Semaphorin 6D drives anti-tumor type I interferon responses to reprogram the tumor microenvironment in colorectal cancer
Wei Shi, Fan Zhang, Wei-Qing Sun, Li-Qi Liang, Shamalagowri Krishnan
TL;DR
This study shows that Semaphorin 6D (SEMA6D) is a tumor suppressor in colorectal cancer that enhances anti-tumor immunity and could improve treatment strategies.
Contribution
The study identifies SEMA6D as a tumor suppressor in CRC and reveals its role in activating type I interferon signaling to reprogram the tumor microenvironment.
Findings
SEMA6D is underexpressed in CRC and associated with poor prognosis due to promoter hypermethylation.
SEMA6D enhances anti-tumor immunity by promoting T cell infiltration and suppressing tumor growth and metastasis.
Demethylating agents restore SEMA6D expression and improve immunotherapy efficacy in CRC.
Abstract
Colorectal cancer (CRC) persists as the third most prevalent cause of cancer-associated mortality worldwide, largely due to late diagnosis, limited treatment efficacy, and poor response to immunotherapy. However, the underlying molecular mechanisms remain incompletely characterized. This study identified Semaphorin 6D (SEMA6D) as a potential tumor suppressor that was markedly underexpressed in CRC and associated with poor prognosis. Promoter hypermethylation emerges as the primary mechanism underlying its transcriptional silencing, with notably low expression observed in CIMP-H, MSI-H, CMS4 and CMS1 subtypes. Functional experiments demonstrated that SEMA6D overexpression significantly attenuated cellular proliferation, epithelial-mesenchymal transition (EMT), and migratory capacity in vitro, while concurrently suppressing tumor growth and metastatic spread in vivo. Conversely, SEMA6D…
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Figure 9- —https://doi.org/10.13039/501100001809National Natural Science Foundation of China (National Science Foundation of China)
- —the Joint Special Funds for the Department of Science and Technology of Yunnan Province – Kunming Medical University [No.: 2018FE001(-151)]
- —CAS Youth Innovation Promotion Association, Yunnan Revitalization Talent Support Program in Yunnan Province and Nanjing Program for High-Level Talent of Yunnan Minzu University.
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Taxonomy
TopicsAxon Guidance and Neuronal Signaling · Hippo pathway signaling and YAP/TAZ · Angiogenesis and VEGF in Cancer
Introduction
Colorectal cancer (CRC) ranks among the most prevalent and lethal malignancies globally, with over 1.85 million new cases annually. A concerning epidemiological shift is the rising morbidity and mortality rates among younger populations, particularly in industrialized nations [1, 2]. Despite advancements in current therapeutic strategies, efficacy remains limited [3], particularly as over 60% of patients are diagnosed at late stages, resulting in an unfavorable prognosis [4], and nearly 40% of surgically resected cases relapse within five years [5]. Accumulating evidence suggests that CRC progression involves dynamic dysregulation of gene expression profiles, elevated mutational burden, and profound molecular heterogeneity [6], yet the molecular drivers of CRC progression remain elusive.
Semaphorins are secreted and transmembrane glycoproteins characterized by a conserved extracellular sema domain mediating ligand-receptor interactions. Initially identified as axon guidance regulators acting through plexin receptors [7, 8], semaphorins are now recognized to regulate diverse biological processes, including angiogenesis [9], cardiac morphogenesis [10, 11], immunomodulation, and tumorigenesis. Semaphorin 6D (SEMA6D), a class 6 transmembrane semaphorin, exhibits bidirectional signaling: forward signaling through Plexin-A1 regulates cardiac development and immune homeostasis, while reverse signaling recruits c-Abl kinase to mediate cytoskeletal remodeling during avian cardiogenesis [12]. Beyond embryogenesis, SEMA6D contributes to immunoregulation [13], tissue remodeling [14] and tumorigenesis [15]. Notably, Kang et al. confirmed the necessity of SEMA6D for the differentiation of intestinal CX3CR1^hi^ macrophages to prevent colitis [16]. However, the mechanistic and clinical relevance of SEMA6D in colorectal carcinogenesis remains undefined, necessitating systematic exploration.
Interferon (IFN) signaling regulates antiviral defense [17, 18], cell proliferation, and apoptosis, thereby orchestrating the immune response. Attenuation of IFN pathways promotes tumorigenesis, highlighting their tumor-suppressive functions [19], while IFN-I/II/III signaling cascades (IFNα-λ) in the TME orchestrate context-dependent immunoregulatory effects, including enhanced cytotoxic T lymphocyte (CTL)-mediated tumor clearance. As principal adaptive immune effectors, T cells mediate direct cytolytic activity against neoplastic populations [20–22]. IFN-Is activate the IFN-stimulated gene factor 3 (ISGF3) complex (IRF9/p-STAT1/STAT2), which translocates to the nucleus, binds interferon-sensitive response elements (ISREs), and initiates interferon-stimulated genes (ISGs) transcription [23]. Impaired IFN-I signaling promotes oncogenesis [24], whereas IFN-I-primed dendritic cell maturation enables antitumor T cell activation [25]. IFN-I secretion by tumor cells and plasmacytoid dendritic cells initiates an antiviral inflammatory response, enhancing immune surveillance. Both T cells and tumor cells are critical targets for IFN-I-mediated antitumor immunity.
This study identifies SEMA6D as a novel tumor suppressor in CRC, silenced by promoter hypermethylation and associated with poor prognosis. SEMA6D overexpression suppresses tumor growth and metastasis while promoting T-cell infiltration into the tumor microenvironment. Mechanistically, Plexin A4 mediates the SEMA6D-IRF9 interaction, activating the type I interferon signaling cascade and reprogramming the tumor microenvironment. Furthermore, we demonstrate that SEMA6D expression can be restored by hypomethylating agents, which in turn potentiates immunotherapy efficacy. These findings uncover the Plexin A4/SEMA6D/IRF9 axis as a critical regulator of CRC progression and a promising target for epigenetic and immunomodulatory intervention.
Results
SEMA6D downregulation indicates tumor aggressiveness and poor outcomes of CRC patients
Multi-cohort analyses revealed significant SEMA6D downregulation in CRC. GSE32323 identified SEMA6D as the most significantly downregulated semaphorin family member in CRC tissues versus normal mucosa (P < 0.0001, Fig. 1A, B). In GSE28702, SEMA6D was further reduced in metastatic lesions relative to primary tumors (Fig. 1C). Consistent downregulation was confirmed across 15 Oncomine cohorts and UALCAN analysis (Figs. S1A–I and S2A), independent of tumor stage (Fig. S2B). High SEMA6D expression linked with prolonged disease-free survival (DFS; Fig. 1D) and trended toward improved overall survival (OS) in GEPIA (P = 0.17, Fig. S2C). Kaplan-Meier analyses demonstrated significantly improved OS and RFS with high SEMA6D expression (Fig. 1E, F).Fig. 1. Reduced SEMA6D expression correlates with poor clinical outcomes in CRC patients.A Heatmap depicting differential expression of vertebrate semaphorins in 17 paired CRC and adjacent normal tissues (GSE32323). B SEMA6D mRNA levels in 17 paired CRC and adjacent normal tissues (GSE32323). C SEMA6D expression in primary (n = 33) versus metastatic CRC lesions (n = 21) (GSE28702). D Disease-free survival (DFS) in the TCGA CRC cohort stratified by SEMA6D expression (high vs. low, n = 135 each). E, F Kaplan-Meier analysis of overall survival (OS; n = 207 high, 607 low) and recurrence-free survival (RFS; n = 295 high, 872 low). G SEMA6D mRNA expression in 19 paired fresh-frozen CRC and normal tissues. H Immunoblot analysis of SEMA6D protein in four paired CRC/normal tissues. I Representative immunohistochemical (IHC) staining of SEMA6D in CRC and adjacent mucosa. Scale bar: 100 µm. J Histoscore distribution of SEMA6D expression in 101 CRC and 80 normal tissues (score: 0–3). K, L Significantly reduced OS (P < 0.001) and disease-free survival (DFS; P < 0.001) in patients with low SEMA6D expression (n = 80) versus high expression (n = 101). Data are presented as mean ± SD of three independent experiments. Statistical significance was determined using Student’s t test. ^^P < 0.01, ^^P < 0.001, ^***^P < 0.0001.
