Hypoxia-induced Semaphorin 6B promotes the development of cervical cancer through regulating macrophage polarization

Hypoxia-induced Semaphorin 6B promotes the development of cervical cancer through regulating macrophage polarization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hypoxia-induced Semaphorin 6B promotes the development of cervical cancer through regulating macrophage polarization Shuyan Yi, Sha Hu, Wenhan Li, Jing Cai, Liqiong Cai, lufang wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5304636/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jul, 2025 Read the published version in Journal of Translational Medicine → Version 1 posted 5 You are reading this latest preprint version Abstract The progression of cervical cancer is closely related to the tumor microenvironment (TME) (which includes hypoxia and macrophages). However, the impact of hypoxia on macrophages remains to be determined. In the present study, mRNA sequencing was used to detect differential gene expression in macrophages induced from peripheral blood mononuclear cells of cervical cancer patients under hypoxic and normoxic conditions, and 236 genes were upregulated in macrophages exposed to hypoxia; these genes were mainly enriched in response to chemokines and the actin cytoskeleton. The expression of semaphorin 6B (SEMA6B) significantly increased after hypoxia treatment, and high expression of SEMA6B was related to poorer survival in cervical cancer patients. Multicolor immunofluorescence revealed that abundant CD206 + SEMA6B + TAMs were associated with poor prognosis, late clinical stage, lymph node metastasis, poor differentiation, and lymphovascular space invasion in cervical cancer patients. TIMER database analysis revealed that SEMA6B expression was positively correlated with the infiltration of M2 macrophages and Tregs and negatively correlated with the infiltration of CD4 + and CD8 + T cells. In vitro, knocking down SEMA6B in TAMs inhibited macrophage M2 polarization and the migration of macrophages. Furthermore, after coculture of macrophages with SEMA6B knockdown and cervical cancer cells, the proliferation, migration and invasion of SiHa and HeLa cells was significantly reduced. In conclusion, SEMA6B is a promoting factor for the development of cervical cancer. Targeting SEMA6B may be a potential immunotherapy approach for treating cervical cancer. Hypoxia Semaphorin 6B cervical cancer macrophage polarization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Cervical cancer is a common malignant tumor of the female reproductive system and is the third most common cause of cancer death in women. According to global cancer statistics, in 2020, there were approximately 600000 new cases of cervical cancer each year, and 340000 women died from cervical cancer [ 1 ]. Although surgery and chemotherapy are currently the main treatment and prevention measures for cervical cancer, patients with advanced and recurrent metastatic cervical cancer still have poor prognoses [ 2 ]. The rise of immunotherapy provides hope for improving the treatment and survival of cervical cancer patients. However, due to the complexity of immune regulatory mechanisms in the tumor microenvironment (TME) and the heterogeneity of malignant tumors, tumor cells evade immune effects through various pathways [ 3 ]. Therefore, finding a new immunotherapy target for cervical cancer is crucial for improving the prognosis of cervical cancer. Hypoxia, the main characteristic of the tumor microenvironment, can not only enhance the invasion and metastasis ability of tumors but also promote tumor cells to enter a "dormant state" to avoid immune surveillance, leading to immunotherapy failure [ 4 ]. TAMs intricately regulate antitumor immune responses in the tumor immune microenvironment through interactions with different immune cell subsets. TAMs can activate immune checkpoints, downregulate antigen presentation, and secrete regulatory factors to coordinate CD8 + T-cell responses [ 5 ]. In addition, TAMs inhibit dendritic cell antigen presentation and infiltration. TAMs that secrete TGF-β CSF-1 can promote the expansion of MDSCs, while TAMs recruit immunosuppressive Treg cells and inhibit the function of NKT cells, leading to inhibitory effects [ 6 ]. However, the impact of hypoxia on macrophages is currently unclear, and understanding the mechanism by which hypoxia affects macrophages is crucial for identifying new immunotherapy targets for clinical cancer treatment. We analyzed the differential gene expression of macrophages induced from peripheral blood mononuclear cells of cervical cancer patients under hypoxic and normoxic conditions by mRNA sequencing and revealed a significant increase in SEMA6B expression. Recent research has confirmed that high SEMA6B expression is associated with poor prognosis and a tumor immunosuppressive microenvironment in colorectal cancer (CRC) patients [ 7 ]. Herein, we confirmed that high SEMA6B is associated with poor prognosis in cervical cancer patients and provided evidence that SEMA6B induces M2-polarized immune infiltration of macrophages to promote cervical cancer progression in vitro through the use of clinical data and experimental models combined with bioinformatics methods, indicating that SEMA6B could be a new immunotherapy target in cervical cancer. Materials and methods 2.1 Cell culture and transfection HeLa cells (cervical adenocarcinoma, CADC), SiHa cells (cervical squamous cell carcinoma, CSCC), and the human monocyte line THP-1 were purchased from the American Type Culture Library (ATCC, Manassas, Virginia, USA) and cultured according to its guidelines. All cell lines were identified within 2 years via short tandem repeat analysis. The cells were placed under low oxygen tension (1% O 2 , 5% carbon monoxide 2, and 94% N 2 ). After the cells were placed in a normoxic incubator (21% O2, 5% carbon monoxide 2, and 74% N 2 ), SEMA6B small interfering RNA (siRNA) was purchased from Tsingke Biotechnology Co., Ltd., and transfected with Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's protocol. All the siRNA target sequences are listed in Table S1 . 2.2 Human blood and tissue samples The human body research plan was reviewed and approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China) (UHCT-IEC-SOP-016-03-01), and informed consent was obtained from all patients. This study conducted tissue microarray, immunofluorescence, and survival analyses on paraffin-embedded surgical specimens from 58 cervical cancer patients admitted from September 2020 to May 2021 who underwent surgery in our hospital before initiating antitumor therapy and who did not receive radiotherapy, chemotherapy, or antiangiogenic treatment. All patients were pathologically diagnosed with primary cervical cancer, with no other malignant diseases or symptoms related to immune or hematopoietic dysfunction. The pathological characteristics of these cervical cancer patients are summarized in Table 1 . Patients with a follow-up period exceeding 6 months were included in the survival analysis. OS and DFS were defined as the time interval from surgery to the first tumor recurrence and death or the time interval to the last follow-up when no events occurred. Table 1 The relation of CD206 + SEMA6B + TAM to clinical pathological parameters in cervical cancer (n = 58). Clinicopathological parameters N (%) The density value of CD206 + SEMA6B + TAM P High(N = 32) Low(N = 26) Age (years) < 45 28 15 13 0.813 ≥ 45 30 17 13 BMI < 24 41 23 18 0.826 ≥ 24 17 9 8 FIGO IA1-IB1 31 13 18 0.030 IB2-IIB 27 19 8 Histological type Squamous cell carcinoma 47 28 19 0.163 Adenocarcinoma 11 4 7 Histologic grade G1/G2 40 16 24 0.001 G3 18 16 2 Lymph-vascular space invasion Negative 50 25 25 0.048 Positive 8 7 1 Lymph node metastasis Negative 45 21 24 0.015 Positive 13 11 2 2.3 Ficoll density gradient centrifugation for isolating peripheral blood mononuclear cells and inducing macrophages The human body research plan was reviewed and approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China), and informed consent was obtained from all patients. We collected peripheral blood from cervical cancer patients and separated peripheral blood mononuclear cells (PBMCs) using Ficoll Paque (TBD Science, Tianjin, China) density centrifugation. The collected PBMCs were placed in an incubator overnight cultivation; the cells were washed with RPMI 1640 (50 ng/mL M-CSF) once the next day to remove mixed lymphocytes; and a normal oxygen incubator (21% O2, 5% carbon monoxide 2, and 74% N2) was used. After 7 days of cultivation, monocytes develop into macrophages (Supplementary Fig. 1A). After 24 hours of hypoxia and normoxia culture, the collected macrophages were collected for hematocrit determination and mRNA sequencing. 2.4 mRNA sequencing (RNA-seq) The collected TAMs treated with hypoxia or normoxia as described in Section 2.3 above were sent to Servicebio (Wuhan, China) for RNA-seq analysis via an Illumina NovaSeq 6000 platform. DEGs were identified with a fold change ≥ 1.5 and p < 0.05 as the cutoff criteria. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were further conducted to interpret the biological significance of the DEGs. 2.5 Database analysis From the Cancer Genome Map (TCGA) database (available at https://portal.gdc.cancer.gov/ ), RNA-Seq spectra and clinical information from CCa patients were downloaded to determine their correlation with clinical case characteristics and the prognosis of patients with SEMA6B and cervical cancer. It is a web-based platform for systematic analysis of immune infiltration in different cancer types in the TCGA. The platform uses deconvolution of previously published computational methods to infer TIICs from gene expression profiles. In this study, the expression of SEMA6B in tumor-related macrophages and its correlation with tumor purity, M2 macrophages, Treg infiltration, CD8 + T cells, and CD4 + T cells in cervical cancer were analyzed in the TCGA Cancer Service. 2.6 Tissue immunofluorescence Immunofluorescence assays were performed on CD206 and SEMA6B cells using tissue microarrays. Briefly, after dewaxing, the slices were placed in antigen repair buffer under microwave irradiation at 15°C for 90 minutes. Then, they were washed and sealed at 10°C for 26 minutes and incubated overnight at 4°C with anti-CD206 (# ab313398; 1:1000; Abcam) and anti-semaphorin 6B (SEMA6B) (sc-390928; 1:200 dilution; Santa Cruz Biotechnology) antibodies. The slides were incubated with Alexa Fluor 488- and 594-conjugated secondary antibiotics (A11034, A21125; Invitrogen, Carlsbad, CA, USA). Immunostating was visualized under a slice scanner (Pannoramic MIDI: 3Dhistech, Hungary) on a scanning slide and evaluated by Image-Pro Plus Software (Media Cybernetics, Silver Springs, MD). The fluorescence intensities of SEMA6B, CD206, and DAPI were measured using a Zeiss scanning microscope. Using the SEMA6B/CD206&SEMA6B ratio, a value < 0.3 was defined as a low content of CD206 + SEMA6B + TAMs, and a value ≥ 0.3 was defined as a high content of CD206 + SEMA6B + TAMs. 2.7 Cellular immunofluorescence THP-1 cells were inoculated at a density of 24 or 30 cells/well into a 24-well plate, induced to differentiate into macrophages by adding PMA, and incubated overnight at 37°C. Then, the plate was fixed with 4% paraformaldehyde for 30 minutes. After washing twice with PBS and 1% BSA, the antigen was blocked at room temperature for 1 h and then incubated overnight with primary antibody (SEMA6B, sc-390928; 1:50 dilution; Santa Cruz Biotechnology) at 4°C. Next, the plate was incubated with a secondary antibody (goat anti-rabbit IgG Cy1600:1 dilution, ab2, Abcam) at room temperature for 30 hours and then incubated with DAPI for 15 minutes to stain the nucleus. Images were obtained using a fluorescence microscope (Olympus), and the average fluorescence intensity was quantified using ImageJ. 2.8 Coculture determination A Transwell system (6 Transwell boards, 0.4 µM aperture, Corning Life Sciences, USA) was used for indirect cocultivation. After macrophages (2.5 × 10 4 cells) were inoculated into the insert, cervical cancer cells (2.5 × 10 4 cells) were cultured in the basal chamber. The cocultures were incubated for 48 to 72 hours, after which the cells were collected for subsequent experiments. 2.9 RNA extraction and quantitative real-time fluorescence quantitative PCR (qRT‒PCR) Total cell RNA was extracted using TRIzol reagent (TaKaRa), and reverse transcription was performed (HiScript® III RT SuperMix Vazyme). The primers were synthesized by Qingke Biotechnology Co., Ltd., and their sequences are shown in Table S2 . qRT‒PCR was performed using qPCR SYBR Green premix (Vazyme) on a Step-One Plus real-time fluorescence quantitative PCR system (Thermo Fisher). Three biological replicates were conducted. 2.10 Western blotting Whole-cell protein extraction and Western blotting were performed according to previous methods [ 8 , 9 ]. The antibodies used for protein blotting are listed in Table S3 . The grayscale values of the protein bands were checked by ImageJ (ImageJ software, National Institutes of Health, Bethesda, Maryland). Each experiment was conducted at least three times. 2.11 5-Ethynyl-20-deoxyuridine (EdU) proliferation assay EdU determination was carried out according to the manufacturer's instructions for the EdU experimental kit (RiboBio). The transfected cells were inoculated into a 000-well plate at a density of 96 to 10 cells per well. The proportion of EdU-positive cells is the proportion of proliferating cells. Images were obtained using a fluorescence microscope (Olympus). The results were obtained from three biological replicates. Each experiment was repeated in triplicate. 2.12 Wound healing and Transwell measurements For the wound healing assay, cells were inoculated into a 3-well plate at × 105 cells per well. After transfection with siRNA, the cells were cultured for 48 hours. When the cells formed a converging monolayer, a 200 µL pipette suction head was used to scrape them. An inverted optical microscope (Olympus, Japan) was used to visualize the wound area at 0 and 24 hours. ImageJ (version 1.51) software was used to measure the proportion of wound healing areas and represent the migration characteristics of cancer cells. For Transwell measurements, 200 µL of serum-free medium containing 5 × Inoculate cells onto the upper surface of the chamber coated without (migration assay) or with (invasion assay) matrix glue, and 600 mL of complete culture medium was added to the bottom chamber. After incubation at 37°C for 24 hours, the cells were fixed through 8x using 4% paraformaldehyde. Then, the cells were stained with 0.1% crystal violet. The number of cells in five random fields of view for each insert was calculated to evaluate the migration and invasion characteristics of the cancer cells at 200× magnification. Each experiment was conducted three times. 2.13 Statistical analysis All the statistical analyses were conducted using the R environment (version 3.6.3) and GraphPad Prism (version 8.0.2). Kaplan–Meier (KM) analysis was performed on the total survival period (OS, time from initial diagnosis to death or end of follow-up) using survival and survival packages, and logarithmic ranking tests were used. Survival packages for univariate and multivariate Cox regression analyses were used to screen for survival-related genes. Pearson correlation analysis was performed to determine the relationships between survival-related genes. The associations between SEMA6B expression and clinicopathological features were evaluated using the Mann‒Whitney U test. Significant differences between different groups were evaluated through Student’s t tests. A P value < 0.05 was considered to indicate statistical significance. Results 3.1 Hypoxia induced increased expression of SEMA6B in macrophages We collected peripheral blood from cervical cancer patients and separated peripheral blood mononuclear cells (PBMCs) and induced to macrophages. After 24 hours of hypoxia and normoxia culture and the RNA sequencing results showed that 236 genes were upregulated and 385 genes were downregulated (Fig. 1 A, S1B). The 236 upregulated genes were subjected to GO and KEGG enrichment analyses. These genes were mainly enriched in chemokines, neutral migration, cytokine receptors and activity (Fig. 1 B, S1D, 1 E). Thirty-one genes whose expression was significantly upregulated were identified (Fig. 1 C) and the upregulated DEGs was associated with OS through the TCGA database (Fig. 1 D). To evaluate the correlation between the 31 OS-related genes, Pearson correlation analysis was conducted, and 31 identified key genes exhibited a coexpression pattern (S1C). Semaphorins play important roles in regulating various immune system responses. Tian et al. revealed that Sema3A drives alternative macrophage activation in the resolution of periodontitis via PI3K/AKT/mTOR Signaling [ 10 ]. Semaphorin 3G exacerbates joint inflammation through the accumulation and proliferation of macrophages in the synovium [ 11 ].Cellular immunofluorescence revealed that SEMA6B was expressed in both the cytoplasm and cell membrane of TAMs and that the expression of SEMA6B was significantly increased after hypoxia treatment (Fig. 1 E). 3.2 Validation of the association between CD206 + SEMA6B + TAMs and the progression of cervical cancer The results of tissue polychromatic immunostaining showed that CD206 + SEMA6B + TAMs were mainly located in the cervical cancer tissue matrix (Fig. 2 A). We confirmed that the greater the percentage of CD206 + SEMA6B + TAMs was, the worse the OS and DFS were for cervical cancer patients (Fig. 2 B, 2 C). We further revealed that abundant CD206 + SEMA6B + TAMs were associated with poor prognosis, late clinical stage, lymph node metastasis, poor differentiation, and lymphovascular space invasion in cervical cancer tissues (Table 1 , Fig. 2 D-G). 3.3 Databases confirm that SEMA6B is associated with poor prognosis and immune infiltration in cervical cancer patients According to the analysis of the TCGA dataset, the overall survival, disease-specific survival, and progression-free survival of cervical cancer patients with high expression of SEMA6B were significantly reduced (P = 0.008, 0.025 and 0.038, respectively) (Fig. 3 A). Multivariate analysis revealed that high SEMA6B expression was closely related to lymph node metastasis and late pathological T stage in cervical cancer patients (P = 0.002 and < 0.003, respectively) (Fig. 3 B). To further determine the correlation between SEMA6B and immune infiltration, we analyzed the relationships between SEMA6B and different immune cell marker genes using the TIMER database. After correlation adjustment through purity, the results showed that SEMA6B mainly expressed in Neutrophils, Mast cell, Eosinophils, Tem and Macrophages(Fig. 3 C, 3 D) and was positively correlated with the infiltration of M2 macrophages and Tregs and negatively correlated with the infiltration of CD8 + T cells and CD4 + T cells (Fig. 3 E), SEMA6B indicating that SEMA6B plays a crucial role in immune escape by promoting the immunosuppressive tumor microenvironment. 