Abstract

   Neutrophils are integral components of the bone marrow and stromal cell
   network. They express the immune checkpoint molecule PD-L1 and can
   induce T cell exhaustion, thereby promoting immunosuppression. In this
   study, we investigated whether tumor-derived Cldn7 deficiency could
   recruit polymorphonuclear neutrophils (PMNs), induce their metabolic
   reprogramming, and consequently drive their transition toward a
   pro-tumor phenotype, leading to the establishment of an
   immunosuppressive tumor microenvironment (TME). Using single-cell RNA
   sequencing, clinical sample validation, and both in vivo and in vitro
   experiments, we found that Cldn7 deficiency in colorectal cancer (CRC)
   results in a tumor microenvironment characterized by significantly
   increased infiltration of CD11b⁺ Ly6G⁺ neutrophils. This is accompanied
   by neutrophil metabolic reprogramming that facilitates their phenotypic
   shift toward a tumor-promoting state, which in turn suppresses the
   cytotoxic function of CD8⁺ T cells and contributes to the formation of
   an immunosuppressive TME, thereby accelerating CRC progression.
   Mechanistically, Cldn7 deficiency indirectly activates the NF-κB
   signaling pathway, leading to elevated secretion of chemokines such as
   CXCL1 that are responsible for PMN recruitment. Inhibition of the
   NF-κB/CXCL1 axis reduces PMN infiltration, decreases PD-L1 expression
   on neutrophils, suppresses neutrophil glycolysis and histone
   lactylation, reverses the exhausted phenotype of CD8⁺ T cells, thereby
   mitigating the immunosuppressive microenvironment. Furthermore,
   overexpression of Cldn7 enhances the efficacy of immune checkpoint
   blockade (ICB) therapy.Collectively, our findings indicate that Cldn7
   deficiency not only contributes to immune evasion and malignant
   progression in CRC but also plays a critical role in immune modulation.
   Targeting PMN metabolic reprogramming and immunosuppressive function
   associated with Cldn7 loss may offer a promising strategy to improve
   the therapeutic efficacy of immunotherapy in CRC.

   Subject terms: Immune evasion, Gastrointestinal cancer

Introduction

   Colorectal cancer (CRC) is one of the most common malignant tumors of
   the digestive system, ranking third globally in incidence and leading
   in mortality, posing a severe threat to human health. The development
   of CRC is a multi-stage, complex biological process involving various
   factors contributing to its progression and malignant transformation
   [[46]1–[47]3]. Current treatments for CRC include traditional surgery,
   radiotherapy, chemotherapy, and emerging biological immunotherapies
   [[48]4]. Immune checkpoint inhibitor therapy (ICT) for CRC has been
   challenging to achieve effective results due to the formation of an
   immunosuppressive tumor microenvironment (TME) [[49]5, [50]6].
   Therefore, studying the molecular mechanisms underlying the formation
   of the immunosuppressive microenvironment in CRC may provide a
   theoretical basis for improving ICT treatment for CRC.

   Cldn7 is a member of the Claudins family of tight junction proteins,
   expressed in the apical and lateral membranes of intestinal epithelial
   cells, as well as in the basal crypt stem cell regions of the intestine
   [[51]7, [52]8]. Studies have shown that, in addition to its classic
   tight junction functions such as barrier maintenance and cell polarity,
   which are crucial for the physiological functions of various organs,
   Cldn7 also plays significant roles in the initiation of inflammation
   and tumorigenesis [[53]9, [54]10]. Downregulation of Cldn7 expression
   has been observed in both inflammatory and tumor tissues. Our data
   indicate that low expression of Cldn7 in tumor tissues is a biomarker
   for poor prognosis in CRC patients. Previous research has also
   confirmed that Cldn7 deletion significantly affects the inflammatory
   microenvironment of tumors in mice, primarily through changes in
   neutrophil infiltration. However, the specific mechanisms remain
   unclear and require further investigation.

   The tumor microenvironment (TME) consists of various cells and
   extracellular components, including endothelial cells, fibroblasts,
   immune cells, and extracellular components such as cytokines, growth
   factors, hormones, and extracellular matrix [[55]11, [56]12]. These
   elements surround tumor cells, are nourished by an intricate network of
   microvessels, and form a localized biological microenvironment that
   influences tumor progression [[57]13–[58]15]. The role of
   tumor-associated polymorphonuclear neutrophils (PMNs) in tumor
   development has been underexplored. Recent studies have gradually
   revealed the role of PMNs in tumorigenesis and progression [[59]16,
   [60]17]. Despite the diversity of their functions, a high density of
   PMNs in tumor tissue is usually associated with a poor prognosis. Under
   the influence of the tumor microenvironment, these cells are induced to
   transform into subtypes with immunosuppressive features, which suppress
   the body’s immune response to the tumor and thus contribute to the
   spread of cancer cells and their resistance to immunotherapy [[61]18,
   [62]19]. However, the specific molecular mechanisms driving neutrophil
   phenotypic changes have not been fully elucidated. PMNs can secrete
   cytokines or form neutrophil extracellular traps (NETs) to regulate
   tumor metastasis [[63]20]. At the primary tumor site, PMNs promote
   tumor cell migration and invasion by secreting cytokines or directly
   interacting with tumor cells [[64]21, [65]22]. Research indicates that
   PMNs play a critical regulatory role in the pathological processes of
   tumor initiation, progression, invasion, and metastasis [[66]23,
   [67]24]. Additionally, clinical studies have reported that the
   neutrophil/lymphocyte ratio in peripheral blood is an independent
   prognostic factor for CRC patients [[68]25, [69]26]. However, due to
   the phenotypic diversity and highly heterogeneous functions of PMNs,
   their biological role in the malignant progression of CRC remains to be
   fully elucidated.

   Neutrophils are part of the bone marrow and stromal cell network,
   express PDL1, and drive immune checkpoint engagement and T cell
   exhaustion [[70]27]. In this study, we investigated whether
   tumor-derived Cldn7 deletion attracts PMNs infiltration and promotes
   their shift to a pro-tumorigenic phenotype, resulting in an
   immunosuppressive microenvironment. Using single-cell sequencing,
   clinical sample validation, and in vivo and in vitro experiments, we
   found that Cldn7 deletion leads to increased recruitment of
   CD11b^+Ly6G^+ neutrophils in the CRC microenvironment and accelerates
   malignant progression of CRC.Cldn7 deletion indirectly activates the
   NF-kB signaling pathway, leading to increased secretion of
   PMN-recruitment-associated chemokines (e.g. CXCL1). Furthermore,
   deletion of Cldn7 expression in the CRC microenvironment leads to
   adaptive metabolic reprogramming of PMNs, as evidenced by elevated
   glycolysis mediated by histone lactylation, a higher proportion of
   PDL1^+ PMNs expression, and an antiapoptotic phenotype of neutrophils
   (increased expression of the Bcl2). Inhibition of the NF-kB/CXCL1 axis
   in Cldn7-deficient tumor cells ameliorates the immunosuppressive
   microenvironment by decreasing PMN recruitment, decreasing PD-L1
   expression on PMNs, and reversing the CD8^+ T cell exhaustion
   phenotype. In addition, overexpression of Cldn7 improved the efficacy
   of ICB therapy. In conclusion, our experimental results suggest that
   Cldn7 deficiency plays an initiating role in the malignant progression
   of CRC and has an immunomodulatory function, and that the study of
   related biological processes may improve the efficacy of CRC
   immunotherapy in the future.