Matched tumor-normal pairs confirmed significant SEMA6D downregulation at mRNA (Fig. 1G) and protein levels (Fig. 1H). Immunohistochemical analysis of 181 CRC specimens demonstrated cytoplasmic SEMA6D localization with reduced tumor staining (histoscores: 0–3; Figs. 1I, J and S2D). SEMA6D downregulation occurred in 44.2% (80/181) of cases (Table 1) and correlated inversely with tumor diameter (P = 0.033), invasion depth (P = 0.005), metastasis (M0; P < 0.001), and TNM stage (P < 0.001). High SEMA6D expression correlated with improved OS (P < 0.001; Fig. 1K) and PFS (P < 0.001; Fig. 1L). Multivariate analysis identified SEMA6D as an independent prognostic predictor for PFS and OS (P < 0.001, Table S4). These findings establish SEMA6D as a tumor suppressor and independent prognostic biomarker in CRC.Table 1. Clinicopathologic characteristics of 181 CRC patients and the correlation with SEMA6D expression.VariablesNo. (%)SEMA6D lowSEMA6D highP valueTotal cases18180 (44.2)101 (55.8)Age (years) <6592 (50.8)46 (25.4)46 (25.4) ≥6589 (49.2)34 (18.8)55 (30.4)0.110Gender Male107 (59.1)46 (25.4)61 (33.7) Female74 (40.9)34 (18.8)40 (22.1)0.694Tumor location Colon97 (53.6)41 (22.7)56 (30.9) Rectum84 (46.4)39 (21.5)45 (24.9)0.574Tumor size (cm) <5102 (56.4)38 (21)64 (35.4) ≥579 (43.6)42 (23.2)37 (20.4)0.033Histology Adenocarcinoma165 (91.2)71 (39.2)94 (52) Mucinous16 (8.8)9 (5.0)7 (3.8)0.309Tumor invasive depth T1-T280 (44.2)26 (14.4)54 (29.8) T3-T4101 (55.8)54 (29.8)47 (26)0.005Lymph node status <182 (45.3)30 (16.6)52 (28.7) ≥199 (56.7)50 (27.6)49 (27.1)0.061Distant metastasis No metastasis108 (59.7)33 (18.2)75 (41.5) Metastasis73 (40.3)47 (26.0)26 (14.4)<0.001AJCC/TNM stage I-II87 (48.1)25 (13.8)62 (34.3) III-IV94 (51.9)55 (30.4)39 (21.5)<0.001Preoperative CA199 (ng/mL) <=35112 (61.9)53 (29.3)59 (32.6) >3569 (38.1)27 (14.9)42 (23.2)0.112Preoperative CEA (ng/mL) ≤5137 (75.7)56 (30.9)81 (44.8) >544 (24.3)24 (13.3)20 (11.0)0.281The numbers in parentheses indicate the percentages of tumors with a specific clinical or pathologic feature for a given SEMA6D subtype.Abbreviation: CA199, carbohydrate antigen 199.CEA, carcinoembryonic antigen.*Statistically significant, P < 0.05.
SEMA6D suppresses CRC cell growth and metastatic phenotype in vitro
qPCR (Fig. S3A) and immunoblotting (Fig. 2A) demonstrated reduced SEMA6D expression in CRC cell lines versus NCM460 normal colonic epithelial cells. Stable SEMA6D-knockdown HCT116 cells and SEMA6D-overexpressing RKO/DLD1 cells were established and validated (Fig. S3B–D and Fig. 2B). SEMA6D depletion enhanced HCT116 proliferation (Fig. 2C), whereas overexpression suppressed DLD1 (Fig. 2D) and RKO viability (Fig. 2E), confirmed by colony formation (Fig. 2F, H) and soft agar assays (Fig. 2G, H). SEMA6D overexpression increased apoptotic populations (Fig. 2I) and upregulated cleaved caspase-3 and cleaved caspase-8 (Fig. 2J), indicating intrinsic apoptotic pathways activation.Fig. 2SEMA6D suppresses CRC proliferation and induces apoptosis in vitro.A Western blot analysis of SEMA6D protein expression in human CRC cell lines versus normal colonic epithelial NCM460 cells. B Validation of SEMA6D knockdown (HCT116) and overexpression (DLD1, RKO) efficiency. C CCK-8 assay demonstrates enhanced proliferation in SEMA6D-depleted HCT116 cells. D, E SEMA6D overexpression suppresses proliferation in DLD1 and RKO cells (CCK-8 assay). F Colony formation assays show reduced clonogenicity in SEMA6D-overexpressing cells (representative images and quantification). G Soft-agar-colony formation assay confirms impaired anchorage-independent growth in SEMA6D-overexpressing cells (left: representative images; right: quantification). H SEMA6D knockdown in HCT116 cells increases colony formation and anchorage-independent growth (left: representative images; right: quantification). I Flow cytometric analysis (Annexin V/PI staining) reveals increased apoptosis in SEMA6D-overexpressing DLD1 and RKO cells (left: representative plots; right: quantification). J Western blot analysis shows elevated cleaved Caspase-3 and Caspase-8 levels in SEMA6D-overexpressing DLD1 and RKO cells. K SEMA6D overexpression alters tumor organoid morphology and number in patient-derived CRC models (left: representative images, scale bar: 200 μm; right: quantification). Data represent mean ± SD of three independent experiments. Statistical significance was determined by two-way ANOVA: ^^P < 0.05, ^^P < 0.01, ^^P < 0.001, ^****^P < 0.0001.
Given the inverse correlation between SEMA6D expression and metastasis, its role in metastatic progression was examined. Transwell assays showed SEMA6D overexpression attenuated migration and invasion, while knockdown enhanced these phenotypes (Fig. 3A). Wound-healing assays confirmed delayed wound closure with overexpression and accelerated repair with depletion (Fig. 3B). Considering the critical role of EMT in metastasis, we evaluated SEMA6D’s regulation of EMT markers [26]. SEMA6D depletion increased N-cadherin and vimentin while reducing E-cadherin, β-catenin and claudin-1 (Fig. 3C–E), with overexpression producing opposite effects (Figs. 3D, E, and S3E). Immunofluorescence confirmed reduced membranous E-cadherin with knockdown and restored epithelial integrity with overexpression (Fig. 3F).Fig. 3SEMA6D suppresses CRC cell migration, invasion, and EMT in vitro.A Migration and invasion assays in SEMA6D-knockdown HCT116 and SEMA6D-overexpressing DLD1/RKO cells. Left: representative images, scale bar: 200 μm. Right: quantification. B Wound healing assay in SEMA6D-depleted HCT116 cells and SEMA6D-overexpressing DLD1/RKO cells. Left: representative images, scale bar: 200 μm. Right: quantification. qPCR analysis of EMT-associated genes in SEMA6D-knockdown HCT116 cells (C) and SEMA6D-overexpressing DLD1 cells (D). E Western blot analysis of EMT markers in SEMA6D-knockdown HCT116, SEMA6D-overexpressing DLD1/RKO cells. F Immunofluorescence staining of E-cadherin (green) in HCT116 (SEMA6D knockdown) and DLD1 (SEMA6D overexpression) cells. Nuclei are counterstained with DAPI (blue). Scale bar, 20 µm. Data represent mean ± SD from three independent experiments. Statistical significance was determined by two-way ANOVA: ^^P < 0.05, ^^P < 0.01, ^^P < 0.001, ^****^P < 0.0001.
These findings were validated in patient-derived CRC organoids, with confirmed SEMA6D modulation (Fig. S4A). SEMA6D overexpression reduced organoid number and size (Fig. 2K), whereas knockdown promoted growth (Fig. S4B, D), and overexpression inhibited expansion (Fig. S4C, E). SEMA6D-silenced organoids exhibited enhanced Matrigel invasion (Fig. S4F). EMT marker analysis in organoids paralleled cell line results (Fig. S4H, I). Collectively, SEMA6D inhibits CRC cell proliferation and metastatic potential in vitro.