3.4 Knocking down SEMA6B inhibits macrophage M2 polarization and migration by inhibiting AKT phosphorylation The effect of knocking down SEMA6B on polarization was detected by flow cytometry, and the results confirmed that knocking down SEMA6B can inhibit CD206 + M2 polarization (Fig. 4 A, 4 B). PCR revealed an increase in the expression of M1 markers (CD86 and iNOS) and M2 markers (Arg-1 and CD206) after knocking down SEMA6B (Fig. 4 C). The migration ability of macrophages was evaluated by transwell assays, and as expected, SEMA6B-knockdown macrophages exhibited reduced migration ability (Fig. 4 D). Previous studies have confirmed that AKT phosphorylation are associated with macrophage polarization [ 12 ]. We further determined that knocking down SEMA6B in macrophages inhibited AKT phosphorylation (Fig. 4 E). Chemokines serve as the main cytokines involved in communication between TAMs and cervical cancer cells. Previous studies have confirmed that CXCL2 and CXCL8 are downstream genes of AKT [ 13 , 14 ]. We further determined that knocking down SEMA6B in macrophages can reduce the expression of CXCL2 and CXCL8 (Fig. 4 F). These results suggested that SEMA6B could regulate macrophage polarization in cervical cancer. 3.5 Knocking down SEMA6B in macrophages inhibits the proliferation of cervical cancer cells After coculture of macrophages with SEMA6B knockdown and cervical cancer cells, the proliferation of SiHa and HeLa cells was significantly reduced according to EdU proliferation assay (Fig. 5 A, 5 B). 3.5 Knocking down SEMA6B in macrophages inhibits the migration and invasion of cervical cancer cells After coculture of macrophages with SEMA6B knockdown and cervical cancer cells, the invasion and migration abilities of SiHa and HeLa cells were significantly reduced according to Transwell assays (Figs. 6 A, 6 C). The migration ability of SiHa and HeLa cells was significantly reduced according to the scratch assay (Fig. 6 B, 6 D). Western blot analysis revealed that E-cadherin expression increased, while N-cadherin and vimentin expression decreased in SiHa and HeLa cells after coculture with macrophages with SEMA6B knockdown (Fig. 6 E). These results suggested that SEMA6B could be a new prognostic immunological biomarker in cervical cancer that promotes tumor progression. Discussion Although immunotherapy for cervical cancer has achieved significant clinical success, its clinical application is greatly limited due to its low response rate, low applicability and severe adverse reactions [ 15 ]. The development of novel immune biomarkers and targets for the diagnosis and immunotherapy of cervical cancer is highly important. This study is the first to show that hypoxia induces increased expression of SEMA6B in macrophages induced from peripheral blood mononuclear cells of cervical cancer patients, and SEMA6B could be a new immunotherapy target in cervical cancer that regulates macrophage polarization and promotes tumor progression. Hypoxia plays an important role in the recurrence and metastasis of malignant tumors. Multiple studies have confirmed that hypoxia stimulates cancer cells to undergo mitotic arrest, allowing them to escape immune monitoring and form an immunosuppressive microenvironment, leading to recurrence, chemotherapy resistance, and distant metastasis [ 16 – 18 ]. Multiple studies have confirmed that a large number of tumor-related macrophages aggregate in the hypoxic zone of tumor tissue and play important roles in the occurrence, growth, invasion, metastasis, angiogenesis, and lymphangiogenesis of tumors [ 19 ]. TAMs are attractive targets for tumor immunotherapy, but the lack of specific biomarkers hinders their research and clinical application. Understanding the mechanism by which hypoxia affects TAMs and identifying specific biomarkers are highly important for the diagnosis, recurrence, and metastasis of cervical cancer. From a structural point of view, these semaphorin proteins contain a highly conserved N-terminal semaphorin domain of approximately 500 amino acids, a plexin–semaphorin–integrin domain and distinct protein domains that further define semaphorins, including immunoglobulin-like, thrombospondin, and basic C-terminal domains [ 20 ]. In this study, we found a significant increase in SEMA6B expression in macrophages induced from peripheral blood mononuclear cells of cervical cancer patients under hypoxia conditions. Previous studies have confirmed that SEMA4B is a direct target of hypoxia inducible Factor 1 (HIF-1) α, a hypoxia responsive element (HRE) that can recognize the SEMA4B gene [ 21 ]. However, Sema3A is expressed at low levels in hypoxia-treated cardiomyocytes due to hypoxia-induced inflammatory factor (TNF)-α and IL-1β, which are related to IL-6 secretion, decreased cell viability, myocardial cell apoptosis, ROS release, decreased ATP production, and a decreased GSH/GSSG ratio [ 22 ]. Therefore, different SEMA proteins may play different roles in hypoxic microenvironments. To our knowledge, this is the first report to confirm that hypoxia induces increased expression of SEMA6B in cervical cancer, but the mechanisms involved need to be further explored. According to the analysis of the TCGA dataset, the prognosis of cervical cancer patients with high expression of SEMA6B is poor. High expression of SEMA6B was closely related to lymph node metastasis and late pathological T stage in cervical cancer patients. Another noteworthy finding of this study is that SEMA6B is positively correlated with the infiltration of M2 macrophages and Tregs and negatively correlated with CD8 + T cells and CD4 + T cells, indicating that SEMA6B plays a crucial role in immune escape by promoting the immunosuppressive tumor microenvironment. These results are consistent with those of previous studies by Li et al., who confirmed a moderate to strong positive correlation between SEMA6B expression levels and infiltration levels in macrophages, MDSCs, NK cells, Tregs and Th6 cells in colorectal cancer and a significant positive correlation between infiltration levels of CD4 T cells, neutrophils, and dendritic cells and SEMA6B expression in colorectal cancer [ 7 ]. We found that SEMA6B is expressed in both the nucleus and cytoplasm of macrophages in cervical cancer tissue, and we further revealed that abundant CD206 + SEMA6B + TAMs were associated with poor prognosis, late clinical stage, lymph node metastasis, poor differentiation, and lymphovascular space invasion in cervical cancer tissues. The proliferation, invasion and migration ability of SiHa and HeLa cells significantly decreased after coculture with macrophages with SEMA6B knockdown. In summary, our research suggested that high SEMA6B expression predicts poor overall survival (OS) and may serve as a potential prognostic immune marker for cervical cancer patients. Previous studies have confirmed that the SEMA family mainly acts on the integrins β1, NRP and plexin C1 receptors to regulate various physiological and pathological processes [ 23 – 25 ]. Kang et al. revealed the important roles of the mTOR-Sema6D-PPARγ signaling pathway in controlling macrophage polarization [ 26 ]. Another study suggested that the association of SEMA4D with M2 macrophages may help stimulate tumor angiogenesis in epithelial ovarian cancer [ 27 ]. SEMA3A can bind to the NP1-1 receptor of TAMs, leading to downstream PI3K/Akt phosphorylation, continuous recruitment of monocytes and M2 polarization, thereby promoting cancer cell resistance to androgen deprivation therapy [ 28 ]. A recent study confirmed that hypoxia therapy can polarize macrophages toward the M2 phenotype and promote the development of keloids, mainly by activating the PTEN-PI3K/AKT pathway [ 29 ]. This research result is consistent with our findings. Previous studies have confirmed that recombinant SEMA3A can induce AKT or NF by binding to a complex of the T-cell surface receptors NRP-1 and PlexinA1- κ to inhibit B signal transduction [ 30 ]. AKIP1 regulates NFκB via the CXCL1, CXCL8 and AKT pathways and promotes GBM activity, migration, and chemical radiation resistance [ 31 ]. Previous studies have confirmed that malaria parasite infection in female BWF1 lupus mice regulates the CXCL12/CXCR4 axis and its downstream signals PI3K/AKT, NF-κB and ERK to weaken B-cell autoreactivity [ 32 ]. Atelectasis causes an alveolar hypoxia-induced inflammatory response, mainly through the secretion of NF-κB by pulmonary epithelial cells, which is dependent on increased CXCL-1 secretion [ 33 ]. Our study confirmed that knocking down SEMA6B in macrophages can inhibit macrophage polarization toward M2 and inhibit macrophage migration by inhibiting AKT phosphorylation. In summary, this study revealed the important role of SEMA6B in the poor prognosis of cervical cancer patients. We speculate that SEMA6B can serve as a new immunotherapy target in cervical cancer, providing new ideas for in-depth research on the effectiveness and safety of immunotherapy. However, the mechanism of SEMA6B in the hypoxic tumor immune microenvironment of cervical cancer and its potential target for targeting TAMs still need further exploration. In addition, these findings require additional preclinical models, clinical patient cohorts, and clinical trials for further validation. Declarations Ethics statement The study was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (UHCT-IEC-SOP-016-03-01). Written informed consent was obtained from all patients. Consent for publication Not applicable. Funding No fundings. Authors’ contributions Lufang Wang: Conceptualization, Methodology, Software. Shuyan Yi: Data curation, Writing- Original draft preparation. Sha Hu: Visualization, Investigation. Wenhan Li: Supervision. Jing Cai: Software, Validation. Liqiong Cai: Writing- Reviewing and Editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Availability of data The datasets analyzed in this study are available at TCGA (http://www.cancer.gov/tcga), GEO (https://www.ncbi.nlm.nih.gov/gds/) and TIMER database (http://timer.comp-genomics.org/ ). Acknowledgements Not applicable. Data availability The datasets used and/or analyzed during the current study are available from the corresponding authors upon reasonable request. References Singh D, Vignat J, Lorenzoni V, Eslahi M, Ginsburg O, Lauby-Secretan B, Arbyn M, Basu P, Bray F, Vaccarella S. Global estimates of incidence and mortality of cervical cancer in 2020: a baseline analysis of the WHO Global Cervical Cancer Elimination Initiative. Lancet Glob Health. 