Results

Single-cell analysis reveals a potential regulatory role of tumor-derived
Cldn7 in polymorphonuclear neutrophils (PMNs) within colorectal cancer

   Claudin7 (Cldn7) has previously been recognized as a tumor suppressor
   gene, yet its specific functional mechanisms in colorectal cancer (CRC)
   progression remain largely unclear [[71]10, [72]28]. To systematically
   investigate the biological role of Cldn7, we performed single-cell RNA
   sequencing on tumor samples from five clinically diagnosed CRC
   patients. Using UMAP-based clustering analysis, we classified and
   annotated 12 major cell types, including NK cells, neutrophils,
   epithelial cells, myeloid cells, smooth muscle cells, T cells,
   endothelial cells, antigen-presenting cells, plasma cells,
   proliferating cells, and B cells (Fig. [73]1A, B, Supplementary Fig.
   [74]1A, B). Subsequently, we conducted a more detailed analysis of the
   epithelial cells using inferCNV, identifying 14 epithelial cell
   subpopulations and obtaining inferCNV scores for each (Supplementary
   Fig. [75]1C, D). Based on these scores, we classified epithelial
   subpopulations in the CRC single-cell dataset into malignant and
   non-malignant epithelial cells and compared Cldn7 expression levels
   between these two groups. The results showed that Cldn7 was
   significantly downregulated in malignant epithelial cells, with some
   malignant CRC epithelial subpopulations exhibiting a complete loss of
   Cldn7 expression (Fig. [76]1C, D). In further analysis, we also
   observed the heterogeneity of neutrophils in the CRC microenvironment.
   Through subpopulation clustering, we identified seven distinct
   tumor-associated neutrophil (TAN) subgroups (Fig. [77]1E) [[78]29].
   Among the seven TAN subpopulations identified in CRC, TAN-1 exhibited
   high expression of genes related to neutrophil transendothelial
   migration, such as VNN2, SELL, and ITGB2, suggesting that this cluster
   may represent a transitional state from PMNs to TANs. TAN-4 was
   characterized as a pro-tumorigenic subpopulation, showing elevated
   expression of multiple pro-angiogenic and pro-metastatic factors,
   including VEGFA, PLAU, and LGALS3. This subset also displayed a
   distinct transcriptional profile, with upregulation of a series of
   interferon-stimulated genes (ISGs), such as IFIT1, IFIT2, IFIT3, ISG15,
   and RSAD2 (Fig. [79]2F). TAN-7 belonged to an inflammation-related
   subcluster, with strong expression of inflammatory regulators NLRP3 and
   PDE4B, along with high levels of the neutrophil activation marker CD69.
   Additionally, TAN-6 demonstrated marked expression of tumor-promoting
   molecules, including IL1RN and adrenomedullin (ADM) (Fig. [80]2D),
   indicating its potential role in modulating the tumor microenvironment.
   Subsequently, ligand–receptor interaction analysis revealed
   intercellular communication differences between malignant cells with
   high and low Cldn7 expression and different TAN subpopulations.
   Notably, several chemokine pathways—including CXCL1–CXCR1, CXCL2–CXCR1,
   and CXCL8–CXCR1—were significantly involved, suggesting that Cldn7 may
   play a role in regulating neutrophil recruitment and function (Fig.
   [81]1G). Furthermore, hallmark enrichment analysis indicated distinct
   signaling pathway enrichments in CRC malignant epithelial cells with
   high versus low Cldn7 expression. Low Cldn7 expression was potentially
   associated with enrichment in signaling pathways such as
   TNFα-signaling-via-NFκB, PI3K-AKT-mTOR-signaling, and the p53-pathway
   (Fig. [82]1H). Cell interaction analysis also indicated a strong
   interaction between Cldn7-low malignant epithelial cell subpopulations
   and TAN subgroups (Fig. [83]1I). These findings suggest that Cldn7
   expression is significantly downregulated in malignant epithelial cells
   in colorectal cancer and that it may play a key role in reshaping the
   tumor microenvironment and promoting CRC progression by affecting
   neutrophil heterogeneity and functional state through aberrant
   communication networks with TANs.

Fig. 1. Single-cell sequencing data of colorectal cancer illustrates the
negative correlation between tumor suppressor gene Cldn7 expression levels
and neutrophil infiltration within the tumor microenvironment.

   [84]Fig. 1
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   A UMAP clustering of single cells showing distinct cell clusters
   labeled 1–18. B Cell-type annotation based on clusters, identifying
   various cell types including NK cells, neutrophils, plasma cells,
   proliferating cells, epithelial cells, smooth muscle cells, endothelial
   cells, fibroblasts, and mast cells. C UMAP plot displaying malignant
   (red) versus non-malignant (blue) cell populations, with CLDN7
   expression levels indicated by a color gradient. D Violin plot
   comparing CLDN7 expression levels between non-malignant and malignant
   cells, showing significantly lower expression in malignant cells. E
   UMAP plot identifying subclusters of tumor-associated neutrophils
   (TANs), labeled TAN-1 through TAN-7. F Dot plot showing the expression
   of selected genes across TAN subclusters, with dot size representing
   the proportion of expressing cells and color indicating average
   expression levels. G Dot plot of ligand-receptor interactions between
   CLDN7-expressing cells and TAN subclusters, with color indicating
   maximum communication probability and significant interactions marked.
   H Pathway enrichment analysis of genes associated with CLDN7 and TAN
   subclusters, highlighting significantly enriched pathways and their
   direction (upregulated or downregulated). I Network diagram showing the
   interaction weights and strengths among TAN subclusters, with red and
   blue lines representing stronger or weaker interactions, respectively.

Fig. 2. Cldn7 is expressed at low levels in CRC tumor tissues and is
negatively correlated with PMN infiltration.

