhibited a higher proportion of M2 macrophages compared to MKN-74 group, which decreased after anti-SULF1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Exhaustion levels of CD8⁺ T cells were also examined. Mice with Hs746T-SULF1-KD exhibited reduced CD8⁺ T cell exhaustion, whereas those with Hs746T-SULF1-KD\u0026thinsp;+\u0026thinsp;SULF1-His showed increased exhaustion levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG \u0026amp; I). Similarly, MKN-74-SULF1-OE group had higher CD8⁺ T cell exhaustion levels, which decreased following anti-SULF1 antibody treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH \u0026amp; L). ELISA assays indicated that SULF1 KD led to decreased levels of IL-10 and TGF-β, while SULF1-His treatment increased these cytokine levels. IFN-γ levels were elevated in SULF1 KD mice but decreased upon SULF1-His treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). In MKN-74-SULF1-OE group, IFN-γ levels were reduced, but increased after anti-SULF1 antibody treatment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWB analysis showed that in Hs746T cells, SULF1 KD inhibited P-STAT3-Tyr705 and reduced its nuclear localization. SULF1-His treatment activated P-STAT3-Tyr705 and increased its nuclear presence (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK \u0026amp; O). In contrast, SULF1-OE in MKN-74 cells led to opposite changes in IL-10, TGF-β, IFN-γ, and P-STAT3-Tyr705 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM \u0026amp; K \u0026amp; P). In summary, these in vivo experimental results further confirmed the observations from our in vitro studies, demonstrating that SULF1 significantly affects tumor formation and the immune microenvironment in gastric cancer by modulating various cytokines and signaling pathways.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe progression of gastric cancer is contingent upon the dynamic interplay between tumor cells and immune cells within the microenvironment, with the polarization state of TAMs being a pivotal factor in regulating immune evasion\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This study, for the first time, elucidates that SULF1 secreted by gastric cancer cells promotes the polarization of TAMs towards the immunosuppressive M2 phenotype by activating the STAT3 signaling pathway, thereby inducing CD8⁺ T cell exhaustion. This finding offers a novel perspective for understanding the regulatory mechanisms of the gastric cancer immune microenvironment.\u003c/p\u003e\u003cp\u003eBy analyzing data from the TCGA database and conducting cellular functional experiments, we discovered that SULF1 is highly expressed in gastric cancer tissues, with its levels positively correlated with tumor progression and patient prognosis. Additionally, our study found that SULF1-KD significantly inhibits the viability of gastric cancer cells and induces apoptosis, whereas overexpression enhances their invasive capacity. These findings further confirm the central role of SULF1 in tumor evolution. This is consistent with previous studies showing SULF1's oncogenic role in breast and ovarian cancers but contrasts sharply with its tumor-suppressive effects in liver and pancreatic cancers\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. This tissue specificity likely arises from differences in the baseline sulfation levels of HS and the growth factor networks in different tumor microenvironments. For instance, in liver cancer, SULF1 inhibits angiogenesis by suppressing FGF signaling, whereas in gastric cancer, SULF1 indirectly activates TAM polarization by relieving HS-mediated inhibition of the JAK2/STAT3 pathway. This suggests that the functional plasticity of SULF1 is closely related to the \"sulfation background\" in its microenvironment, and future research should employ single-cell sulfation genomics to further elucidate this relationship.\u003c/p\u003e\u003cp\u003eTumor cells reshape macrophage polarization through secreted factors as an important strategy for immune evasion. Our study found that the SULF1-STAT3 axis complements previously reported pathways such as HIF-1α and NF-κB. Notably, SULF1-His can directly induce the nuclear translocation of macrophage p-STAT3-Tyr705, while SULF1-KD in tumor cells indirectly inhibits macrophage STAT3 activation through a secretion defect. This indicates a \"secretion-response\" positive feedback loop between tumor cells and macrophages. This mechanism, together with the finding that SPP1⁺ TAMs induce T cell exhaustion through the GDF15-TGFβ axis\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, reveals the multi-dimensional characteristics of TAM polarization regulation. However, the extracellular enzymatic nature of SULF1 endows it with the potential for \"remote regulation\" of the microenvironment, making it a potentially more universal intervention target.\u003c/p\u003e\u003cp\u003eRegarding the mechanism of CD8⁺ T cell exhaustion, our study confirmed that the effects of SULF1 are entirely mediated by TAMs, which is different from the previously reported mode of tumor cells directly secreting PD-L1 to inhibit T cells\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Importantly, M2 macrophages not only inhibit T cell proliferation by secreting IL-10 and TGF-β but also enhance exhaustion signals by upregulating PD-L1 expression, forming a dual-inhibition network. Animal experiments in our study showed that SULF1-KD in tumor tissues increased IFN-γ levels and reduced exhaustion markers, further confirming the central role of TAM polarization in T cell functional remodeling. This finding provides a theoretical basis for the combined treatment strategy of \"reprogramming TAM polarization\u0026thinsp;+\u0026thinsp;restoring T cell function,\" such as the potential synergistic effect of using SULF1 neutralizing antibodies with PD-1 inhibitors.