2023, 11(2): e197-e206. Polgár C, Major T and Varga S: Radiotherapy and radio- chemotherapy of cervical cancer. 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Yang Y, Wang Q, Wang W, Wei S, Zeng Q, Zhang A. Semaphorin 4A antibody alleviates arsenic-induced hepatotoxicity in mice via inhibition of AKT2/NF-κB inflammatory signaling. Toxicol Appl Pharmacol 410:115364, 2021. Kang S, Nakanishi Y, Kioi Y, Okuzaki D, Kimura T, Takamatsu H, Koyama S, Nojima S, Nishide M, Hayama Y, Kinehara Y, Kato Y, Nakatani T, Shimogori T, Takagi J, Toyofuku T, Kumanogoh A. Semaphorin 6D reverse signaling controls macrophage lipid metabolism and anti-inflammatory polarization. Nat Immunol, 2018,19(6):561-570. Chen Y, Zhang L, Lv R, Zhang WQ. Overexpression of Semaphorin4D indicates poor prognosis and prompts monocyte differentiation toward M2 macrophages in epithelial ovarian cancer. Asian Pac J Cancer Prev 14:5883-90,2013. Liu F, Wang C, Huang H, Yang Y, Dai L, Han S, Xing N, Ren S. SEMA3A-mediated crosstalk between prostate cancer cells and tumor-associated macrophages promotes androgen deprivation therapy resistance. Cell Mol Immunol 18:752-754, 2021. Dai S, Xu M, Pang Q, Sun J, Lin X, Chu X, Guo C, Xu J. Hypoxia macrophage-derived exosomal miR-26b-5p targeting PTEN promotes the development of keloids. Burns Trauma, 2024, 12:tkad036. Sumi C, Hirose N, Yanoshita M, Takano M, Nishiyama S, Okamoto Y, Asakawa YK. Tanimoto Semaphorin 3A inhibits inflammation in chondrocytes under excessive mechanical stress Mediat. Inflamm 2018: 5703651, 2018. Han D, Zhang N, Zhao S, Liu H, Wang X, Yang M, Wang S, Li Y, Liu Z, Teng L. AKIP1 promotes glioblastoma viability, mobility and chemoradiation resistance via regulating CXCL1 and CXCL8 mediated NF-κB and AKT pathways. Am J Cancer Re 11:1185-1205, 2021. Badr G, Sayed A, Abdel-Maksoud MA, Mohamed AO, El-Amir A, Abdel-Ghaffar FA, Al-Quraishy S, Mahmoud MH. Infection of Female BWF1 Lupus Mice with Malaria Parasite Attenuates B Cell Autoreactivity by Modulating the CXCL12/CXCR4 Axis and Its Downstream Signals PI3K/AKT, NFκB and ERK. PLoS One 10: e0125340, 2015. Tojo K, Nagamine Y, Yazawa T, Mihara T, Baba Y, Ota S, Goto T, Kurahashi K. Atelectasis causes alveolar hypoxia-induced inflammation during uneven mechanical ventilation in rats. Intensive Care Med Exp 3:56, 2015. Supplementary Files Suplementarytable.docx SupplementFigure1.jpg Supplementary Figure 1. Hypoxia induces an increase in SEMA6B expression in TAMs in cervical cancer. (A) Schematic diagram of the process of collecting peripheral blood from cervical cancer patients to extract monocytes and induce them into macrophages. (B) Heatmap of the differentially expressed genes identified via transcriptional sequencing in cervical cancer-related macrophages treated with normoxia and hypoxia. (C) Pearson correlation analysis was used to determine the correlation between 31 differentially upregulated genes in OS. The depth of color and the size of the circle represent the correlation coefficient between paired genes. Red represents a positive correlation, blue represents a negative correlation, and * represents a statistically significant difference. (D) Network diagram and histogram (E) of 236 upregulated genes enriched and analyzed through GO and KEGG analyses. Cite Share Download PDF Status: Published Journal Publication published 10 Jul, 2025 Read the published version in Journal of Translational Medicine → Version 1 posted Editorial decision: Major revision 25 Dec, 2024 Reviewers agreed at journal 05 Nov, 2024 Reviewers invited by journal 05 Nov, 2024 Editor assigned by journal 01 Nov, 2024 First submitted to journal 21 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5304636","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":374567010,"identity":"6ffb270e-608e-4548-9517-3ab11e3acab6","order_by":0,"name":"Shuyan Yi","email":"","orcid":"","institution":"Wuhan Union Hospital Department of Obstetrics and Gynecology: Huazhong University of Science and Technology Tongji Medical College First Clinical College Union Hospital Department of Obstetrics and Gynecology","correspondingAuthor":false,"prefix":"","firstName":"Shuyan","middleName":"","lastName":"Yi","suffix":""},{"id":374567011,"identity":"083e5d57-0fc2-4007-ae1e-9f14907a5a60","order_by":1,"name":"Sha Hu","email":"","orcid":"","institution":"Wuhan Union Hospital Department of Obstetrics and Gynecology: Huazhong University of Science and Technology Tongji Medical College First Clinical College Union Hospital Department of Obstetrics and Gynecology","correspondingAuthor":false,"prefix":"","firstName":"Sha","middleName":"","lastName":"Hu","suffix":""},{"id":374567012,"identity":"777f58b8-a7a0-4b04-af75-3d01f628aae4","order_by":2,"name":"Wenhan Li","email":"","orcid":"","institution":"Wuhan Union Hospital Department of Obstetrics and Gynecology: Huazhong University of Science and Technology Tongji Medical College First Clinical College Union Hospital Department of Obstetrics and Gynecology","correspondingAuthor":false,"prefix":"","firstName":"Wenhan","middleName":"","lastName":"Li","suffix":""},{"id":374567013,"identity":"d81fa784-4f88-4eb4-8d5c-514777631e70","order_by":3,"name":"Jing Cai","email":"","orcid":"","institution":"Wuhan Union Hospital Department of Obstetrics and Gynecology: Huazhong University of Science and Technology Tongji Medical College First Clinical College Union Hospital Department of Obstetrics and Gynecology","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Cai","suffix":""},{"id":374567014,"identity":"fdfdf26d-a08d-4959-b248-fd87e13c3b5f","order_by":4,"name":"Liqiong Cai","email":"","orcid":"","institution":"Wuhan Union Hospital Department of Obstetrics and Gynecology: Huazhong University of Science and Technology Tongji Medical College First Clinical College Union Hospital Department of Obstetrics and Gynecology","correspondingAuthor":false,"prefix":"","firstName":"Liqiong","middleName":"","lastName":"Cai","suffix":""},{"id":374567015,"identity":"4368061d-0457-4749-9ce9-8746097da555","order_by":5,"name":"lufang wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIie3OsWrDMBCAYQmBvJzr9YyL+wpXDJkMfpWagicX+gg2gsvS7M3Qh+jSZmwItIseIGNCwFOWLIWAhyahW2q73TroRwhO8KETwuX6hwVK1itZIWil3r/fbvpJODaGZJVeXnhcHGYaJmQ/GGVVpDHY0e+IWOYm8WcL0Fh+bqBtReCVJPazbiEf83o9tUdy95r4TCJ82JKc2G6iMDe04xN5ifyKBC1LUpK7icacD+e0WBNBSyIbIgBzxh0XoMHqCPThFxwg6NXmesopaI9H4RMngLa5n096SLbw1hufMbsyqsFtG8fB+PZ5te8h55ser7c/AJfL5XL90Bf6rkv6AwBPWgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-6670-0021","institution":"Wuhan Union Hospital Department of Obstetrics and Gynecology: Huazhong University of Science and Technology Tongji Medical College First Clinical College Union Hospital Department of Obstetrics and Gynecology","correspondingAuthor":true,"prefix":"","firstName":"lufang","middleName":"","lastName":"wang","suffix":""}],"badges":[],"createdAt":"2024-10-21 12:56:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5304636/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5304636/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12967-025-06692-z","type":"published","date":"2025-07-10T15:57:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71001231,"identity":"cc3365b6-2a47-4019-8216-2217dd38a4e0","added_by":"auto","created_at":"2024-12-10 05:40:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1692310,"visible":true,"origin":"","legend":"\u003cp\u003eHypoxia increased the expression of SEMA6B macrophages. (A) Volcano map of differentially expressed genes identified via transcriptional sequencing in cervical cancer-related macrophages treated with normoxia and hypoxia. (B) Bubble chart of 236 upregulated genes enriched and analyzed through GO and KEGG analyses. (C) Heatmap of 31 genes with significantly upregulated differential gene expression screened through the TCGA database for upregulated genes. (D) A forest plot of 31 genes with significantly upregulated DEGs associated with OS. (E) Cell localization of SEMA6B in TAMs and the effect of hypoxia on the expression of SEMA6B.