   [86]Fig. 2
   [87]Open in a new tab

   A Comparison of CLDN7 mRNA expression levels in normal colorectal
   tissues and colorectal cancer tissues from TCGA-CRC data, showing a
   significant reduction in cancer tissues (****p < 0.0001). B
   Kaplan–Meier survival analysis showing a significantly worse overall
   survival (OS) in colorectal cancer patients with low CLDN7 expression
   compared to those with high CLDN7 expression (p = 0.024). C Correlation
   between CLDN7 expression (log2 TPM) and neutrophil infiltration levels
   using TIMER and CIBERSORT-ABS methods. Negative correlations were
   observed in both models, where the infiltration level increases as
   CLDN7 expression decreases. D, E Immunohistochemical staining of CLDN7
   in normal (upper panel) and colorectal cancer tissues (lower panel) at
   different magnifications, showing that CLDN7 is primarily localized in
   the epithelial cells in normal tissue and is significantly reduced in
   cancer tissues (****p < 0.0001). F Western blot analysis of CLDN7
   expression in colorectal cancer patient samples from tumor and adjacent
   normal tissues, the image suggests that CLDN7 expression is reduced in
   CRC tumor tissues compared to the adjacent normal tissues. G–I
   Immunofluorescence staining of colorectal cancer tissues showing immune
   cell infiltration in low (upper panel) and high (lower panel) CLDN7
   expression tissues. Tissues with low CLDN7 expression exhibit fewer
   CD8-positive T cells (red) and more CD66B-positive neutrophils (green),
   while high CLDN7 expression associated with significantly reduced
   neutrophil infiltration and increased CD8-positive T cell infiltration
   (****p < 0.0001).

Cldn7 deletion constructs an immunosuppressive microenvironment in CRC in a
PMN-dependent manner

   We first performed paired transcriptomic analysis using the TCGA-COAD
   dataset and observed a significant downregulation of Cldn7 expression
   in CRC tumor tissues (Fig. [88]2A). This finding was further validated
   by immunohistochemistry and Western blot analysis of CRC patient
   samples collected from our center, confirming the reduced expression of
   Cldn7 in tumor tissues (Fig. [89]2D–F). Moreover, Kaplan–Meier survival
   analysis revealed that low Cldn7 expression was significantly
   associated with poor prognosis in CRC patients (Fig. [90]2B). Immune
   infiltration analysis showed a negative correlation between Cldn7
   expression and neutrophil infiltration (Fig. [91]2C). Multiplex
   immunofluorescence staining further demonstrated that regions with high
   Cldn7 expression exhibited enhanced CD8⁺ T cell infiltration and
   reduced infiltration of CD66B⁺ polymorphonuclear neutrophils (PMNs)
   (Fig. [92]2G–I), suggesting a potential role of Cldn7 in modulating
   immune cell distribution and function within the CRC tumor
   microenvironment. Our previous research has partially confirmed that
   the expression level of Cldn7 is closely associated with tumor staging
   and metastasis in CRC, further supporting its potential biological role
   in CRC [[93]30, [94]31]. To investigate the functional role of Cldn7 in
   CRC progression, we established two syngeneic tumor-bearing mouse
   models. One model employed MC38 cells transduced with either a vector
   control (Ctrl) or Cldn7 overexpression (Cldn7-oe), and the other used
   CMT93 cells transduced with either a control shRNA (shNC) or Cldn7
   knockdown (shCldn7). In the MC38 model, Cldn7 overexpression
   significantly inhibited tumor growth, reduced tumor weight, and
   prolonged mouse survival (Fig. [95]3A–D). In contrast, Cldn7 knockdown
   in the CMT93 model promoted tumor growth, increased tumor weight, and
   shortened survival (Fig. [96]3K–N). Based on our previous single-cell
   RNA sequencing and multiplex imaging results, we hypothesized that
   Cldn7 may influence CRC progression by regulating immune cell function
   within the tumor microenvironment. Flow cytometry analysis revealed
   that Cldn7 overexpression markedly reduced the infiltration of CD11b⁺
   Ly6G⁺ neutrophils, with no obvious effect on other myeloid cell
   populations (Fig. [97]3E, Supplementary Fig. [98]2A–B), and enhanced
   CD8⁺ T cell infiltration (Fig. [99]3F). Further analysis demonstrated
   that CD8⁺ T cells in the Cldn7-overexpressing group exhibited
   significantly increased production of effector cytokines IFN-γ and
   TNF-α, indicating enhanced antitumor immune activity (Fig. [100]3G–I).
   Moreover, the expression levels of the inhibitory receptors PD-1 and
   Tim-3 on CD8⁺ T cells were markedly reduced in the Cldn7-oe group (Fig.
   [101]3J), suggesting alleviation of T cell exhaustion. Conversely, in
   the CMT93 model, Cldn7 knockdown led to increased accumulation of
   CD11b⁺ Ly6G⁺ neutrophils within the tumor (Fig. [102]3O, Supplementary
   Fig. [103]3A–B) and reduced CD8⁺ T cell infiltration (Fig. [104]3P,
   Supplementary Fig. [105]2C). Additionally, IFN-γ and TNF-α production
   by CD8⁺ T cells was significantly impaired in the shCldn7 group (Fig.
   [106]3Q–S, Supplementary Fig. [107]3C), indicating weakened effector
   function. Inhibitory receptor analysis further revealed that PD-1 and
   Tim-3 expression was elevated in CD8⁺ T cells upon Cldn7 knockdown
   (Fig. [108]3T), suggesting enhanced T cell exhaustion. To further
   elucidate the role of tumor-derived Cldn7 in regulating PMN
   infiltration and malignant progression, we performed immune cell
   depletion experiments in the CMT-93 tumor model (Fig. [109]4A). In mice
   with Cldn7 knockdown, depletion of Ly6G⁺ neutrophils using anti-Ly6G
   antibodies abolished the pro-tumor effects of Cldn7 loss, resulting in
   tumor growth comparable to the Cldn7-oe group (Fig. [110]4B–D).
   Moreover, when CD8⁺ T cells were depleted using anti-CD8 antibodies,
   the tumor-promoting effect of Cldn7 knockdown was markedly attenuated,
   restoring tumor growth to levels observed in the shNC + anti-CD8
   control group (Fig. [111]4I–K). Flow cytometry showed that Ly6G⁺ cell
   depletion in Cldn7-deficient tumors significantly enhanced CD8⁺ T cell
   activation, as evidenced by increased IFN-γ (Fig. [112]4E) and TNF-α
   (Fig. [113]4F–H) expression. In summary, loss of Cldn7 facilitates CRC
   progression by suppressing CD8⁺ T cell cytotoxicity in a PMN-dependent
   manner. These findings highlight Cldn7 as a key modulator of the immune
   microenvironment in CRC and suggest its potential as a therapeutic
   target for restoring anti-tumor immunity.

Fig. 3. Cldn7 deficiency promotes neutrophil accumulation in the tumor
microenvironment of tumor-bearing mice.