SEMA6D suppresses CRC proliferation and metastasis in vivo
To validate SEMA6D tumor-suppressive function in vivo, DLD1 cells overexpressing SEMA6D or control, and HCT116 cells with SEMA6D knockdown or control were subcutaneously implanted into BALB/c nude mice. SEMA6D overexpression markedly reduced tumor volume and weight, while depletion increased these parameters (Figs. 4A–C, and S5A–C). H&E staining revealed decreased cellular density in SEMA6D-overexpressing tumors, with IHC confirming persistent SEMA6D expression and lower Ki-67 positivity. SEMA6D knockdown increased cellularity and Ki67 levels (Figs. 4D and S5D).Fig. 4SEMA6D suppresses colorectal cancer progression in vivo.A Representative images of subcutaneous tumors in nude mice injected with SEMA6D-overexpressing (SEMA6D) or control (Ctrl) CRC cells (n = 8 for each group). B Tumor growth curves. C Tumor weight at endpoint. D Representative H&E and immunohistochemical staining for SEMA6D and Ki-67 in subcutaneous tumors. Scale bar: 100 μm. E Representative images (left) with quantification (right) of pulmonary metastatic nodules in a tail vein injection model (n = 8 per group). Scale bar 100 μm. F Representative images (left) and quantification (right) of hepatic metastatic nodules in an intrasplenic injection model (n = 6 per experimental group). Scale bar: 100 μm. The graphs represent means ± SD. Statistical analysis was performed using Student’s t test. ^*^P < 0.001, ^**^P < 0.0001.
Tail vein and intrasplenic injections established pulmonary and hepatic metastasis models. SEMA6D overexpression significantly inhibited metastatic colonization, with fewer and smaller nodules in the lungs (Fig. 4E) and livers (Fig. 4F), while knockdown enhanced metastatic burden (Fig. S5E–G). These findings demonstrate that SEMA6D suppresses colorectal cancer growth and metastatic dissemination in vivo.
Epigenetic silencing of SEMA6D in CRC via promoter hypermethylation
To elucidate the mechanism underlying SEMA6D downregulation in CRC, promoter methylation was examined. MethPrimer [27] analysis identified two CpG islands within the SEMA6D promoter (Fig. 5A). Methylation-Specific PCR (MSP) revealed pronounced hypermethylation in CRC cell lines and tissues, while normal colonic epithelium exhibited unmethylated profiles (Fig. 5B, C). Bisulfite sequencing of 11 CpG dinucleotides confirmed hypermethylation in CRC cells and specimens (Fig. 5B, C). Treatment with the demethylating agent 5-Aza-2’-deoxycytidine (decitabine, DAC) reduced promoter methylation (Fig. 5D) and restored SEMA6D expression (Fig. 5E).Fig. 5SEMA6D promoter is hypermethylated in CRC cell lines and tissues.A Schematic of CpG clusters in the SEMA6D promoter region identified by MethPrimer analysis. Shaded regions indicate CpG-rich domains. Bisulfite sequencing of CpG islands in the SEMA6D promoter in CRC cell lines (B) and matched CRC tissue pairs (C). Open and filled circles represent unmethylated and methylated CpG sites, respectively. D Methylation-specific PCR (MSP) analysis demonstrating the impact of 5-aza-2’-deoxycytidine (DAC) treatment on SEMA6D promoter methylation status in CRC cell lines. E qRT-PCR analysis of SEMA6D expression following DAC treatment in CRC cell lines. Data represent mean ± SD from three independent experiments. Statistical significance was determined by Two-way ANOVA: ^****^P < 0.0001.
TCGA-COAD analysis demonstrated elevated SEMA6D promoter methylation in CRC, inversely correlating with mRNA expression (Fig. S6A, B). SEMA6D expression was progressively diminished in CpG island methylator phenotype-high (CIMP-H) and microsatellite instability-high (MSI-H) subtypes (Fig. S6C, D). GSE39582 analysis revealed the lowest SEMA6D expression in CMS4, followed by CMS1 (Fig. S6E). CMS1 tumors exhibit MSI-high status and CIMP positivity, while CMS4 tumors display mesenchymal features and promoter hypermethylation. Reduced SEMA6D in CMS4 aligns with its EMT-suppressive role. These findings indicate that promoter hypermethylation mediates SEMA6D silencing in hypermethylated and aggressive CRC subtypes.
Plexin A4 serves as the receptor for SEMA6D to regulate CRC growth and progression
Plexins function as primary semaphorin receptors, coordinating intracellular signaling. Canonically, SEMA6D engages Plexin A1 during cardiac morphogenesis and immune homeostasis [13], while recent evidence demonstrates SEMA6D-Plexin A4 interactions in macrophage polarization and colitis, indicating context-dependent receptor specificity [16].
To delineate the receptor mediating SEMA6D antitumor effects in CRC, systematic analyses were performed. GEPIA analysis revealed significant SEMA6D-Plexin A4 co-expression (Fig. S7A). qPCR and western blot of 19 matched tumor-normal pairs demonstrated PLXNA4 downregulation in tumor (Fig. S7B, C), confirmed in CRC cell lines versus NCM460 (Fig. S7D). Functional validation showed SEMA6D overexpression inhibited clonogenicity, proliferation (Fig. S7E, G, H) and migration (Fig. S7J, L–M) in RKO and DLD1 cells. PlXNA4 knockdown, but not Plexin A1 depletion, reversed these effects. Conversely, Plexin A4 overexpression rescued enhanced proliferation, clonogenicity, and migration in SEMA6D-knockdown HCT116 cells (Fig. S7F, I, K, N). EMT marker analysis confirmed these observations (Fig. S7O–Q). These data establish Plexin A4 as the indispensable receptor mediating SEMA6D-driven inhibition of CRC progression.
SEMA6D activates type I interferon signaling to suppress CRC
To delineate SEMA6D tumor-suppressive mechanisms, RNA-seq analysis of SEMA6D-knockdown (HCT116^shSEMA6D^ vs. HCT116^shNC^) and -overexpressing (DLD1^SEMA6D^ vs. DLD1^EV^) models revealed distinct transcriptional changes (Fig. 6A). GSEA identified significant IFN-I pathway enrichment in SEMA6D-overexpressing cells (NES = 1.60, P = 0.002; Fig. 6B), with no other pathways enriched (Fig. S8A, B). Dual-luciferase assays demonstrated that SEMA6D overexpression potentiated IFNα-induced ISRE activation (Figs. 6C and S9A), while suppression attenuated it (Fig. 6C). SEMA6D specifically amplified IRF9-mediated ISRE activation (Fig. S9B). Transcriptomic and qPCR validation confirmed SEMA6D-upregulated IFN-I pathway components (Figs. 6D, E and S9C), whereas knockdown suppressed their expression (P < 0.01; Fig. 6F). GEPIA analysis revealed positive associations between SEMA6D and IRF9 (Fig. 6G), STAT2, JAK1, and IFITM1 (Fig. S9D–F). Western blot confirmed SEMA6D enhanced IRF9 expression and STAT1/STAT2 phosphorylation, effects abrogated by SEMA6D depletion (Fig. 6H). These findings establish SEMA6D as a critical activator of the IFN-I signaling cascade, driving tumor suppression in CRC.Fig. 6SEMA6D activates the type I interferon signaling in CRC cells.A Heatmap of differentially expressed genes (DEGs) between SEMA6D-knockdown HCT116 cells and SEMA6D-overexpressing DLD1 cells. Red and green indicate upregulated and downregulated genes, respectively. Black lines highlight interferon signaling-related genes. B GSEA plot showing enrichment of IFN-α response genes in SEMA6D-overexpressing DLD1 cells. C IFN-α-induced luciferase activity in HCT116 (SEMA6D knockdown) and 293 T cells (SEMA6D overexpression) with or without IFN-α treatment. D Top six IFN-related genes upregulated in SEMA6D-overexpressing cells. qRT-PCR analysis of IFN signaling genes in SEMA6D-overexpressing RKO cells (E) and SEMA6D-depleted HCT116 cells (F). G Correlation between SEMA6D and IRF9 expression in the GEPIA COAD cohort. H Immunoblot analysis of IFN signaling proteins in SEMA6D-manipulated cells. Values represent mean ± SD from three independent experiments. Statistical significance was determined by two-way ANOVA: ^^P < 0.05, ^^P < 0.01, ^^P < 0.001, ^****^P < 0.0001.