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5304636/v1/2a4c1e4e160f590a48b57966.jpg"},{"id":71003569,"identity":"8a5a9620-bb21-4b5c-b4a9-0015b1737916","added_by":"auto","created_at":"2024-12-10 06:04:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1359130,"visible":true,"origin":"","legend":"\u003cp\u003eRelationships between CD206+SEMA6B+ TAM content and the clinicopathological characteristics and prognosis of cervical cancer patients (A) Tissue polychromatic immunofluorescence showed that CD206+SEMA6B+ TAMs were mainly located in the matrix of cervical cancer tissue CD206+ TAMs (green), SEMA6B+ TAMs (red), and DAPI+ TAMs (blue). In cervical cancer tissue, we selected representative images with low CD206+SEMA6B+TAM content (Patient ID: #1121 and density of CD206+SEMA6B+TAMs=0.25) and high CD206+SEMA6B+TAM content (Patient ID: # 1040 and density of CD206+SEMA6B+TAMs=0.36). (B) OS and DFS (C) in patients with high CD206+SEMA6B+ TAM content and low CD206+SEMA6B+ TAM content. The scatter plot shows the distribution of CD206+SEMA6B+ TAMs according to clinical stage (D), lymph node metastasis (LNM) (E), tumor grade (F), and lymphovascular space invasion (LVSI) (G). * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5304636/v1/110646e3b7f3509fd186b992.jpg"},{"id":71001235,"identity":"36873c5d-74b0-4933-aae8-36eae49cf716","added_by":"auto","created_at":"2024-12-10 05:40:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2237027,"visible":true,"origin":"","legend":"\u003cp\u003eDatabases confirmed that SEMA6B is associated with poor prognosis and immune infiltration in cervical cancer patients. (A) The relationships between SEMA6B expression and overall survival, progression-free survival and disease-specific survival in cervical cancer patients were analyzed using the TCGA dataset. (B) Univariate and multivariate analyses of the correlations between SEMA6B expression and pathological T stage, pathological N stage, histologic grade, age and histologic type in cervical cancer patients. (C) Rod diagram of immune infiltrating cell expression. (D) Stacked bar chart of immune infiltration differences between high and low expression groups of SEMA6B. (E) Analysis of the correlation between SEMA6B expression in tumor-related macrophages and tumor purity, as well as M2 macrophages, Treg infiltration, CD8+ T cells, and CD4+ T cells, in cervical cancer through the TIMER database.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5304636/v1/5fa592a06b3674cdbdba7dd4.jpg"},{"id":71001240,"identity":"dba949cb-3268-467d-87b1-d0343ed9780c","added_by":"auto","created_at":"2024-12-10 05:40:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1900221,"visible":true,"origin":"","legend":"\u003cp\u003eKnocking down SEMA6B inhibits macrophage M2 polarization and migration by inhibiting AKT phosphorylation. (A and B) Flow cytometry detection of the effect of SEMA6B knockdown on M2 macrophage polarization. (C) PCR analysis revealed the expression of M1 markers (CD86 and iNOS) and M2 markers (Arg-1 and CD206) after knocking down SEMA6B in macrophages. (D) Transwell assays were used to measure the impact of SEMA6B knockdown on the migration ability of macrophages. (E) Western blotting was used to determine the protein expression of AKT and pAKT in TAMs after SEMA6B knockdown in TAMs. (F) PCR analysis of changes in the mRNA expression of CXCL2 and CXCL8 in TAMs after SEMA6B knockdown. * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5304636/v1/4281962cca1d33e457101f92.jpg"},{"id":71001237,"identity":"60768f47-c272-4a0b-86e8-a0a20189c566","added_by":"auto","created_at":"2024-12-10 05:40:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1145590,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of knocking down SEMA6B in macrophages on the proliferation, of cervical cancer cells. EdU detection of the impact on the proliferation ability of SiHa cells (A) and HeLa cells (B) after coculture with macrophages with SEMA6B knockdown. * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5304636/v1/9af21347a8a291274df3aeec.jpg"},{"id":71001236,"identity":"28aa7f3e-55c3-4e61-ae30-880a7de035af","added_by":"auto","created_at":"2024-12-10 05:40:42","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3966702,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of knocking down SEMA6B in macrophages on the the invasion and migration ability of cervical cancer cells. (A) Transwell assays were used to determine the impact of SEMA6B knockdown on the invasion and migration ability of SiHa cells after coculture with macrophages. (B) A scratch assay was used to determine the impact of macrophage-mediated knockdown of SEMA6B on the migration ability of SiHa cells. (C) Transwell assays were used to determine the impact of SEMA6B knockdown on the invasion and migration ability of HeLa cells after coculture with macrophages. (D) A scratch assay was used to determine the impact of macrophage-mediated knockdown of SEMA6B on the migration ability of HeLa cells. (E) Western blot was used to determine E-cadherin, N-cadherin and Vimentin of SiHa and HeLa cells after co-cultured with macrophages knocked down SEMA6B. * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5304636/v1/cb6166eabf1f14be2db7a5c2.jpg"},{"id":86700105,"identity":"983a2232-478c-4d51-8230-92fcd9cbe6d8","added_by":"auto","created_at":"2025-07-14 16:11:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12888354,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5304636/v1/c3761c93-1d34-46f0-aac3-4aa58aca7868.pdf"},{"id":71001233,"identity":"03680fd7-5e65-4329-8b00-e35fb40a3090","added_by":"auto","created_at":"2024-12-10 05:40:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18392,"visible":true,"origin":"","legend":"","description":"","filename":"Suplementarytable.docx","url":"https://assets-eu.researchsquare.com/files/rs-5304636/v1/b910bd700f9828689495e578.docx"},{"id":71002231,"identity":"2adb333a-6816-4d35-9d29-c9c1194086a4","added_by":"auto","created_at":"2024-12-10 05:56:42","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1958430,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. \u0026nbsp;\u003c/strong\u003eHypoxia induces an increase in SEMA6B expression in TAMs in cervical cancer.\u003cstrong\u003e \u003c/strong\u003e(A) Schematic diagram of the process of collecting peripheral blood from cervical cancer patients to extract monocytes and induce them into macrophages. (B) Heatmap of the differentially expressed genes identified via transcriptional sequencing in cervical cancer-related macrophages treated with normoxia and hypoxia. (C) Pearson correlation analysis was used to determine the correlation between 31 differentially upregulated genes in OS. The depth of color and the size of the circle represent the correlation coefficient between paired genes. Red represents a positive correlation, blue represents a negative correlation, and * represents a statistically significant difference. (D) Network diagram and histogram (E) of 236 upregulated genes enriched and analyzed through GO and KEGG analyses.\u003c/p\u003e","description":"","filename":"SupplementFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5304636/v1/98060d72389dd15d884e44a6.jpg"}],"financialInterests":"","formattedTitle":"Hypoxia-induced Semaphorin 6B promotes the development of cervical cancer through regulating macrophage polarization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCervical cancer is a common malignant tumor of the female reproductive system and is the third most common cause of cancer death in women. According to global cancer statistics, in 2020, there were approximately 600000 new cases of cervical cancer each year, and 340000 women died from cervical cancer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although surgery and chemotherapy are currently the main treatment and prevention measures for cervical cancer, patients with advanced and recurrent metastatic cervical cancer still have poor prognoses [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The rise of immunotherapy provides hope for improving the treatment and survival of cervical cancer patients. However, due to the complexity of immune regulatory mechanisms in the tumor microenvironment (TME) and the heterogeneity of malignant tumors, tumor cells evade immune effects through various pathways [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, finding a new immunotherapy target for cervical cancer is crucial for improving the prognosis of cervical cancer.\u003c/p\u003e \u003cp\u003eHypoxia, the main characteristic of the tumor microenvironment, can not only enhance the invasion and metastasis ability of tumors but also promote tumor cells to enter a \"dormant state\" to avoid immune surveillance, leading to immunotherapy failure [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. TAMs intricately regulate antitumor immune responses in the tumor immune microenvironment through interactions with different immune cell subsets. TAMs can activate immune checkpoints, downregulate antigen presentation, and secrete regulatory factors to coordinate CD8\u0026thinsp;+\u0026thinsp;T-cell responses [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In addition, TAMs inhibit dendritic cell antigen presentation and infiltration. TAMs that secrete TGF-β CSF-1 can promote the expansion of MDSCs, while TAMs recruit immunosuppressive Treg cells and inhibit the function of NKT cells, leading to inhibitory effects [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the impact of hypoxia on macrophages is currently unclear, and understanding the mechanism by which hypoxia affects macrophages is crucial for identifying new immunotherapy targets for clinical cancer treatment.