   [114]Fig. 3
   [115]Open in a new tab

   (A–D) In the MC38 tumor model, Cldn7 overexpression (Cldn7⁺⁺)
   significantly reduced tumor volume (B), tumor weight (C), and prolonged
   mouse survival (D) compared to the control group (Ctrl); A shows
   representative tumor images. (n = 5 per group). Flow cytometry analysis
   shows that Cldn7 overexpression significantly decreased the proportion
   of CD11b⁺ Ly6G⁺ neutrophils in the tumor tissue (E), while increasing
   the infiltration of CD8⁺ T cells (F). Further analysis revealed that
   Cldn7 overexpression significantly increased IFN-γ (G, H) and TNF-α (I)
   production by CD8⁺ T cells. J Inhibitory receptor analysis shows that
   Cldn7 overexpression reduces the expression of PD-1 and Tim-3 on CD8⁺ T
   cells, suggested reduced T cell exhaustion. K–N In the CMT93 model,
   Cldn7 knockdown (shCldn7) promotes tumor growth (L), increases tumor
   weight (N), and significantly shortens mouse survival (M); K shows
   representative tumor images. Cldn7 knockdown leads to an increased
   proportion of CD11b⁺ Ly6G⁺ neutrophils in the tumor tissue (O), while
   decreasing CD8⁺ T cell infiltration (P). Compared to the shNC control
   group, shCldn7 significantly reduces IFN-γ (Q, R) and TNF-α (S)
   production by CD8⁺ T cells. T Inhibitory receptor analysis shows that
   Cldn7 knockdown increases PD-1 and Tim-3 expression levels, suggesting
   enhanced T cell exhaustion. Data are presented as mean ± SEM, with
   statistical significance indicated as follows: *p < 0.05, **p < 0.01,
   ***p < 0.001, ****p < 0.0001.

Fig. 4. Neutrophil depletion partially restores CD8^+ T cell function and
inhibits tumor progression in CLDN7-knockdown tumors.

   [116]Fig. 4
   [117]Open in a new tab

   A Schematic of the experimental timeline. C57BL/6J mice (n = 5 per
   group) bearing subcutaneous CMT93 tumors (transfected with shNC or
   shCldn7) were treated intraperitoneally with anti-CD8 or anti-Ly6G
   antibodies every 4 days starting on day 5 post-inoculation. B Tumor
   growth curves showing that Ly6G⁺ neutrophil depletion significantly
   reduced tumor volume in the shCldn7 group. Representative tumor images
   are shown on the right. C Tumor weights on day 21; no significant
   difference was observed between groups. D Flow cytometry plots
   validating effective depletion of CD11b⁺Ly6G⁺ neutrophils after
   anti-Ly6G antibody treatment. E, F Representative flow cytometry plots
   and quantification of IFN-γ⁺CD8⁺ T cells. Anti-Ly6G treatment
   significantly increased the frequency of IFN-γ⁺ CD8⁺ T cells in the
   shCldn7 group. G, H Representative plots and quantification of
   TNF-α⁺CD8⁺ T cells. shCldn7 + anti-Ly6G tumors showed restored TNF-α
   production comparable to controls. I Tumor growth curves after CD8⁺ T
   cell depletion showing no significant difference between shNC and
   shCldn7 groups. Representative tumor images are shown. J Tumor weights
   on day 21 after CD8⁺ T cell depletion; no significant difference. K
   Flow cytometry confirming efficient depletion of CD3⁺CD8⁺ T cells by
   anti-CD8 antibody. Data are presented as mean ± SEM (n = 5 mice per
   group). Tumor growth curves were analyzed using two-way ANOVA with
   Bonferroni post hoc test. Tumor weight and flow cytometry data were
   analyzed using an unpaired two-tailed Student’s t test. P < 0.05 was
   considered statistically significant. ns， not significant; p < 0.05;
   p < 0.01.

Cldn7 deficiency mediates PMN recruitment and phenotypic conversion through
the NF-κB/CXCL1 pathway

   To further investigate the effect of tumor-derived Cldn7 on the
   recruitment of PMN, we performed transcriptome sequencing on the
   Ctrl/Cldn7-oe mc38 cell lines. The results showed that Cldn7
   overexpression significantly downregulated the expression of several
   chemokines related to PMN recruitment, including Cxcl1, Ccl2, Cxcl10
   and Ccl7 (Fig. [118]5A). Further ELISA validation demonstrated that
   CXCL1 secretion was significantly reduced in MC38 Cldn7-overexpressing
   cells, while it was significantly increased upon Cldn7 knockdown (Fig.
   [119]5B–D). Additionally, qPCR results confirmed that Cldn7 negatively
   regulates the expression of these chemokines in various CRC cell lines
   MC38, CMT93, and HCT116 (Fig. [120]5E–G). The in vitro neutrophil
   chemotaxis assay showed that conditioned medium (CM) from Cldn7-low
   expression cells significantly enhanced PMN migration (Fig. [121]5H,
   I), with no obvious impact on PMN apoptosis (Supplementary Fig.
   [122]4A). This suggests that the increased PMN infiltration in CRC
   primarily results from enhanced chemotaxis due to the downregulation of
   tumor-derived Cldn7. Functional experiments further demonstrated that
   PMNs induced by Cldn7 deficiency significantly inhibited CD8⁺ T cell
   proliferation and the secretion of IFN-γ and TNF-α (Fig. [123]5J–L).
   These results indicate that Cldn7-deficient CRC cells promote PMN
   recruitment and induce their conversion to a pro-tumor phenotype,
   thereby creating an immunosuppressive microenvironment that enhances
   CRC progression by inducing CD8⁺ T cell exhaustion. Next, we performed
   further analysis of transcriptome data from MC38 Cldn7-oe/sh cell
   lines. Gene Set Enrichment Analysis (GSEA) revealed that the NF-κB
   signaling pathway was significantly suppressed in Cldn7-overexpressing
   cells, suggesting that Cldn7 may exert its effects by inhibiting NF-κB
   pathway activation (Fig. [124]6A, Supplementary Fig. [125]5A, B).
   Further Western blot experiments showed that NF-κB activity was
   elevated in Cldn7 knockdown CMT93 cells (Fig. [126]6B, C), while
   overexpression of Cldn7 inhibited this effect (Fig. [127]6D, E).
   Migration assays showed that CM from Cldn7 knockdown CMT93 and HCT116
   cells significantly enhanced PMN migration, an effect that could be
   partially reversed by the NF-κB inhibitor PDTC or CXCL1-neutralizing
   antibodies. Conversely, CM from Cldn7-oe MC38 cells significantly
   suppressed PMN migration, even under LPS-induced NF-κB activation
   conditions (Fig. [128]6F–H). These results suggest that Cldn7
   negatively regulates PMN recruitment by inhibiting NF-κB-driven
   chemokine secretion (e.g., CXCL1). Further in vitro co-culture
   experiments showed that PMNs treated with CM from Cldn7 knockdown cells
   significantly inhibited CD8⁺ T cell proliferation (Fig. [129]6I) as
   well as the secretion of cytokines such as IFN-γ (Fig. [130]6J) and
   TNF-α (Fig. [131]6K). The immunosuppressive effect on CD8⁺ T cells
   could be partially reversed by PDTC or CXCL1-neutralizing antibodies,
   further confirming the key role of the NF-κB/CXCL1 axis in this
   process. Additionally, Cldn7 knockdown also induced an increase in
   PD-L1 expression on PMN surfaces, an effect that could be reversed by
   PDTC or CXCL1-neutralizing antibodies (Fig. [132]6L, M). Even under
   LPS-induced NF-κB activation conditions, Cldn7-oe still suppressed
   PD-L1 expression (Fig. [133]6N), further suggesting that Cldn7
   modulates PMN’s immunosuppressive phenotype through negative regulation
   of the NF-κB/CXCL1 axis.