SEMA6D binds to IRF9 to activate type I interferon signaling in CRC
Co-immunoprecipitation assays confirmed direct SEMA6D-IRF9 interaction in 293T cells (Flag-SEMA6D/HA-IRF9; Fig. 7A) and endogenous interaction in DLD1 cells (Fig. 7B). IRF9 knockdown abrogated SEMA6D-mediated suppression of proliferation, colony formation, and migration in DLD1/RKO cells, while IRF9 depletion alone enhanced these properties (Fig. 7C–H). IRF9 overexpression in DLD1 and RKO cells (Fig. 7I, J) or knockdown in HCT116 cells (Fig. S10A) did not alter SEMA6D expression. IRF9 overexpression reversed SEMA6D knockdown-induced increases in proliferation, colony formation, and migration in HCT116 cells (Fig. S10B–E). Organoids validation showed SEMA6D knockdown reduced IRF9 expression, whereas IRF9 overexpression did not affect SEMA6D levels (Fig. S11A). IRF9 overexpression reversed SEMA6D knockdown-induced enhancement of organoid growth and invasion (Fig. S11B–E). The SEMA6D-IRF9 axis was Plexin A4-dependent, as Plexin A4 knockdown attenuated IRF9 upregulation (Fig. S12A) and SEMA6D-IRF9 interaction (Fig. S12B). Analysis of 115 CRC specimens revealed a strong positive correlation between SEMA6D and IRF9 protein levels (Pearson’s r = 0.3074, P < 0.001; Fig. 7K, L). These findings demonstrate that SEMA6D regulates IFN-I-dependent antitumor immunity in CRC through direct binding to IRF9.Fig. 7SEMA6D suppresses CRC progression via IRF9-dependent interferon pathway activation.A, B Co-immunoprecipitation (Co-IP) confirms exogenous and endogenous SEMA6D-IRF9 interaction in 293 T and DLD1 cells. C, D CCK-8 assay demonstrating IRF9 silencing abrogates SEMA6D-mediated growth suppression in DLD1 and RKO cells. E IRF9 knockdown reversed SEMA6D-mediated suppression of colony formation. F Transwell migration assays showing IRF9 knockdown reverses SEMA6D-dependent migration inhibition. Scale bar: 200 μm. G, H IRF9 knockdown attenuated SEMA6D-mediated inhibition of wound healing. Scale bar: 200 μm. qRT-PCR showing SEMA6D overexpression does not alter IRF9 mRNA levels in DLD1 (I) and RKO (J) cells. K Representative IHC staining of SEMA6D and IRF9 in CRC specimens. Scale bar: 100 μm. L Positive correlation between SEMA6D and IRF9 protein expressions in 115 CRC tissues (Pearson’s r = 0.3074, P < 0.001). Values represent mean ± SD from three independent experiments. Statistical significance was determined by two-way ANOVA: ns not significant, ^^P < 0.05, ^^P < 0.01, ^^P < 0.001, ^****^P < 0.0001.
SEMA6D activates IFN-I signaling to augment antitumor immunity in CRC
Interferon signaling is essential for CTL-mediated tumor elimination, with neoantigen presentation regulated by this pathway influencing CTL activity and immune checkpoint inhibitors (ICIs) efficacy. Building on evidence of SEMA6D-mediated immune regulation [13, 16], we investigated its role in IFN-I-driven antitumor immunity. TIMER analysis revealed positive correlations between SEMA6D expression and CD4^+^/CD8^+^ T cell infiltration in CRC (Fig. 8A).Fig. 8SEMA6D overexpression suppresses xenograft tumor growth via IRF9-mediated activation of type I interferon signaling in immunocompetent mice.A TIMER2.0 analysis showing positive correlation between SEMA6D expression and CD4^+^/CD8^+^ T cell infiltration in CRC cohorts. B Representative images of tumor-bearing BALB/c mice following bilateral subcutaneous inoculation with CT26^Ctrl^ (left flank) and CT26^OE^ (right flank). C Representative images of excised xenograft tumors from CT26^Ctrl^ and CT26^OE^ groups. D Tumor growth curves demonstrating suppressed tumor volume in CT26^OE^ versus CT26^Ctrl^. E Quantification of final tumor weights. Validation of SEMA6D overexpression in CT26^OE^ cells by qPCR (F) and Western blot (G). H Flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) showing enhanced CD4⁺/CD8⁺ T-cell populations in CT26^OE^ tumors versus controls. I H&E staining and IHC for SEMA6D and Ki-67 in subcutaneous tumors, revealing reduced Ki-67 positivity in CT26^OE^ tumors. Scale bar: 100 μm. The graphs represent means ± SD. Statistical significance was assessed using Student’s t test: ^^P < 0.05, ^^P < 0.01, ^^P < 0.001, ^****^P < 0.0001.
In immunocompetent BALB/c mice bearing CT26 cells overexpressing SEMA6D (CT26^OE^) or controls (CT26^CTRL^), SEMA6D overexpression inhibited tumor growth (Fig. 8B–E), upregulated SEMA6D and IRF9 expression (Fig. 8F, G), enhanced CD4^+^/CD8^+^ T cell infiltration (Fig. 8H) and reduced Ki-67 level (Fig. 8I). SEMA6D knockdown increased tumor growth, reversed by IRF9 overexpression; IRF9 depletion alone produced the most rapid growth (Fig. S13A–C). SEMA6D knockdown enhanced Ki-67 and reduced T cell infiltration, both rescued by IRF9 overexpression (Fig. S13D–F). Orthotopic CT26^OE^ implantation showed reduced bioluminescence (Fig. S14A), decreased tumor burden (Fig. S14B–D) in SEMA6D-overexpressing cohorts, elevated SEMA6D and IRF9 expression (Fig. S14E) and enriched CD4^+^ and CD8^+^ T cells (Fig. S14F).
CD8^+^ T cells exhibited increased migration toward CT26^OE^-conditioned media (Fig. S15A, B) and enhanced cytotoxicity (Fig. S15C). CD8^+^ T cells from SEMA6D-primed mice induced greater apoptosis (Fig. S15D–F) with elevated Granzyme B/Perforin (Fig. S15G). In mice treated with αCD8a, αCD4, or αIgG (Fig. S16A), SEMA6D-mediated suppression was reversed by CD8^+^ depletion and partially attenuated by CD4^+^ depletion (Fig. S16B–D). SEMA6D overexpression increased IFN-γ and TNF-α, reduced by T cell depletion (Fig. S16E, F). Immunohistochemistry confirmed enhanced T cell infiltration and effective antibody-mediated depletion (Fig. S16G, H). These findings position SEMA6D as an enhancer of IFN-I-driven antitumor immunity in CRC.
Hypomethylating agents enhance CRC sensitivity to immunotherapy in vivo
We previously identified SEMA6D, a promoter of anti-tumor immunity, as hypermethylated in CRC and reactivatable by demethylating agents. To assess whether restoring SEMA6D expression improves immunotherapy response, BALB/c mice bearing CT26 tumors were randomized into four groups: (1) PBS + αIgG, (2) decitabine (DAC) + αIgG, (3) PBS + αPD-1, and (4) DAC + αPD-1, with DAC administered three days prior to antibody treatment (Fig. S17A).
While monotherapies reduced tumor burden, DAC + αPD-1 combination produced significantly greater tumor growth inhibition, demonstrating synergistic efficacy (Fig. S17B–D). qPCR and Western blot confirmed DAC-mediated SEMA6D restoration (Fig. S17E, F). H&E and Ki67 analyses revealed that combination therapy suppressed proliferation more effectively than monotherapies (Fig. S17G). These findings suggest that hypomethylating agents restore SEMA6D expression, activate IFN-I signaling, and sensitize CRC tumors to PD-1 blockade.
Discussion
CRC persists as a major global health challenge, characterized by late-stage diagnosis [28], poor metastatic survival [29], and limited immunotherapeutic responsiveness [30] predominantly restricted to MSI-H subsets [31], underscoring the need for novel therapeutic approaches [32, 33]. In this study, we identified SEMA6D as a crucial, epigenetically regulated tumor suppressor and delineated a novel Plexin A4/SEMA6D/IRF9 signaling axis that orchestrates potent anti-tumor immunity in CRC.