\u003c/p\u003e\u003cp\u003eCompared with similar studies, our research has completely constructed the regulatory axis of \"tumor cell secreted factors - macrophage polarization - effector T cell function\" through in vivo and in vitro experiments and has verified its clinical transformation potential in animal models. However, several scientific questions remain to be addressed: First, as a secreted protein, the specific binding receptors of SULF1 on macrophages in the microenvironment (LRP1, CD44) have not been identified, which may affect the development of targeted drugs. Second, the spatial co-localization evidence of SULF1 expression with TAM polarization and CD8⁺ T cell exhaustion in clinical samples still needs to be supplemented by double immunofluorescence experiments. Moreover, whether SULF1 indirectly affects the microenvironment by regulating other immune cell subsets (NK cells, Treg cells) also needs further exploration.\u003c/p\u003e\u003cp\u003eFrom the perspective of translational medicine, the SULF1-STAT3 axis revealed in this study provides a dual intervention target for gastric cancer immunotherapy: inhibiting SULF1 can block the upstream signal of TAM polarization, while inhibiting STAT3 can directly reverse the M2 phenotype. Existing research has shown that STAT3 inhibitors (OPB-31121) can enhance the efficacy of PD-1 inhibitors in preclinical models, and our study further suggests that combined blockade of SULF1 may produce a synergistic effect. Additionally, designing small-molecule inhibitors or monoclonal antibodies based on the enzymatic activity of SULF1 holds promise as a new strategy for \"de-immunosuppressive\" therapy.\u003c/p\u003e\u003cp\u003eIn summary, by integrating multi-omics data with functional experiments, our study systematically elucidates the molecular mechanisms by which SULF1 regulates the immune microenvironment in gastric cancer. This not only expands our understanding of the tumor-immune interaction network but also provides a new direction for overcoming current immunotherapy resistance. Future work should validate this mechanism in larger clinical cohorts and explore personalized targeting strategies using organoid models to promote the translation of basic research into clinical applications.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study has uncovered the complete molecular pathway of the \"GC-SULF1-TAM-CD8⁺ T cell\" immunosuppressive axis, offering new insights for the precision immunotherapy of GC. Future targeting of SULF1 or combination with STAT3 inhibitors may help overcome current immunotherapy resistance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003efunding statement\u003c/p\u003e\n\u003cp\u003eThe authors did not receive support from any organization for the submitted work.\u003c/p\u003e\n\u003cp\u003eConflict of interest disclosure\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed Consent: All authors have read and approved the final manuscript and consent to its submission for publication.\u003c/p\u003e\n\u003cp\u003eRegistry and the Registration No. of the study/trial: N/A.\u003c/p\u003e\n\u003cp\u003eAnimal Studies: All animal experiments conformed to the internationally accepted principles for the care and use of laboratory animals (Medical Ethics Committee of Henan Provincial People\u0026apos;s Hospital).\u003c/p\u003e\n\u003cp\u003eAuthor Contributions: Conceptualization, Xiaodan Lu and Di Lu; Methodology, Xiaodan Lu; Validation, Xiaodan Lu and Di Lu; Formal Analysis, Xiaodan Lu; Investigation, Xiaodan Lu and Di Lu; Data Curation, Xiaodan Lu; Writing \u0026ndash; Original Draft Preparation, Di Lu; Writing \u0026ndash; Review \u0026amp; Editing, Xiaodan Lu; Visualization, Di Lu; Supervision, Xiaodan Lu; Project Administration, Xiaodan Lu; Funding Acquisition, Xiaodan Lu. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A \u003cem\u003eet al.\u003c/em\u003e Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. \u003cem\u003eCA Cancer J Clin\u003c/em\u003e 2021; 71(3): 209\u0026ndash;249.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl Halabi M, Horanieh R, Tamim H, Mukherji D, Jdiaa S, Temraz S \u003cem\u003eet al.\u003c/em\u003e The impact of age on prognosis in patients with gastric cancer: experience in a tertiary care centre. \u003cem\u003eJournal of gastrointestinal oncology\u003c/em\u003e 2020; 11(6): 1233\u0026ndash;1241.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim S, Han IH, Lee S, Park D, Lee H, Kim J \u003cem\u003eet al.\u003c/em\u003e The Combination of CD300c Antibody with PD-1 Blockade Suppresses Tumor Growth and Metastasis by Remodeling the Tumor Microenvironment in Triple-Negative Breast Cancer. \u003cem\u003eInternational journal of molecular sciences\u003c/em\u003e 2025; 26(11).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe Y, Hong Q, Chen S, Zhou J, Qiu S. 