Fig. 5. Cldn7 deficiency recruits PMN aggregation and promotes their
phenotypic transformation.

   [134]Fig. 5
   [135]Open in a new tab

   A Heatmap of RNA-seq data showing that overexpression of Cldn7
   (Cldn7-oe) in MC38 cells significantly downregulates the expression of
   several chemokines involved in neutrophil recruitment, including
   CX3CL1, CXCL10, CCL7, CXCL1, and CCL2, compared to control (ctrl) cells
   (n = 3 per group). B–D ELISA analysis of CXCL1 secretion in MC38,
   CMT93, and HCT116 models. Cldn7 overexpression markedly reduces CXCL1
   levels, while Cldn7 knockdown (shCldn7) increases it secretion. E–G
   qPCR analysis of chemokine expression in cell lines with different
   levels of Cldn7 expression confirms that Cldn7 overexpression
   suppresses genes such as CX3CL1, CXCL10, and CCL7. H Schematic of the
   neutrophil migration assay: neutrophils were exposed to conditioned
   media (CM) from tumor cells with varying Cldn7 expression to assess
   their migratory response. I Quantification of neutrophil migration in
   response to CM derived from MC38, CMT93, and HCT116 cells with
   different Cldn7 levels. High Cldn7 expression impairs neutrophil
   migration, whereas low Cldn7 expression enhances it. J In vitro
   co-culture assay: CD8^+ T cells were cultured with neutrophils (T + NE)
   in the presence of CM from either shNC or shCldn7 tumor cells.
   CFSE-based proliferation analysis revealed that CM from Cldn7-deficient
   cells suppresses CD8^+ T cell proliferation. K, L Neutrophils
   pre-conditioned with CM from Cldn7-deficient cells reduced the
   proportion of IFN-γ^+CD8^+ T cells and TNF-α^+ CD8 +T cells, suggesting
   a shift toward an immunosuppressive neutrophil phenotype.

Fig. 6. Cldn7 modulates the NF-kappa B/CXCL1 axis to influence neutrophil
recruitment and CD8^+ T cell activation.

   [136]Fig. 6
   [137]Open in a new tab

   A Gene Set Enrichment Analysis (GSEA) showed that Cldn7 overexpression
   is associated with suppression of NF-κB signaling pathway activity.
   Western blot analysis demonstrated a negative correlation between Cldn7
   expression and the levels of key NF-κB signaling proteins, including
   phospho-p65 (P-p65) and p65, in CMT-93 (B, C) and MC38 (D, E) cells. In
   CMT-93 (F), HCT116 (G), and MC38 (H) cells, CD8+ T cells were
   co-cultured with neutrophils and treated with either NF-κB pathway
   activators or inhibitors. The results showed that Cldn7 deficiency
   suppressed CD8+ T cell proliferation and activation, as indicated by
   the proportions of TNF-α⁺ and IFN-γ⁺ CD8+ T cells. These effects were
   partially reversed by NF-κB activators and enhanced by NF-κB
   inhibitors. Neutrophil migration assays revealed that Cldn7
   overexpression inhibited neutrophil migration in response to
   conditioned media from CMT-93 (I), HCT116 (J), and MC38 (K) cells. This
   inhibitory effect was modulated by the NF-κB pathway: NF-κB activators
   restored neutrophil migration, while inhibitors further enhanced the
   suppression. L–N Analysis of PD-L1 expression in neutrophils cultured
   with conditioned media from CMT-93, HCT116, and MC38 cells indicated
   that Cldn7 knockdown increased of PD-L1 expression in neutrophils.

Tumor-derived Cldn7 regulates metabolic reprogramming of neutrophils in the
CRC microenvironment

   To investigate how tumor-derived Cldn7 expression modulates neutrophil
   function and contributes to immune suppression in CRC, we performed
   bulk RNA sequencing on CD11b^+Ly6G^+ neutrophils sorted from tumor
   tissues of CMT93 tumor-bearing mice with either shNC or shCldn7. The
   results revealed that in neutrophils from the shCldn7 group, genes
   associated with metabolic reprogramming and immunosuppression were
   significantly upregulated, including anti-apoptotic genes such as
   Argef2, Hmox1, Higd1a, Arg1, and Bcl2 (Fig. [138]7A). Gene Set
   Enrichment Analysis (GSEA) further indicated enhanced metabolic
   activity in tumor-derived neutrophils from the shCldn7 group, involving
   key pathways such as hypoxia, oxidative stress, glycolysis, and the
   reactive oxygen species (ROS) pathway (Fig. [139]7B).To validate the
   effect of Cldn7 on neutrophil metabolism, we established Cldn7
   knockdown and overexpression models in CMT-93, HCT116, and MC38 cell
   lines, and collected their conditioned media (CM) for co-culture with
   in vitro–sorted neutrophils. CM from Cldn7 knockdown cells
   significantly increased lactate production in neutrophils, an effect
   that could be reversed by the NF-κB pathway inhibitor (PDTC).
   Conversely, CM from Cldn7-overexpressing cells suppressed lactate
   production, and this suppression was counteracted by the NF-κB
   activator (LPS) (Fig. [140]7C–E). qPCR analysis revealed that
   neutrophils cultured in Cldn7 knockdown tumor CM exhibited marked
   upregulation of genes associated with glycolysis, anti-apoptosis, and
   immunosuppression (Fig. [141]7F). Moreover, extracellular acidification
   rate (ECAR) assays confirmed that CM from Cldn7 knockdown tumors
   promoted increased glycolytic activity in neutrophils (Fig. [142]7G–J).
   In parallel, Western blot analysis showed significantly elevated PD-L1
   expression in neutrophils under Cldn7 knockdown conditions, indicating
   a more immunosuppressive phenotype (Fig. [143]7K, L). To further
   explore whether metabolic reprogramming regulates neutrophil immune
   function via epigenetic mechanisms, we assessed histone lactylation
   levels. Neutrophils under Cldn7 knockdown conditions exhibited
   increased levels of Pan-Kla and H3K18la, both of which were markedly
   reduced by treatment with the glycolysis inhibitor 2-DG. In contrast,
   CM from Cldn7-overexpressing cells suppressed histone lactylation in
   neutrophils (Fig. [144]7M, N). In summary, these results demonstrate
   that tumor-derived Cldn7 modulates neutrophil glycolytic metabolism via
   the NF-κB signaling pathway and further regulates their
   immunosuppressive phenotype through histone lactylation. This mechanism
   highlights a novel role for Cldn7 in orchestrating immune modulation
   within the CRC tumor microenvironment.