The identification of SEMA6D as a tumor suppressor refines our understanding of semaphorin signaling in cancer biology. Although semaphorins are established neuronal guidance regulators, their oncogenic functions are complex and context-dependent. We demonstrate that SEMA6D is frequently epigenetically silenced through promoter hypermethylation in CRC, and its loss correlates with unfavorable prognosis, supporting tumor-suppressive roles for specific semaphorins in solid malignancies. This aligns with the established paradigm that tumorigenesis is driven by both genetic mutations and epigenetic dysregulation [34], with DNA methylation serving as a principal mechanism governing tumor-related gene expression [35, 36]. SEMA6D silencing associates with CIMP-H, MSI-H, and CMS4 molecular subtypes, suggesting its inactivation may drive aggressive biology in these subtypes, potentially explaining their poor outcomes and therapeutic resistance. Pronounced downregulation in CIMP-H tumors suggests SEMA6D silencing may represent an early event in serrated carcinogenesis [37], while reduced expression in MSI-H tumors indicates decreased immunogenicity. Particularly noteworthy is markedly reduced SEMA6D expression in CMS4 tumors, characterized by pronounced TGF-β activation, extensive stromal invasion, and enhanced angiogenesis, representing the mesenchymal phenotype with the poorest prognosis. This downregulation aligns with our findings that SEMA6D inhibits EMT and metastatic progression, suggesting SEMA6D loss promotes aggressive mesenchymal characteristics of CMS4. This molecular signature underscores SEMA6D’s potential as a biomarker for molecular classification and personalized therapeutic strategies. Therapeutically, targeting DNA methylation in SEMA6D-silenced tumors may restore immunogenicity and enhance immunotherapy response, offering a promising strategy for overcoming resistance in non-MSI-H colorectal cancers.
One of the most significant findings is the elucidation of the Plexin A4/SEMA6D/IRF9 signaling axis and its role in modulating the tumor microenvironment. Plexins serve as semaphorin receptors, mediating cell migration, survival, and immune responses. While the SEMA6D-Plexin-A1 axis orchestrates T cell responses and immune memory, SEMA6D exhibits context-dependent roles—driving tumorigenesis in gastric cancer [38, 39] and serving as a metastatic prognostic marker in triple-negative breast cancer (TNBC) [40]. Its role in CRC remained unclear. We demonstrated that SEMA6D exerts tumor-suppressive effects via apoptosis induction and EMT inhibition.
EMT initiates tumor migratory programs [41], enabling epithelial-derived malignancies to acquire invasive properties through cytoskeletal reorganization and adhesion molecule dysregulation. SEMA6D is markedly downregulated in CMS4 CRC, a subtype defined by mesenchymal and metastatic features, and its restoration suppresses EMT and metastasis. We propose that SEMA6D regulates EMT through three interconnected mechanisms. First, SEMA6D activates type I interferon signaling, which suppresses EMT across multiple cancers [42]. Second, SEMA6D remodels the TME, reciprocally influencing EMT through cytokine signaling, immune cell recruitment, and stromal interactions [43]. Third, as a semaphorin family member, SEMA6D intrinsically modulates cytoskeletal reorganization to regulate cell migration—a core EMT component—demonstrating context-dependent control over metastatic phenotypes [14]. Future studies will elucidate molecular mechanisms underlying SEMA6D-mediated EMT suppression to identify additional biomarkers and therapeutic targets for CRC metastasis.
SEMA6D mediates biological functions through specific plexin receptors, demonstrating context-dependent partnerships across cancer types [44]. While SEMA6D interacts with plexin-A1 in malignant pleural mesothelioma (MPM) [45] and plexin-A4 during macrophage polarization [16], we identified plexin-A4 as the exclusive SEMA6D-binding partner in CRC. Both SEMA6D and plexin-A4 exhibited concomitant downregulation in CRC tissues and cell lines. Functional validation confirmed plexin-A4 knockdown abrogated SEMA6D-mediated suppression of proliferation and migration, while plexin-A4 overexpression rescued the tumor-promoting phenotype induced by SEMA6D depletion, establishing the SEMA6D-plexin-A4 axis as essential for tumor suppressor activity.
Our study identifies IFN-I signaling activation as the primary mechanism by which SEMA6D suppresses CRC progression. IFN-I cytokines exhibit dual roles -potentiating antitumor immunity while paradoxically fostering immunosuppression in chronic inflammation [46]. Within the TME, IFN-I upregulates ISGs such as IFI16, CCL5, and CXCL11, mediating immunogenic cell death and recruiting cytotoxic effectors [47]. Mechanistically, SEMA6D amplified IFN-I pathway activity through direct interaction with IRF9, while SEMA6D silencing abrogated this signaling. IRF9, an integral transcription factor in the ISGF3 complex, mediates type I interferon responses by regulating ISG expression and participates in regulating cell proliferation, tumor formation and immune regulation [48]. Our results demonstrate that IRF9 acts downstream of SEMA6D, rescuing tumor growth upon SEMA6D loss and being upregulated by SEMA6D without affecting its expression. Their interaction requires Plexin-A4, which likely functions as an essential scaffold for the SEMA6D-IRF9 complex, enabling IRF9 upregulation and pathway initiation. Absence of Plexin-A4 abrogates this interaction, diminishing IRF9 levels and impairing IFN-I signaling. These findings position the SEMA6D/plexin-A4-IRF9 axis as a novel regulatory mechanism linking semaphorin signaling to IFN-I-mediated tumor surveillance in CRC, representing a promising therapeutic target to enhance TME immunogenicity and combat metastatic resistance.
Interferons are crucial immune regulators with significant influence across diseases, including cancer [49]. Immune cell infiltration within the TME is a well-established prognostic and therapeutic response biomarker in CRC [50, 51]. IFN-I signaling mediates antitumor effects in CRC, including p21-mediated cell cycle arrest [52, 53] and caspase-3-dependent apoptosis [54]. While CRC is generally immunogenically quiescent, mismatch repair-deficient (MMR-d) tumors exhibit lymphocyte-rich TMEs driven by high neoantigen burden [55]. IFN-α enhanced EGFR trafficking, supporting combination regimens with EGFR inhibitors [56], and suppresses metastasis by inhibiting hepatic colonization, attenuating angiogenesis, and reducing primary tumor growth [57]. IFN-α/β upregulation triggers STAT1-STAT2-IRF9 complex assembly, impeding tumor progression [58]. Here, we demonstrate that SEMA6D overexpression suppresses tumor growth by promoting CD4^+^ and CD8^+^ T-cell infiltration, whereas SEMA6D loss accelerates tumor progression but is rescued by IRF9 restoration. Both in vitro and in vivo analysis demonstrate that SEMA6D strengthens immune-mediated cytotoxicity, underscoring its role in augmenting antitumor immunity. These findings propose a therapeutic strategy combining IFN-I agonists with ICIs to amplify antitumor immunity and impede disease progression.
Hypermethylation of the SEMA6D promoter in CRC reduces SEMA6D expression and attenuates IFN-I pathway activation, impairing antitumor immunity. While DNA methyltransferase inhibitors such as decitabine demonstrate efficacy in hematologic malignancies, their application in solid tumors remains limited [59]. Given SEMA6D promoter hypermethylation and its immunomodulatory genes in CRC, we evaluated decitabine (DAC) combined with ICIs in vivo. DAC restored SEMA6D expression and enhanced immunotherapy efficacy, demonstrating proof-of-concept for epigenetic reactivation of immunomodulatory genes in CRC. These results suggest demethylating agents may exert antitumor effects through re-expression of immunomodulatory genes such as SEMA6D, consistent with emerging evidence supporting epigenetic modulator-immunotherapy combinations across malignancies [60, 61]. This finding links tumor-intrinsic suppression to extrinsic antitumor immunity, suggesting a strategy to epigenetically “prime” immunologically “cold” CRC tumors for enhanced checkpoint blockade sensitivity. By reversing epigenetic silencing of SEMA6D and other immune-related genes, demethylating agents may expand the CRC patient population eligible for immunotherapy benefits.