\u003c/p\u003e \u003cp\u003eWe analyzed the differential gene expression of macrophages induced from peripheral blood mononuclear cells of cervical cancer patients under hypoxic and normoxic conditions by mRNA sequencing and revealed a significant increase in SEMA6B expression. Recent research has confirmed that high SEMA6B expression is associated with poor prognosis and a tumor immunosuppressive microenvironment in colorectal cancer (CRC) patients [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Herein, we confirmed that high SEMA6B is associated with poor prognosis in cervical cancer patients and provided evidence that SEMA6B induces M2-polarized immune infiltration of macrophages to promote cervical cancer progression in vitro through the use of clinical data and experimental models combined with bioinformatics methods, indicating that SEMA6B could be a new immunotherapy target in cervical cancer.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e2.1 Cell culture and transfection\u003c/p\u003e \u003cp\u003e HeLa cells (cervical adenocarcinoma, CADC), SiHa cells (cervical squamous cell carcinoma, CSCC), and the human monocyte line THP-1 were purchased from the American Type Culture Library (ATCC, Manassas, Virginia, USA) and cultured according to its guidelines. All cell lines were identified within 2 years via short tandem repeat analysis. The cells were placed under low oxygen tension (1% O\u003csub\u003e2\u003c/sub\u003e, 5% carbon monoxide 2, and 94% N\u003csub\u003e2\u003c/sub\u003e). After the cells were placed in a normoxic incubator (21% O2, 5% carbon monoxide 2, and 74% N\u003csub\u003e2\u003c/sub\u003e), SEMA6B small interfering RNA (siRNA) was purchased from Tsingke Biotechnology Co., Ltd., and transfected with Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's protocol. All the siRNA target sequences are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e2.2 Human blood and tissue samples\u003c/p\u003e \u003cp\u003e The human body research plan was reviewed and approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China) (UHCT-IEC-SOP-016-03-01), and informed consent was obtained from all patients.\u003c/p\u003e \u003cp\u003eThis study conducted tissue microarray, immunofluorescence, and survival analyses on paraffin-embedded surgical specimens from 58 cervical cancer patients admitted from September 2020 to May 2021 who underwent surgery in our hospital before initiating antitumor therapy and who did not receive radiotherapy, chemotherapy, or antiangiogenic treatment. All patients were pathologically diagnosed with primary cervical cancer, with no other malignant diseases or symptoms related to immune or hematopoietic dysfunction. The pathological characteristics of these cervical cancer patients are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Patients with a follow-up period exceeding 6 months were included in the survival analysis. OS and DFS were defined as the time interval from surgery to the first tumor recurrence and death or the time interval to the last follow-up when no events occurred.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe relation of CD206 + SEMA6B + TAM to clinical pathological parameters in cervical cancer (n = 58).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eClinicopathological parameters\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN (%)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eThe density value of CD206 + SEMA6B + TAM\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigh(N = 32)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow(N = 26)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt; 45\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.813\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e≥ 45\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt; 24\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.826\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e≥ 24\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFIGO\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIA1-IB1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.030\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIB2-IIB\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistological type\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSquamous cell carcinoma\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.163\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdenocarcinoma\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistologic grade\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG1/G2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymph-vascular space invasion\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.048\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymph node metastasis\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.015\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e2.3 Ficoll density gradient centrifugation for isolating peripheral blood mononuclear cells and inducing macrophages\u003c/p\u003e \u003cp\u003e The human body research plan was reviewed and approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China), and informed consent was obtained from all patients. We collected peripheral blood from cervical cancer patients and separated peripheral blood mononuclear cells (PBMCs) using Ficoll Paque (TBD Science, Tianjin, China) density centrifugation. The collected PBMCs were placed in an incubator overnight cultivation; the cells were washed with RPMI 1640 (50 ng/mL M-CSF) once the next day to remove mixed lymphocytes; and a normal oxygen incubator (21% O2, 5% carbon monoxide 2, and 74% N2) was used. After 7 days of cultivation, monocytes develop into macrophages (Supplementary Fig.\u0026nbsp;1A). After 24 hours of hypoxia and normoxia culture, the collected macrophages were collected for hematocrit determination and mRNA sequencing.\u003c/p\u003e \u003cp\u003e2.4 mRNA sequencing (RNA-seq)\u003c/p\u003e \u003cp\u003eThe collected TAMs treated with hypoxia or normoxia as described in Section 2.3 above were sent to Servicebio (Wuhan, China) for RNA-seq analysis via an Illumina NovaSeq 6000 platform. DEGs were identified with a fold change ≥ 1.5 and p \u0026lt; 0.05 as the cutoff criteria. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were further conducted to interpret the biological significance of the DEGs.\u003c/p\u003e \u003cp\u003e2.5 Database analysis\u003c/p\u003e \u003cp\u003eFrom the Cancer Genome Map (TCGA) database (available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov/\u003c/span\u003e\u003cspan address=\"https://portal.gdc.cancer.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), RNA-Seq spectra and clinical information from CCa patients were downloaded to determine their correlation with clinical case characteristics and the prognosis of patients with SEMA6B and cervical cancer. It is a web-based platform for systematic analysis of immune infiltration in different cancer types in the TCGA. The platform uses deconvolution of previously published computational methods to infer TIICs from gene expression profiles. In this study, the expression of SEMA6B in tumor-related macrophages and its correlation with tumor purity, M2 macrophages, Treg infiltration, CD8 + T cells, and CD4 + T cells in cervical cancer were analyzed in the TCGA Cancer Service.\u003c/p\u003e \u003cp\u003e2.6 Tissue immunofluorescence\u003c/p\u003e \u003cp\u003eImmunofluorescence assays were performed on CD206 and SEMA6B cells using tissue microarrays. Briefly, after dewaxing, the slices were placed in antigen repair buffer under microwave irradiation at 15°C for 90 minutes. Then, they were washed and sealed at 10°C for 26 minutes and incubated overnight at 4°C with anti-CD206 (# ab313398; 1:1000; Abcam) and anti-semaphorin 6B (SEMA6B) (sc-390928; 1:200 dilution; Santa Cruz Biotechnology) antibodies. The slides were incubated with Alexa Fluor 488- and 594-conjugated secondary antibiotics (A11034, A21125; Invitrogen, Carlsbad, CA, USA). Immunostating was visualized under a slice scanner (Pannoramic MIDI: 3Dhistech, Hungary) on a scanning slide and evaluated by Image-Pro Plus Software (Media Cybernetics, Silver Springs, MD). The fluorescence intensities of SEMA6B, CD206, and DAPI were measured using a Zeiss scanning microscope. Using the SEMA6B/CD206\u0026amp;SEMA6B ratio, a value \u0026lt; 0.3 was defined as a low content of CD206 + SEMA6B + TAMs, and a value ≥ 0.3 was defined as a high content of CD206 + SEMA6B + TAMs.\u003c/p\u003e \u003cp\u003e2.7 Cellular immunofluorescence\u003c/p\u003e \u003cp\u003eTHP-1 cells were inoculated at a density of 24 or 30 cells/well into a 24-well plate, induced to differentiate into macrophages by adding PMA, and incubated overnight at 37°C. Then, the plate was fixed with 4% paraformaldehyde for 30 minutes. After washing twice with PBS and 1% BSA, the antigen was blocked at room temperature for 1 h and then incubated overnight with primary antibody (SEMA6B, sc-390928; 1:50 dilution; Santa Cruz Biotechnology) at 4°C. Next, the plate was incubated with a secondary antibody (goat anti-rabbit IgG Cy1600:1 dilution, ab2, Abcam) at room temperature for 30 hours and then incubated with DAPI for 15 minutes to stain the nucleus. Images were obtained using a fluorescence microscope (Olympus), and the average fluorescence intensity was quantified using ImageJ.