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Advantages of targeting the tumor immune microenvironment over blocking immune checkpoint in cancer immunotherapy. \u003cem\u003eSignal Transduct Target Ther\u003c/em\u003e 2021; 6(1): 72.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBiernacki K, Ciupak O, Daśko M, Rachon J, Kozak W, Rak J \u003cem\u003eet al.\u003c/em\u003e Development of Sulfamoylated 4-(1-Phenyl-1H-1,2,3-triazol-4-yl)phenol Derivatives as Potent Steroid Sulfatase Inhibitors for Efficient Treatment of Breast Cancer. \u003cem\u003eJ Med Chem\u003c/em\u003e 2022; 65(6): 5044\u0026ndash;5056.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhurana A, Beleford D, He X, Chien J, Shridhar V. Role of heparan sulfatases in ovarian and breast cancer. \u003cem\u003eAmerican journal of cancer research\u003c/em\u003e 2013; 3(1): 34\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLai JP, Thompson JR, Sandhu DS, Roberts LR. 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HIF1α promotes tumor chemoresistance via recruiting GDF15-producing TAMs in colorectal cancer. \u003cem\u003eExperimental cell research\u003c/em\u003e 2021; 398(2): 112394.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDammeijer F, van Gulijk M, Mulder EE, Lukkes M, Klaase L, van den Bosch T \u003cem\u003eet al.\u003c/em\u003e The PD-1/PD-L1-Checkpoint Restrains T cell Immunity in Tumor-Draining Lymph Nodes. \u003cem\u003eCancer Cell\u003c/em\u003e 2020; 38(5): 685\u0026ndash;700.e8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"genes-and-immunity","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"genes","sideBox":"Learn more about [Genes \u0026 Immunity](http://www.nature.com/gene/)","snPcode":"41435","submissionUrl":"https://mts-gene.nature.com/cgi-bin/main.plex","title":"Genes \u0026 Immunity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Gastric cancer, SULF1, Tumor-associated macrophages, CD8⁺T cell exhaustion, STAT3 signaling","lastPublishedDoi":"10.21203/rs.3.rs-8132447/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8132447/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGastric cancer (GC) progression is linked to immune escape in the tumor microenvironment, yet the molecules regulating tumor-associated macrophage polarization and CD8⁺ T-cell exhaustion are unclear. This study analyzed TCGA data to examine SULF1 expression and its prognostic role. It used CRISPR/Cas9 and lentiviral methods in GC cells to test proliferation, invasion, and apoptosis, plus co-culture and flow cytometry to assess SULF1’s impact on macrophages and CD8⁺ T-cells. STAT3 signaling was studied via immunoblotting and nuclear translocation assays, and a mouse model tested SULF1’s therapeutic relevance. Results showed SULF1 was up-regulated in GC, tied to advanced stages and poor survival. SULF1 knockdown inhibited GC cell traits, while overexpression boosted them. SULF1 activated macrophage STAT3, promoting M2 polarization and CD8⁺ T-cell dysfunction. In mice, SULF1 silencing reduced tumors and T-cell exhaustion, while supplementation reversed this. Conclusions: GC-secreted SULF1 creates an immunosuppressive microenvironment via STAT3-dependent pathways, and targeting SULF1–STAT3 may improve GC immunity.\u003c/p\u003e","manuscriptTitle":"Secreted SULF1 Protein Modulates CD8+ T Cell Exhaustion by Promoting TAM Polarization in Gastric Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-05 17:18:25","doi":"10.21203/rs.3.rs-8132447/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-01-19T19:24:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-01-07T00:42:54+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-06T23:16:55+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-12-15T19:15:53+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-12-03T18:04:24+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-12-03T16:52:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-18T16:22:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-17T08:00:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genes \u0026 Immunity","date":"2025-11-17T08:00:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genes-and-immunity","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"genes","sideBox":"Learn more about [Genes \u0026 Immunity](http://www.nature.com/gene/)","snPcode":"41435","submissionUrl":"https://mts-gene.nature.com/cgi-bin/main.plex","title":"Genes \u0026 Immunity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b4e83249-2eb1-479f-9f21-4c9482dd8701","owner":[],"postedDate":"December 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":59046107,"name":"Biological sciences/Immunology/Tumour immunology/Immunosurveillance/Immunoediting"},{"id":59046108,"name":"Biological sciences/Immunology/Immune cell death"}],"tags":[],"updatedAt":"2026-04-16T07:12:37+00:00","versionOfRecord":{"articleIdentity":"rs-8132447","link":"https://doi.org/10.1038/s41435-026-00399-x","journal":{"identity":"genes-and-immunity","isVorOnly":false,"title":"Genes \u0026 Immunity"},"publishedOn":"2026-04-15 04:00:00","publishedOnDateReadable":"April 15th, 2026"},"versionCreatedAt":"2025-12-05 17:18:25","video":"","vorDoi":"10.1038/s41435-026-00399-x","vorDoiUrl":"https://doi.org/10.1038/s41435-026-00399-x","workflowStages":[]},"version":"v1","identity":"rs-8132447","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8132447","identity":"rs-8132447","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}