Despite comprehensive investigations, several limitations warrant consideration. The molecular mechanisms governing IRF9 activation by the Plexin A4/SEMA6D axis require further elucidation through proteomics and chromatin immunoprecipitation sequencing. Our in vivo models do not fully recapitulate human CRC microenvironment complexity; patient-derived xenografts would provide more clinically relevant validation. Future investigations should explore SEMA6D interactions with additional immune checkpoints, optimize demethylating agent-immunotherapy combinations, and investigate upstream mechanisms and drivers of promoter methylation. Prospective clinical validation is essential to translate these findings into biomarker-guided therapeutic strategies.
This study establishes SEMA6D as an epigenetically silenced tumor suppressor in CRC and elucidates a novel Plexin A4/SEMA6D/IRF9 signaling axis modulating antitumor immunity. SEMA6D enhances T cell infiltration, and its re-expression through demethylation agents potentiates immunotherapy efficacy. Our findings support SEMA6D as a biomarker for molecular classification, prognosis, and therapeutic decision-making, while highlighting epigenetic therapies in overcoming immunotherapy resistance. These insights provide rationale for clinical trials evaluating demethylating agents combined with immunotherapy in SEMA6D-deficient tumors to improve CRC patient outcomes.
Materials and methods
Bioinformatics analysis
SEMA6D expression was comprehensively analyzed across multiple colorectal cancer datasets. Paired tumor-normal tissues (GSE32323) [62], primary-metastatic comparisons (GSE28702) [63], and bioinformatic platforms (UALCAN) [64], GEPIA and Kaplan Meier plotter revealed consistent SEMA6D downregulation in malignancies, with prognostic significance validated through survival analysis [65]. TCGA colon adenocarcinoma data (n = 278) demonstrated an inverse correlation between promoter methylation across 22 CpG sites and SEMA6D expression. Further stratification showed significant expression differences across CIMP status, microsatellite instability subgroups, and consensus molecular subtypes (GSE39582). All analyses utilized R with standard statistical tests.
Cell culture
Human and murine CRC cells were sourced from ATCC (Manassas, VA, USA). HEK293T cells and NCM460 (a normal colon epithelial cell line) were procured separately from Invitrogen (Thermo Fisher Scientific, Waltham, MA) and ScienCell Research Laboratories (San Diego, CA, USA). These cell lines were cultured in DMEM (Gibco BRL, USA) supplemented with 10% FBS (Thermo Scientific, Waltham, MA) and 100 μg/mL penicillin-streptomycin under standardized conditions (37 °C, 5% CO₂, humidified incubator). Cell line identity was verified by short tandem repeat (STR) profiling, and mycoplasma contamination was routinely assessed by the specific detection kit (Sigma-Aldrich, St. Louis, USA).
Human tumor tissues and organoid culture
A total of 181 samples, including primary CRC tumors, adjacent normal mucosa, and corresponding clinical data, were collected from individuals at Yunnan Cancer Hospital (Kunming, China). Eligible participants were those without metastatic lesions or prior neoadjuvant chemotherapy and with complete follow-up data. Patient health status was monitored through regular follow-ups via telephone or questionnaires. The study obtained approval from the Institutional Review Board of Yunnan Cancer Hospital in accordance with the Declaration of Helsinki guidelines, and written informed consent was obtained from all participants prior to enrollment. Between 2010 and 2015, paraffin-embedded tissue specimens from 181 CRC cases were collected at Yunnan Cancer Hospital. Both tumor and noncancerous tissues were histologically confirmed.
Patient-derived colorectal cancer organoids were established from fresh tumor tissues obtained from treatment-naïve CRC patients undergoing surgical resection at Yunnan Cancer Hospital (Kunming, China). All experimental procedures are conducted in accordance with ethical standards and were approved by the Institutional Review Board (KYLX202124). Furthermore, written informed consent was secured from all participating individuals prior to tissue acquisition. Colorectal tumor organoid culture was performed following an established protocol [66]. Tumor tissues were rinsed with ice-cold PBS supplemented with antibiotics (penicillin 200 U/mL, streptomycin 0.2 mg/mL, primocin 200 µg/mL), mechanically dissected, and digested in DMEM (Gibco) and collagenase IV (Sigma, USA) at 37 °C for 1 h. Isolated organoids were resuspended in Matrigel (Corning) and seeded in 24-well plates. After Matrigel polymerization (30 min at 37 °C), 500 µL of advanced DMEM/F12 medium was added. This complete medium formulation included 10 mM HEPES, 1× N2 supplement (Life Technologies), 2 mM GlutaMAX, 1× B27 supplement, 10 nM gastrin I, 50 ng/mL recombinant Noggin (Peprotech), 500 ng/mL R-spondin-1 (Peprotech), 10 µM SB202190 (Sigma), 10 µM Y-27632, 10 mM nicotinamide, 1 mM N-acetylcysteine, and 100 U/mL penicillin/streptomycin. After 24 h, organoids were transduced with either control or SEMA6D-targeting lentiviral vectors in the presence of polybrene (10 µg/mL, Millipore) for 48 h. Medium was replaced with fresh complete medium and renewed every three days. Organoid was documented by phase-contrast microscopy and quantified by ImageJ (Fiji).
Immunohistochemistry (IHC)
Immunohistochemical evaluation of SEMA6D expression was conducted on 181 paired paraffin-embedded CRC tissues using a standardized protocol. Tissue sections (4 μm) underwent dewaxing, rehydration, and heat-induced epitope retrieval, followed by overnight incubation with primary antibodies: anti-SEMA6D antibody (ab198745, Abcam, Cambridge, UK), anti-CD8a (ab209775, Abcam, Cambridge, UK), anti-CD4 (ab183685, Abcam, Cambridge, UK), anti-IRF9 (ab315277, Abcam, Cambridge, UK). Signal amplification was achieved via an HRP-conjugated anti-rabbit polymer detection system (EnVision+ System, Dako/Agilent Technologies, Santa Clara, CA, USA), followed by DAB chromogen visualization. Immunohistochemical assessment was performed by two independent pathologists. Staining intensity was scored on a four-point scale (0: negative; 1: weak; 2: moderate; 3: strong), and expression was quantified using a histochemical score (H-score). A receiver operating characteristic (ROC) curve constructed from OS data determined the optimal H-score cutoff value (1.65) to divide patients into high and low SEMA6D expression cohorts.
Cell transfection and lentiviral production
The full-length human cDNA sequences for SEMA6D (RefSeq: NM_001358351), Plexin A1(RefSeq:
NM_032242.4), Plexin A4 (RefSeq: NM_020911.2) and IRF9 (RefSeq: NM_006084.5) were individually subcloned into the pLV-EF1α-IRES-Blast lentiviral expression vector (Addgene #85133) to generate lentiviral constructs, with cloning primers detailed in Table S1. For loss-of-function studies, validated shRNA sequences targeting SEMA6D, Plexin A1, Plexin A4 and IRF9 (Table S1) were inserted into the pLKO.1-TRC vector (Addgene #10878). Lentiviruses were generated by co-transfecting 293 T cells with transfer plasmids and packaging vectors (psPAX2, pMD2.G) using Lipofectamine 3000 (Invitrogen, Grand Island, NY, USA). Stable pools were selected under puromycin (Gibco) or blasticidin (Invivogen) for 14 days. Transgene expression and knockdown efficiency were validated via qRT-PCR and immunoblotting. For organoid transduction, organoids cultured in Matrigel were dissociated using a cell dissociation reagent and collected by centrifugation at 240 × g for 10 min. Dissociated organoids were transduced with the appropriate lentiviral particles and incubated at 37 °C for 5 h. Following transduction, organoids were pelleted by centrifugation at 240 × g for 10 min and re-embedded in 50% Matrigel in 24-well plates. Following aggregation, organoids were maintained in the corresponding growth medium.
RNA extraction and RT-qPCR
The extraction of RNA was conducted by TRIzol^TM^ reagent (Thermo Fisher Scientific, Waltham, MA). Subsequently, 1 μg of the isolated RNA was reverse-transcribed into complementary DNA (cDNA) with the PrimeScript RT reagent kit (Promega, Madison, WI, USA). Quantitative PCR (qPCR) assays were performed in triplicate on a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA) using SYBR Green Master Mix kit (Sigma-Aldrich, St. Louis, MO, USA), employing primer pairs listed in Table S2. The 2^-ΔΔCq^ method was employed to quantify mRNA expression. All reactions were performed in triplicate, with melt curve analysis confirming amplification specificity.