\u003c/p\u003e \u003cp\u003e2.8 Coculture determination\u003c/p\u003e \u003cp\u003e A Transwell system (6 Transwell boards, 0.4 µM aperture, Corning Life Sciences, USA) was used for indirect cocultivation. After macrophages (2.5 × 10\u003csup\u003e4\u003c/sup\u003e cells) were inoculated into the insert, cervical cancer cells (2.5 × 10\u003csup\u003e4\u003c/sup\u003e cells) were cultured in the basal chamber. The cocultures were incubated for 48 to 72 hours, after which the cells were collected for subsequent experiments.\u003c/p\u003e \u003cp\u003e2.9 RNA extraction and quantitative real-time fluorescence quantitative PCR (qRT‒PCR)\u003c/p\u003e \u003cp\u003eTotal cell RNA was extracted using TRIzol reagent (TaKaRa), and reverse transcription was performed (HiScript® III RT SuperMix Vazyme). The primers were synthesized by Qingke Biotechnology Co., Ltd., and their sequences are shown in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. qRT‒PCR was performed using qPCR SYBR Green premix (Vazyme) on a Step-One Plus real-time fluorescence quantitative PCR system (Thermo Fisher). Three biological replicates were conducted.\u003c/p\u003e \u003cp\u003e2.10 Western blotting\u003c/p\u003e \u003cp\u003eWhole-cell protein extraction and Western blotting were performed according to previous methods [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The antibodies used for protein blotting are listed in Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e. The grayscale values of the protein bands were checked by ImageJ (ImageJ software, National Institutes of Health, Bethesda, Maryland). Each experiment was conducted at least three times.\u003c/p\u003e \u003cp\u003e2.11 5-Ethynyl-20-deoxyuridine (EdU) proliferation assay\u003c/p\u003e \u003cp\u003eEdU determination was carried out according to the manufacturer's instructions for the EdU experimental kit (RiboBio). The transfected cells were inoculated into a 000-well plate at a density of 96 to 10 cells per well. The proportion of EdU-positive cells is the proportion of proliferating cells. Images were obtained using a fluorescence microscope (Olympus). The results were obtained from three biological replicates. Each experiment was repeated in triplicate.\u003c/p\u003e \u003cp\u003e2.12 Wound healing and Transwell measurements\u003c/p\u003e \u003cp\u003eFor the wound healing assay, cells were inoculated into a 3-well plate at × 105 cells per well. After transfection with siRNA, the cells were cultured for 48 hours. When the cells formed a converging monolayer, a 200 µL pipette suction head was used to scrape them. An inverted optical microscope (Olympus, Japan) was used to visualize the wound area at 0 and 24 hours. ImageJ (version 1.51) software was used to measure the proportion of wound healing areas and represent the migration characteristics of cancer cells. For Transwell measurements, 200 µL of serum-free medium containing 5 × Inoculate cells onto the upper surface of the chamber coated without (migration assay) or with (invasion assay) matrix glue, and 600 mL of complete culture medium was added to the bottom chamber. After incubation at 37°C for 24 hours, the cells were fixed through 8x using 4% paraformaldehyde. Then, the cells were stained with 0.1% crystal violet. The number of cells in five random fields of view for each insert was calculated to evaluate the migration and invasion characteristics of the cancer cells at 200× magnification. Each experiment was conducted three times.\u003c/p\u003e \u003cp\u003e2.13 Statistical analysis\u003c/p\u003e \u003cp\u003eAll the statistical analyses were conducted using the R environment (version 3.6.3) and GraphPad Prism (version 8.0.2). Kaplan–Meier (KM) analysis was performed on the total survival period (OS, time from initial diagnosis to death or end of follow-up) using survival and survival packages, and logarithmic ranking tests were used. Survival packages for univariate and multivariate Cox regression analyses were used to screen for survival-related genes. Pearson correlation analysis was performed to determine the relationships between survival-related genes. The associations between SEMA6B expression and clinicopathological features were evaluated using the Mann‒Whitney U test. Significant differences between different groups were evaluated through Student’s t tests. A P value \u0026lt; 0.05 was considered to indicate statistical significance.\u003c/p\u003e "},{"header":"Results","content":"\u003cp\u003e3.1 Hypoxia induced increased expression of SEMA6B in macrophages\u003c/p\u003e\u003cp\u003eWe collected peripheral blood from cervical cancer patients and separated peripheral blood mononuclear cells (PBMCs) and induced to macrophages. After 24 hours of hypoxia and normoxia culture and the RNA sequencing results showed that 236 genes were upregulated and 385 genes were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, S1B). The 236 upregulated genes were subjected to GO and KEGG enrichment analyses. These genes were mainly enriched in chemokines, neutral migration, cytokine receptors and activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, S1D, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Thirty-one genes whose expression was significantly upregulated were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and the upregulated DEGs was associated with OS through the TCGA database (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). To evaluate the correlation between the 31 OS-related genes, Pearson correlation analysis was conducted, and 31 identified key genes exhibited a coexpression pattern (S1C). Semaphorins play important roles in regulating various immune system responses. Tian et al. revealed that Sema3A drives alternative macrophage activation in the resolution of periodontitis via PI3K/AKT/mTOR Signaling [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Semaphorin 3G exacerbates joint inflammation through the accumulation and proliferation of macrophages in the synovium [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].Cellular immunofluorescence revealed that SEMA6B was expressed in both the cytoplasm and cell membrane of TAMs and that the expression of SEMA6B was significantly increased after hypoxia treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e3.2 Validation of the association between CD206 + SEMA6B + TAMs and the progression of cervical cancer\u003c/p\u003e\u003cp\u003eThe results of tissue polychromatic immunostaining showed that CD206 + SEMA6B + TAMs were mainly located in the cervical cancer tissue matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We confirmed that the greater the percentage of CD206 + SEMA6B + TAMs was, the worse the OS and DFS were for cervical cancer patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). We further revealed that abundant CD206 + SEMA6B + TAMs were associated with poor prognosis, late clinical stage, lymph node metastasis, poor differentiation, and lymphovascular space invasion in cervical cancer tissues (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-G).\u003c/p\u003e\u003cp\u003e3.3 Databases confirm that SEMA6B is associated with poor prognosis and immune infiltration in cervical cancer patients\u003c/p\u003e\u003cp\u003e According to the analysis of the TCGA dataset, the overall survival, disease-specific survival, and progression-free survival of cervical cancer patients with high expression of SEMA6B were significantly reduced (P = 0.008, 0.025 and 0.038, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Multivariate analysis revealed that high SEMA6B expression was closely related to lymph node metastasis and late pathological T stage in cervical cancer patients (P = 0.002 and \u0026lt; 0.003, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To further determine the correlation between SEMA6B and immune infiltration, we analyzed the relationships between SEMA6B and different immune cell marker genes using the TIMER database. After correlation adjustment through purity, the results showed that SEMA6B mainly expressed in Neutrophils, Mast cell, Eosinophils, Tem and Macrophages(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC,\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and was positively correlated with the infiltration of M2 macrophages and Tregs and negatively correlated with the infiltration of CD8 + T cells and CD4 + T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), SEMA6B indicating that SEMA6B plays a crucial role in immune escape by promoting the immunosuppressive tumor microenvironment.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003e3.4 Knocking down SEMA6B inhibits macrophage M2 polarization and migration by inhibiting AKT phosphorylation\u003c/p\u003e\u003cp\u003eThe effect of knocking down SEMA6B on polarization was detected by flow cytometry, and the results confirmed that knocking down SEMA6B can inhibit CD206 + M2 polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). PCR revealed an increase in the expression of M1 markers (CD86 and iNOS) and M2 markers (Arg-1 and CD206) after knocking down SEMA6B (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The migration ability of macrophages was evaluated by transwell assays, and as expected, SEMA6B-knockdown macrophages exhibited reduced migration ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Previous studies have confirmed that AKT phosphorylation are associated with macrophage polarization [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. We further determined that knocking down SEMA6B in macrophages inhibited AKT phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Chemokines serve as the main cytokines involved in communication between TAMs and cervical cancer cells. Previous studies have confirmed that CXCL2 and CXCL8 are downstream genes of AKT [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. We further determined that knocking down SEMA6B in macrophages can reduce the expression of CXCL2 and CXCL8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These results suggested that SEMA6B could regulate macrophage polarization in cervical cancer.