Fig. 7. Tumor-derived Cldn7 modulates metabolic reprogramming and
immune-suppressive phenotype of neutrophils in the CRC microenvironment.

   [145]Fig. 7
   [146]Open in a new tab

   A RNA expression analysis of neutrophils (CD11b^+ Ly6G^+) isolated from
   the tumor microenvironment of Cldn7-overexpressing and Cldn7-shRNA
   tumors revealed differential expression of genes associated with
   metabolic reprogramming and immune suppression. B Gene ontology (GO)
   enrichment analysis indicated significant enrichment of pathways such
   as oxidative phosphorylation and TNF signaling in neutrophils from
   Cldn7-shRNA tumors. Lactate production in neutrophils co-cultured with
   conditioned media (CM) from CMT-93 (C), HCT116 (D), and MC38 (E) tumor
   cell lines was significantly increased in the Cldn7-shRNA groups, which
   was reversed by treatment with the NF-κB pathway inhibitor PDTC (C, D).
   F qPCR analysis of neutrophils after co-culture with tumor CM showed
   that glycolysis-related and anti-apoptotic genes were markedly
   upregulated under Cldn7-shRNA conditions. G–J ECAR (extracellular
   acidification rate) measurements of neutrophils cultured in CM from
   CMT-93 (G), HCT116 (I), and HCT116-Cldn7-shRNA (J) demonstrated
   enhanced glycolytic activity in the Cldn7-shRNA groups. K Western blot
   analysis showed that PD-L1 protein expression was elevated in
   neutrophils exposed to CM from Cldn7-shRNA tumor cells. L
   Quantification of PD-L1 expression confirmed significant upregulation
   in neutrophils treated with CM from Cldn7-shRNA tumors. M, N
   Tumor-derived Cldn7 regulates histone lactylation to mediate neutrophil
   metabolic reprogramming. Western blot analysis showed increased levels
   of histone lactylation markers, including Pan-Kla and H3K18la, in
   neutrophils co-cultured with CM from Cldn7-overexpressing MC38 cells
   (M). Conversely, CM from Cldn7 knockdown CMT-93 cells also led to
   elevated Pan-Kla and H3K18la expression in neutrophils, which was
   reversed by treatment with the glycolysis inhibitor 2-DG (N). These
   results suggest that tumor-derived Cldn7 alters neutrophil metabolic
   status through histone lactylation, thereby promoting their
   immune-suppressive phenotype.

Overexpression of Cldn7 enhances the efficacy of anti-PD-1 therapy in a
subcutaneous tumor model of CRC cells

   Based on in vivo and in vitro evidence showing that Cldn7 deficiency
   induces immune evasion by promoting PMN aggregation and their
   conversion to a pro-tumor phenotype, we aimed to investigate whether
   increasing Cldn7 expression could enhance the response to
   immunotherapy. In this experiment, MC38 and CT26 cells were injected
   subcutaneously into immunocompetent mice, which were then divided into
   four groups: control (Ctrl), anti-PD-1 treatment alone, Cldn7-oe, and
   combination treatment with Cldn7 overexpression and anti-PD-1
   (Cldn7-oe + anti-PD-1). Compared to the other groups, tumor growth was
   significantly reduced in the Cldn7-oe + anti-PD-1 group (Fig. [147]8A,
   D). Survival analysis revealed a significant prolongation of survival
   time in the combination treatment group (Fig. [148]8B, E), accompanied
   by a marked reduction in tumor weight (Fig. [149]8C, F). Flow cytometry
   analysis showed that Cldn7 overexpression combined with anti–PD-1
   therapy significantly reduced the infiltration of CD11b⁺Ly6G⁺
   neutrophils (Fig. [150]8G, K and Supplementary Fig. [151]7A, D), while
   notably increasing CD8⁺ T cell infiltration, particularly the
   proportion of IFN-γ⁺ and TNF-α⁺ CD8⁺ T cells (Fig. [152]8H–J, L–N).
   Moreover, this combination treatment significantly enhanced the
   cytotoxic functionality of CD8⁺ T cells, as evidenced by elevated
   levels of effector cytokine production. In parallel, the expression of
   exhaustion markers such as PD-1 and TIM-3 on CD8⁺ T cells was markedly
   reduced, indicating a substantial improvement in T cell functional
   status and reinforcing their anti-tumor efficacy (Supplementary Fig.
   [153]7B, C, E, F). TUNEL staining and Ki67 IHC staining indicated
   increased apoptosis and reduced proliferation in the tumors of the
   combination treatment group (Fig. [154]8O, P).

Fig. 8. Cldn7 overexpression enhances the therapeutic effect of PD-1 antibody
in MC38 and CT26 tumor models.

   [155]Fig. 8
   [156]Open in a new tab

   Tumor volume growth curves of MC38 (A) and CT26 (D) tumor-bearing mice
   under different treatment conditions, including Vector,
   Vector + anti-PD-1, Cldn7 overexpression (Cldn7-oe), and
   Cldn7-oe + anti-PD-1 groups. Tumor volumes were measured on days 7, 11,
   14, 18, and 21. Cldn7-oe significantly suppressed tumor growth, and
   this effect was further enhanced by anti-PD-1 treatment. Survival
   curves of MC38 (B) and CT26 (E) tumor-bearing mice across the different
   treatment groups. The Cldn7-oe + anti-PD-1 group exhibited
   significantly prolonged survival compared to controls. Final tumor
   weights of MC38 (C) and CT26 (F) models at the end of the experiment.
   Combined Cldn7 overexpression and PD-1 antibody treatment led to a
   marked reduction in tumor burden. Flow cytometry analysis of the
   proportion of CD11b⁺Ly6G⁺ myeloid-derived suppressor cells (MDSCs)
   within CD45⁺ cells in MC38 (G) and CT26 (K) tumor tissues. MDSC
   infiltration was significantly reduced in the Cldn7-oe + anti-PD-1
   group. Flow cytometric analysis of CD8⁺ T cells within CD3⁺ T cells in
   MC38 (H) and CT26 (L) tumor tissues. Cldn7 overexpression significantly
   increased CD8⁺ T cell infiltration, particularly in the combination
   therapy group. Proportion of IFN-γ⁺ cells within CD8⁺ T cells in MC38
   (I) and CT26 (M) models, showing that Cldn7 overexpression combined
   with anti-PD-1 treatment significantly enhanced CD8⁺ T cell activation.
   Proportion of TNF-α⁺ cells within CD8⁺ T cells in MC38 (J) and CT26 (N)
   tumors. TNF-α expression was notably upregulated in the
   Cldn7-oe + anti-PD-1 group, indicating enhanced CD8⁺ T cell effector
   function. TUNEL (O) and Ki67 (P) immunohistochemical staining of MC38
   tumor tissues to assess tumor cell apoptosis and proliferation. Results
   showed increased apoptosis and decreased proliferation in the
   Cldn7-oe + anti-PD-1 group. Statistical significance: *p < 0.05,
   **p < 0.01, ***p < 0.001, ****p < 0.0001.