Western blot analysis
Proteins were extracted by using RIPA lysis buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). After quantification via BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA), equal amounts of protein were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% milk for 2 h subsequently incubated overnight at 4 °C with specific primary antibodies, including: SEMA6D (sc393258) and GAPDH (sc-32233) from Santa Cruz Biotechnology (Dallas, TX, USA); E-cadherin (#3195), Caspase 3 (#3879), Cleaved Caspase 3 (#9585), Cleaved Caspase 8 (#9662), HSP90 (#9715),β-catenin (#8480), Claudin-1 (#13255), N-cadherin (#13116), vimentin (#5741), Plexin A4 (#3816), STAT1 (#14994), p-STAT1 (#9167), IRF9 (#76684), STAT2 (#72604), p-STAT2 (#88410), HA tag (#3724) from Cell Signaling Technology (Massachusetts, USA); FLAG (F1804, Sigma-Aldrich); anti-Granzyme B (ab255598, Abcam, Cambridge, UK) and anti-Perforin (ab256453, Abcam, Cambridge, UK). The membranes were then incubated with the relevant secondary antibodies for 2 h at room temperature. Protein bands were ultimately visualized and imaged using an enhanced chemiluminescence (ECL) detection system (Bio-Rad, Hercules, CA, USA).
Co-immunoprecipitation
Cells were lysed in immunoprecipitation (IP) lysis buffer (CST). Following the determination of the protein concentration using the BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA), 1 mg of the total cell lysate was incubated with either 1 μg of the target primary antibody or a species-matched IgG control overnight at 4 °C under constant rotation. Antigen-antibody complexes were subsequently captured using Pierce^TM^ Protein A/G Magnetic Beads (Thermo Fisher Scientific, Waltham, MA) according to the instructions. After being rinsed with IP lysis buffer five times, the beads were heated with loading buffer at 95 °C for 10 min to elute the bound proteins. Eluates were separated by SDS-PAGE electrophoresis and analysed by immunoblotting as previously described.
RNA sequencing
Total RNA was isolated from designated CRC cell lines utilizing Trizol Reagent (Invitrogen, USA) following the manufacturer’s protocol. Library preparation and RNA sequencing were conducted by Sangon Biotech Co., Ltd. (Shanghai, China). Subsequent to RNA quantification and quality assessment, sequencing libraries were prepared using NEBNext® Ultra^TM^ RNA Library Prep Kit for Illumina® (NEB, USA) according to the supplier’s guidelines. Raw sequencing reads underwent quality control assessment using FastQC v0.11.8 and were subsequently processed with Trimmomatic v0.32 to remove low-quality sequences. Processed reads were aligned to the human reference genome (GRCh38/hg38) employing HISAT2 and Tophat2 alignment algorithms. Differential gene expression analysis was performed using the DESeq2 R package with default parameters. Significantly differentially expressed genes (DEGs) were defined as those exhibiting |log2(fold change)|≥1 and an adjusted p-value (FDR) <0.05. Subsequent DEG analysis encompassed Venn diagram visualization, functional annotation clustering, and pathway enrichment analysis.
Flow cytometry analysis
Tumor-infiltrating lymphocytes (TILs) were harvested from xenografts derived from SEMA6D-overexpressing CT26 (CT26^OE^) and vector control CT26 cells (CT26^CTRL^)-implanted immunocompetent mice. Single-cell suspensions were treated with ammonium-chloride-potassium (ACK) lysis buffer (Beyotime, China) to lyse erythrocytes. Approximately 1 × 10^6^ cells were stained with fluorescence-conjugated monoclonal antibodies targeting CD45 (PE), CD3 (Alexa Fluor 488), CD4 (PE-Cy7) and CD8 (PerCP-Cy5.5) for 20 min at ambient temperature in the dark. Following immunostaining, cells were washed twice with PBS containing 1% FBS and 2 mM EDTA. Flow cytometric analysis was performed using a FACSAria II flow cytometer (BD Biosciences), and data were processed with FlowJo software (Tree Star).
In vitro cell proliferation and clonogenic assays
Cell counting kit-8 (CCK-8) assay
Stable cells were inoculated in 96-well plates with 3 × 10^3^/well and cultured for up to 7 days. At indicated time points, 10% CCK-8 reagent (Dojindo, Japan) replaced the old medium and was cultured for 2 h at 37 °C. Cell viability curves were generated based on optical density (OD) values, which were measured by absorbance at 450 nm via a microplate reader (BioTek, Winooski, VT, USA).
Colony formation assay
Cells (1000 cells/well) were seeded into 6-well plates and grew for 2 weeks. Following fixation with 4% paraformaldehyde (PFA; Sigma-Aldrich, St. Louis, MO, USA) for 15 min, colonies were stained with 0.5% (w/v) crystal violet (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Colonies containing ≥50 cells were quantified using ImageJ software (NIH, Bethesda, MD, USA).
Soft agar anchorage-independent growth assay
To assess malignant transformation, 1 × 10^4^ cells/well SEMA6D-overexpressing cells were suspended in a solution of 0.35% Noble agar (Sigma-Aldrich, St. Louis, MO, USA) dissolved in complete medium and layered onto a base layer of 0.5% agar in 6-well plates. Following incubation at 37 °C for 2 weeks, colonies were stained with 0.005% crystal violet. Colony counts were normalized to control groups and analyzed using ImageJ software.
Cell migration and invasion assays
Cellular migratory and invasive capacities were evaluated using Transwell chambers (BD Falcon, San Jose, CA, USA). For migration assays and invasion assays, 2 × 10^5^ cells in serum-free medium were seeded into Transwell inserts, with inserts for invasion assays pre-coated with Matrigel (Corning #356234). After 24 h, migrated /invaded cells on the lower membrane were fixed with 4% PFA (Sigma-Aldrich, St. Louis, MO, USA), stained with crystal violet (Sigma-Aldrich, St. Louis, MO, USA) and quantified using ImageJ software (NIH, Bethesda, MD, USA).
DNA methylation analysis
The Methylation of the SEMA6D promoter was analyzed via methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP). CpG islands within the SEMA6D promoter region were identified using MethPrimer [27], and primer pairs for MSP/BSP were designed accordingly (Table S3). Genomic DNA was extracted from both CRC patient samples and CRC cell lines. A minimum of 500 ng of DNA underwent sodium bisulfite conversion using EZ DNA Methylation-Lightning™ Kit (Zymo Research, Irvine, CA, USA). For BSP, bisulfite-converted DNA was amplified with BSP primers. The resulting PCR amplicons were ligated into the pGEM-T Easy vector (Promega, Madison, WI, USA). Ten clones per sample were Sanger-sequenced to determine methylation patterns at individual CpG sites. For MSP, bisulfite-modified DNA was amplified using MSP or unmethylation-specific (USP) primers. The PCR products were electrophoresed and visualized using a ChemiDoc™ MP Imaging System (Bio-Rad, Hercules, CA, USA).
For demethylation treatment, CRC cells were administered with 5-aza-2′-deoxycytidine (Decitabine, DAC; Sigma-Aldrich, St. Louis, MO, USA) for 72 h, with fresh drug-containing medium replaced daily. Post-treatment, SEMA6D mRNA expression was quantified via qRT-PCR (primers in Table S2), normalized to GAPDH, and expressed as fold change relative to untreated controls. Statistical significance was determined by Student’s t test (P < 0.05).
Wound-healing assay
Control and SEMA6D-overexpressing (SEMA6D-OE) or SEMA6D-knockdown (SEMA6D-KD) cells were inoculated into six-well plates and cultured to 100% confluency. A linear wound was generated by a sterile pipette tip. Serum-free DMEM replaced the culture medium to minimize proliferative confounding. Wound closure kinetics were imaged at 0, 24, and 48 h post-scratch using an Olympus inverted microscope. Digital images were analyzed using ImageJ, and migration rates were calculated. Data represent mean ± SD from three biological replicates, each performed in triplicate.
Apoptosis analysis
Apoptotic cell populations were quantified by the Annexin V/FITC Apoptosis Detection Kit I (BD Biosciences, #556547, San Jose, CA, USA). Briefly, 5 × 10^5^ stable cells were suspended in 100 μL 1× binding buffer and stained with 5 μL FITC Annexin V and 5 μL propidium iodide (PI) for 15 min at 25 °C in the dark. Cells were analyzed immediately using a BD FACSCanto II flow cytometer (BD Biosciences) equipped with 488 nm excitation. Quadrant gating distinguished viable, early apoptotic, late apoptotic, and necrotic populations. Data were analyzed using FlowJo v10.8 software (BD Biosciences) and are presented as mean ± SD from three independent experiments.
Dual-luciferase reporter assay
The Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) assessed the transcriptional activity of the ISRE. Cells were co-transfected with the ISRE-driven firefly luciferase reporter construct (pGL3-ISRE) and the Renilla luciferase normalization vector (pRL-TK; Promega #E2241) at a 10:1 ratio using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Firefly and Renilla luciferase activities were sequentially quantified 24 h after transfection using a GloMax 20/20 luminometer (Promega). ISRE-driven transcriptional activity was normalized to Renilla luciferase values to account for transfection efficiency. Data were analyzed as fold induction relative to empty vector controls and expressed as mean ± SD from three independent experiments performed in triplicate.
Immunofluorescence assay
Cells (5 × 10^4^) were seeded onto 12-well chamber slides (Thermo Fisher Scientific, Waltham, MA, USA) and adhered overnight. Following fixation with 4% PFA (Sigma-Aldrich, St. Louis, MO, USA), cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) and subsequently blocked with 5% BSA at room temperature. Primary antibodies against E-cadherin (#3195, CST) and vimentin (#5741, CST) were applied at 4 °C for 16–18 h. Following triple washing with PBST, cells were treated with Alexa Fluor 488-conjugated secondary antibody (#4412, CST). Nuclei counterstaining was performed using 4′,6-diamidino-2-phenylindole (DAPI: 1 μg/mL, Thermo Fisher Scientific, #P36935). The Zeiss LSM800 confocal microscope was used to obtain the images.
CD8+ T cell isolation and migration
For functional analyses, the EasySep™ Mouse CD^8+^ T Cell Isolation Kit (Stemcell Technologies) was used to isolate CD8^+^ T cells from spleens or tumors of mice. Then the cells were activated with anti-CD3, anti-CD28 and IL-2 in vitro. In cytotoxicity assays, activated CD8^+^ T cells were co-cultured with CT26 tumor cells at an effector-to-target (E: T) ratio of 1:8 for 48 h, and tumor cell apoptosis was quantified by flow cytometry to determine specific killing. For migration assessment, CD8^+^ T cells were seeded in transwell inserts with tumor-conditioned medium in the lower chamber. After 6 h, migrated cells in the lower chamber were collected and counted to calculate the migration rate.
LDH assay
Cellular cytotoxicity was assessed by quantifying lactate dehydrogenase (LDH) release into the culture supernatant using a commercial detection kit (Roche, Basel, Switzerland). Maximum LDH release was determined by lysing control wells with 1% Triton X-100 (Merck, Darmstadt, Germany). Absorbance was measured at 490 nm with a reference wavelength of 690 nm. The percentage of LDH release was calculated according to the manufacturer’s protocol.
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of IFN-γ and TNF-α were quantified using an IFN-γ ELISA Kit (Beyotime, Shanghai, China) and a TNF-α ELISA Kit (Beyotime, Shanghai, China), respectively, according to the manufacturer’s instructions.
In vivo animal studies
Ethics and housing
All animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Kunming Medical University. Athymic male BALB/c nude mice (4–6 weeks old) and immunocompetent male BALB/c mice (5–6 weeks old) were obtained from Cyagen Biosciences (Suzhou, China) and housed under specific pathogen-free (SPF) conditions. The animal facility maintained controlled environmental parameters (23 °C, 12-h light/dark cycle, ad libitum access to standard chow and sterilized water).
Subcutaneous xenograft model
Subcutaneous xenograft models were established by injecting SEMA6D-overexpressing DLD1 cells (OE), SEMA6D-depleted HCT116 cells (KD), or respective vector controls (1 × 10⁶ cells per mouse) into the dorsal flanks of athymic nude mice. Tumor growth was monitored every 2 days, and tumor volumes were calculated as: V = 0.5 × L × W² (L: longest axis; W: perpendicular axis). Mice were euthanized at 28 days post-inoculation, and tumors were resected, weighed, and subsequently subjected to H&E staining and IHC analysis.
Metastatic dissemination models
For liver metastasis, DLD1 SEMA6D-OE or HCT116 SEMA6D-KD and respective control cells (1 × 10^6^) were orthotopically injected into the spleen of athymic nude mice. For lung metastasis assays, an identical cell number was administered via tail vein intravenous injection. Mice were monitored for 60 days, after which livers and lungs were harvested, immersion-fixed in 4% PFA, and processed for histological sectioning. Metastatic nodules were evaluated through H&E-stained tissue sections.
Syngeneic immunocompetent model
To investigate tumor-immune interactions in a syngeneic, immunocompetent context, CT26 cells with modulated SEMA6D expression (overexpressing, knockdown, or knockdown with IRF9 restoration) and their respective controls were implanted subcutaneously into the dorsal flank of 5- to 6-week-old male BALB/c mice. For T-cell depletion or checkpoint blockade studies, mice received intraperitoneal injections of anti-CD8α (200 μg; Clone 2.43, BioXCell), anti-CD4 (200 μg; Clone GK1.5, BioXCell, Lebanon, USA), anti-PD-1 (200 μg; Clone RMP1-14, Jiangsu Hengri Pharmaceuticals), or isotype control IgG (200 μg; BioXCell, Lebanon, USA). In a separate therapeutic regimen, once tumors reached approximately 100 mm³, mice were randomized to receive either PBS or decitabine (0.2 mg/kg/day, i.p.) for three consecutive days. A combination cohort received anti-PD-1 antibody (200 μg, i.p.) every three days, commencing two days after the decitabine cycle. Tumor volumes were serially calculated using the formula (length × width²)/2.
Concurrently, an orthotopic colorectal cancer mouse model was established via surgical implantation. Briefly, CT26 cells stably transfected with SEMA6D-overexpressing plasmid (CT26^OE^) or control vector (CT26^CTRL^) were prepared as a suspension of 1 × 10⁶ cells in 100 µL of Matrigel-PBS. Under anesthesia, a midline laparotomy was performed on BALB/c mice to externalize the cecum. The cell suspension was injected into the cecal wall, with the needle held in situ for 30 s post-injection, followed by gentle pressure to ensure local containment and homeostasis. The abdominal wall and skin were sutured closed in layers. Following a 28-day period, cecal tumors were resected, weighed, and processed for histological evaluation and flow cytometric analysis of tumor-infiltrating CD4^+^ and CD8^+^ T cell populations.
Statistical analysis
Statistical analyses were conducted using SPSS 25.0 (SPSS, Chicago, IL, USA) and GraphPad Prism 9.0 (GraphPad software, La Jolla, CA, USA). Continuous data are expressed as mean ± SD. All experiments represent at least three independent biological replicates. The homogeneity of variances was confirmed before parametric tests. Group comparisons were assessed using Student’s t test, One-way ANOVA or Two-way ANOVA. Associations between SEMA6D expression and clinicopathological features were determined via the Chi-square test. Correlation analyses measured correlations between two factors in patient samples. Survival probabilities were estimated using the Kaplan-Meier method with the log-rank test (Mantel-Cox). Prognostic independence was assessed using Cox proportional hazards regression (univariate and multivariate analyses). Hazard ratios (HRs) are reported with 95% confidence intervals (CIs). SEMA6D expression was dichotomized (high/low) using receiver operating characteristic (ROC)-optimized H-score thresholds. Statistical significance: ^^P < 0.05, ^^P < 0.01, ^^P < 0.001, ^****^P < 0.0001.
Supplementary information
Supplementary materials Western blot uncropped Table S6
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