   In conclusion, these findings demonstrate that Cldn7 overexpression
   enhances the efficacy of anti-PD-1 therapy in CRC by promoting
   anti-tumor immunity. In both MC38 and CT26 models, Cldn7 overexpression
   combined with anti-PD-1 treatment significantly inhibits tumor growth,
   reduces tumor weight, and prolongs survival. This combined treatment
   decreases the infiltration of immunosuppressive CD11b^+ Ly6G^+
   neutrophils while increasing CD8^+ T cell infiltration and activation,
   as evidenced by higher IFN-γ and TNF-α production. These results
   suggest that targeting Cldn7 may help create a more immune-supportive
   microenvironment, thereby enhancing the effectiveness of immune
   checkpoint therapy in CRC (Fig. [157]9)

Fig. 9. Model of colorectal cancer showing that under conditions of Cldn7
deficiency, recruited neutrophils convert to a pro-tumor phenotype, further
inhibiting CD8^+ T cell function.

   [158]Fig. 9
   [159]Open in a new tab

   Left (Cldn7 high): With high Cldn7 expression (Cldn7 overexpression,
   Cldn7-oe), the NF-κB signaling pathway is suppressed, leading to
   reduced secretion of neutrophil-attracting chemokines such as CXCL1 and
   CCL12, thereby limiting neutrophil recruitment to the tumor site. The
   reduction in neutrophils prevents suppression of CD8^+ T cells and also
   limits the conversion of neutrophils to a pro-tumor phenotype. This
   allows CD8^+ T cells to exert their cytotoxic functions effectively,
   producing IFN-γ and TNF-α, which enhances the anti-tumor immune
   response and leads to colorectal cancer regression. Right (Cldn7 low):
   Under conditions of Cldn7 deficiency (Cldn7 knockdown, Cldn7-sh), the
   NF-κB signaling pathway is activated, leading to increased secretion of
   CXCL1 and CCL12, which promotes neutrophil recruitment to the tumor
   site. These recruited neutrophils convert to a pro-tumor phenotype and
   suppress CD8^+ T cell activation and function (e.g., reduced production
   of IFN-γ and TNF-α), thereby creating an immunosuppressive
   microenvironment that promotes colorectal cancer progression. This
   model suggests that high CLDN7 expression indirectly supports CD8^+ T
   cell anti-tumor function by inhibiting neutrophil recruitment and their
   conversion to a pro-tumor phenotype. In contrast, Cldn7 deficiency
   promotes pro-tumor neutrophil recruitment and suppresses CD8^+ T cell
   function, creating an immunosuppressive microenvironment conducive to
   colorectal cancer growth. This figure was created using BioRender.com.

Discussion

   Colorectal cancer (CRC) is a common malignant tumor, ranking third in
   incidence (6.1%) and second in mortality (9.4%) among all cancers
   [[160]32]. Due to the diversity and heterogeneity of CRC, effective
   treatment strategies have been relatively limited in recent years,
   aside from surgical resection. With the revolutionary advancements
   brought by immunotherapy in postoperative treatment, combined with
   radiotherapy, chemotherapy, and targeted therapy, CRC treatment has
   entered a new phase [[161]33]. Increasing evidence suggests that
   tumor-associated TANs play a significant role in modulating antitumor
   responses [[162]20, [163]34, [164]35]. Due to their high functional
   heterogeneity, pro-tumor TANs are considered a critical factor limiting
   the efficacy of cancer immunotherapy [[165]36–[166]38]. However, the
   lack of markers or targets to specifically identify pro-tumor TANs
   poses a challenge in developing strategies to selectively eliminate
   these cell [[167]21, [168]39–[169]41].

   Neutrophils undergo profound metabolic adaptations within the tumor,
   ultimately leading to distinct pro-tumor or anti-tumor phenotypes
   [[170]42–[171]44]. This study thoroughly investigates how tumor-derived
   Cldn7 alters the CRC tumor microenvironment, inducing functional and
   metabolic reprogramming of polymorphonuclear neutrophils (PMN), which
   in turn affects CD8^+T cell function and influences the malignant
   progression of CRC. Our previous research, utilizing the
   Cldn7^fl/fl;Villin-CreERT2 mouse model, revealed that the loss of Cldn7
   is associated with exacerbated colonic inflammation and carcinogenesis,
   closely linked to increased neutrophil infiltration and elevated
   chemokine expression [[172]10, [173]28, [174]31]. Furthermore,
   single-cell data analysis of clinical surgical specimens and
   transcriptome sequencing of tumor cells indicated that changes in the
   expression level of tumor-derived Cldn7 can significantly regulate the
   secretion of chemokines related to neutrophil recruitment by tumor
   cells. In vitro experiments confirmed that tumor cells with
   knocked-down Cldn7 expression promote PMN migration by enhancing the
   secretion of related chemokines. Conversely, Cldn7 overexpression
   significantly reduces this chemotactic ability. These findings suggest
   that Cldn7 modulates the directional migration of PMN by regulating the
   expression or activity of chemokines, thereby influencing the dynamics
   of immune cells within the tumor microenvironment.

   Moreover, this study found that changes in Cldn7 expression not only
   affect PMN function but also contribute to reshaping their metabolic
   phenotype. PMN within the CRC microenvironment with low Cldn7
   expression exhibit enhanced glycolytic activity, upregulation of
   anti-apoptotic and immunosuppressive genes, and a shift toward a
   glycolysis-driven functional state through histone lactylation,
   ultimately displaying an immunosuppressive neutrophil phenotype.
   Additionally, neutrophils in the Cldn7-deficient CRC microenvironment
   suppress CD8⁺ T cell function by reducing their proliferative capacity
   and antitumor activity—effects that can be partially reversed by
   inhibiting the NF-κB signaling pathway.

   Although PD-1/PD-L1 blockade therapy has been widely used in clinical
   settings, the fundamental mechanisms underlying the restoration of
   dysfunctional CD8^+ T cells in cancer remain incompletely understood
   [[175]45, [176]46]. Increasing evidence suggests that the heterogeneity
   of dysfunctional CD8^+ T cells in cancer is a major obstacle to the
   success of PD-1/PD-L1 blockade therapy [[177]47, [178]48]. In our
   study, after anti-PD-1 treatment using the MC38 and CT26 mouse
   subcutaneous tumor models, we observed that mice with overexpressed
   Cldn7 exhibited better therapeutic outcomes following anti-PD-1
   treatment, with slower tumor growth rates and significantly prolonged
   survival. These findings indicate that Cldn7 enhances the response to
   PD-1 inhibitors by improving the tumor microenvironment, specifically,
   it reduces the infiltration of immunosuppressive TAN and enhances the
   function of tumor-infiltrating CD8^+ T cells, thereby improving the
   response to PD-1 inhibitors (Fig. [179]9).

   Neutrophils are part of the bone marrow and stromal cell network,
   express PDL1, and drive immune checkpoint engagement and T cell
   exhaustion [[180]44]. In this study, we investigated whether
   tumor-derived Cldn7 deletion recruits PMN infiltration, promoting their
   transition to a pro-tumor phenotype and thereby forming an
   immunosuppressive microenvironment. Through single-cell sequencing,
   clinical sample validation, and in vivo and in vitro experiments, we
   found that Cldn7 deletion leads to increased recruitment of CD11b^+
   Ly6G^+ neutrophils in the CRC microenvironment and accelerates CRC
   malignant progression. Cldn7 deletion indirectly activates the NF-κB
   signaling pathway, resulting in increased secretion of PMN
   recruitment-related chemokines such as CXCL1. Inhibiting the NF-κB
   /CXCL1 axis in Cldn7 deficiency tumor cells can reduce PMN recruitment,
   decrease PD-L1 expression on PMN, and reverse the exhausted phenotype
   of CD8 + T cells, thereby improving the immunosuppressive
   microenvironment. Additionally, Cldn7 overexpression can enhance the
   effectiveness of ICB (immune checkpoint blockade) therapy. Overall, our
   experimental results suggest that Cldn7 deletion plays an initiating
   role and has immunoregulatory functions in CRC malignant progression,
   and targeting related biological processes may enhance CRC
   immunotherapy efficacy in the future.

Conclusion

   Our experimental results highlight the pivotal role of Cldn7 in CRC
   progression by modulating the behavior of immune cells, particularly
   TAN and CD8^+ T cells, within the tumor microenvironment, thereby
   influencing tumor immune evasion and antitumor immune responses. This
   provides a deeper understanding of how tumors promote their survival
   and progression by regulating local immune cells. The immunosuppressive
   TAN formed under the condition of Cldn7 deficiency will become the
   focus and challenge of subsequent research.

Material and methods

Ethics approval and consent to participate

   All methods in this study were performed in accordance with the
   relevant guidelines and regulations. The study was approved by the
   Ethics Committee of Beijing Shijitan Hospital Institutional Review
   Board (approval number: sjtkyll-1x-2021(105)). Tumor specimens from
   colorectal cancer radical resection were collected at the Department of
   Gastrointestinal Oncology, Beijing Shijitan Hospital, Capital Medical
   University, for single-cell sequencing or embedding in 4%
   paraformaldehyde. After obtaining informed consent, whole blood samples
   were collected from healthy volunteers for neutrophil separation. All
   animal experiments were conducted in compliance with the ARRIVE
   guidelines and approved by the Animal Ethics Committee of Beijing
   Shijitan Hospital Institutional Review Board (approval number:
   sjtkyll-1x-2021(105)). Informed consent was obtained from all
   participants involved in the study. For any identifiable human images
   included in this article, written informed consent for publication of
   the images was obtained from the participants, which is separate from
   the consent to participate.

Cell cultures

   MC38, CMT-93, CT26, and HCT116 cells were obtained from the American
   Type Culture Collection (ATCC, USA). All cell lines were maintained in
   high-glucose DMEM (GMEM, GIBCO, USA) supplemented with 10% fetal bovine
   serum (GIBCO) and penicillin/streptomycin, at 37 °C with 5% CO[2].
   Cells were routinely tested for mycoplasma contamination. For immune
   cell cultures, primary cells were isolated from peripheral sources
   (such as blood or bone marrow) or spleen, and immediately used for
   functional analyses, such as neutrophil-T cell co-cultures or
   neutrophil migration, with detailed methods found in the supplementary
   materials.

Statistical analysis

   Flow cytometry data were analyzed using FlowJo software (V.12; Tree
   Star), and graphs and statistical analyses were generated using
   GraphPad Prism (V.9). Single-cell and bulk transcriptome data analysis
   were conducted using R 4.2.0. One-way or two-way analysis of variance
   (ANOVA) was used for comparisons among more than two groups, while an
   unpaired two-tailed t-test was used for comparisons between only two
   groups. A p-value of <0.05 was considered statistically significant
   (*p < 0.05, **p < 0.01, ***p < 0.001). Mouse survival was assessed
   using the Kaplan–Meier method and analyzed using the Mantel–Cox
   log-rank test. All experiments were performed at least three times,
   with n representing biological replicates.

Supplementary information

   [181]Supplementary materials^ (2.9MB, pdf)
   [182]Original data for western blot^ (651KB, pdf)
   [183]Original data for qpcr^ (11.4KB, xlsx)

Author contributions

   Xiaoqing Liang, Dajin Yuan, Songyun Zhao and Jianbo Zhou contributed
   equally to this work. Xiaoqing Liang and Dajin Yuan designed the study
   and conducted the experiments. Songyun Zhao and Jianbo Zhou primarily
   performed single-cell data analysis and processing. Kun Wang, Xiaoli
   Liu, Yin Liu, and Huimin Li assisted with data collection and
   experimental procedures. Mengdi Hao, Wenbin Huang and Wenjie Li
   contributed to data interpretation and statistical analysis. Lei Ding
   supervised the research and provided critical insights for manuscript
   preparation. Lei Ding also conceptualization and provided valuable
   infrastructure, guidance, and additional funding the manuscript.

Funding

   This work was supported by grants from the National Natural Science
   Foundation of China (Grant # 82170525 and 82471761).

Data availability

   The TCGA data used in this study were downloaded from The Cancer Genome
   Atlas (TCGA) database ([184]https://portal.gdc.cancer.gov/). All other
   datasets generated and/or analyzed during the current study are
   available from the corresponding author upon reasonable request.

Competing interests

   The authors declare no competing interests.

Footnotes

   Edited by Professor Hans-Uwe Simon

   Publisher’s note Springer Nature remains neutral with regard to
   jurisdictional claims in published maps and institutional affiliations.

   These authors contributed equally: Xiaoqing Liang, Dajin Yuan, Songyun
   Zhao, Jianbo Zhou.

Supplementary information

   The online version contains supplementary material available at
   10.1038/s41419-025-08064-3.

References