\u003c/p\u003e\u003cp\u003e3.5 Knocking down SEMA6B in macrophages inhibits the proliferation of cervical cancer cells\u003c/p\u003e\u003cp\u003eAfter coculture of macrophages with SEMA6B knockdown and cervical cancer cells, the proliferation of SiHa and HeLa cells was significantly reduced according to EdU proliferation assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e3.5 Knocking down SEMA6B in macrophages inhibits the migration and invasion of cervical cancer cells\u003c/p\u003e\u003cp\u003eAfter coculture of macrophages with SEMA6B knockdown and cervical cancer cells, the invasion and migration abilities of SiHa and HeLa cells were significantly reduced according to Transwell assays (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The migration ability of SiHa and HeLa cells was significantly reduced according to the scratch assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Western blot analysis revealed that E-cadherin expression increased, while N-cadherin and vimentin expression decreased in SiHa and HeLa cells after coculture with macrophages with SEMA6B knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). These results suggested that SEMA6B could be a new prognostic immunological biomarker in cervical cancer that promotes tumor progression.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough immunotherapy for cervical cancer has achieved significant clinical success, its clinical application is greatly limited due to its low response rate, low applicability and severe adverse reactions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The development of novel immune biomarkers and targets for the diagnosis and immunotherapy of cervical cancer is highly important. This study is the first to show that hypoxia induces increased expression of SEMA6B in macrophages induced from peripheral blood mononuclear cells of cervical cancer patients, and SEMA6B could be a new immunotherapy target in cervical cancer that regulates macrophage polarization and promotes tumor progression.\u003c/p\u003e \u003cp\u003eHypoxia plays an important role in the recurrence and metastasis of malignant tumors. Multiple studies have confirmed that hypoxia stimulates cancer cells to undergo mitotic arrest, allowing them to escape immune monitoring and form an immunosuppressive microenvironment, leading to recurrence, chemotherapy resistance, and distant metastasis [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Multiple studies have confirmed that a large number of tumor-related macrophages aggregate in the hypoxic zone of tumor tissue and play important roles in the occurrence, growth, invasion, metastasis, angiogenesis, and lymphangiogenesis of tumors [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. TAMs are attractive targets for tumor immunotherapy, but the lack of specific biomarkers hinders their research and clinical application. Understanding the mechanism by which hypoxia affects TAMs and identifying specific biomarkers are highly important for the diagnosis, recurrence, and metastasis of cervical cancer. From a structural point of view, these semaphorin proteins contain a highly conserved N-terminal semaphorin domain of approximately 500 amino acids, a plexin\u0026ndash;semaphorin\u0026ndash;integrin domain and distinct protein domains that further define semaphorins, including immunoglobulin-like, thrombospondin, and basic C-terminal domains [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, we found a significant increase in SEMA6B expression in macrophages induced from peripheral blood mononuclear cells of cervical cancer patients under hypoxia conditions. Previous studies have confirmed that SEMA4B is a direct target of hypoxia inducible Factor 1 (HIF-1) α, a hypoxia responsive element (HRE) that can recognize the SEMA4B gene [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, Sema3A is expressed at low levels in hypoxia-treated cardiomyocytes due to hypoxia-induced inflammatory factor (TNF)-α and IL-1β, which are related to IL-6 secretion, decreased cell viability, myocardial cell apoptosis, ROS release, decreased ATP production, and a decreased GSH/GSSG ratio [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, different SEMA proteins may play different roles in hypoxic microenvironments. To our knowledge, this is the first report to confirm that hypoxia induces increased expression of SEMA6B in cervical cancer, but the mechanisms involved need to be further explored.\u003c/p\u003e \u003cp\u003eAccording to the analysis of the TCGA dataset, the prognosis of cervical cancer patients with high expression of SEMA6B is poor. High expression of SEMA6B was closely related to lymph node metastasis and late pathological T stage in cervical cancer patients. Another noteworthy finding of this study is that SEMA6B is positively correlated with the infiltration of M2 macrophages and Tregs and negatively correlated with CD8\u0026thinsp;+\u0026thinsp;T cells and CD4\u0026thinsp;+\u0026thinsp;T cells, indicating that SEMA6B plays a crucial role in immune escape by promoting the immunosuppressive tumor microenvironment. These results are consistent with those of previous studies by Li et al., who confirmed a moderate to strong positive correlation between SEMA6B expression levels and infiltration levels in macrophages, MDSCs, NK cells, Tregs and Th6 cells in colorectal cancer and a significant positive correlation between infiltration levels of CD4 T cells, neutrophils, and dendritic cells and SEMA6B expression in colorectal cancer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. We found that SEMA6B is expressed in both the nucleus and cytoplasm of macrophages in cervical cancer tissue, and we further revealed that abundant CD206\u0026thinsp;+\u0026thinsp;SEMA6B\u0026thinsp;+\u0026thinsp;TAMs were associated with poor prognosis, late clinical stage, lymph node metastasis, poor differentiation, and lymphovascular space invasion in cervical cancer tissues. The proliferation, invasion and migration ability of SiHa and HeLa cells significantly decreased after coculture with macrophages with SEMA6B knockdown. In summary, our research suggested that high SEMA6B expression predicts poor overall survival (OS) and may serve as a potential prognostic immune marker for cervical cancer patients.\u003c/p\u003e \u003cp\u003ePrevious studies have confirmed that the SEMA family mainly acts on the integrins β1, NRP and plexin C1 receptors to regulate various physiological and pathological processes [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Kang et al. revealed the important roles of the mTOR-Sema6D-PPARγ signaling pathway in controlling macrophage polarization [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Another study suggested that the association of SEMA4D with M2 macrophages may help stimulate tumor angiogenesis in epithelial ovarian cancer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. SEMA3A can bind to the NP1-1 receptor of TAMs, leading to downstream PI3K/Akt phosphorylation, continuous recruitment of monocytes and M2 polarization, thereby promoting cancer cell resistance to androgen deprivation therapy [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A recent study confirmed that hypoxia therapy can polarize macrophages toward the M2 phenotype and promote the development of keloids, mainly by activating the PTEN-PI3K/AKT pathway [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This research result is consistent with our findings.\u003c/p\u003e \u003cp\u003ePrevious studies have confirmed that recombinant SEMA3A can induce AKT or NF by binding to a complex of the T-cell surface receptors NRP-1 and PlexinA1- κ to inhibit B signal transduction [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. AKIP1 regulates NFκB via the CXCL1, CXCL8 and AKT pathways and promotes GBM activity, migration, and chemical radiation resistance [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Previous studies have confirmed that malaria parasite infection in female BWF1 lupus mice regulates the CXCL12/CXCR4 axis and its downstream signals PI3K/AKT, NF-κB and ERK to weaken B-cell autoreactivity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Atelectasis causes an alveolar hypoxia-induced inflammatory response, mainly through the secretion of NF-κB by pulmonary epithelial cells, which is dependent on increased CXCL-1 secretion [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our study confirmed that knocking down SEMA6B in macrophages can inhibit macrophage polarization toward M2 and inhibit macrophage migration by inhibiting AKT phosphorylation.\u003c/p\u003e \u003cp\u003eIn summary, this study revealed the important role of SEMA6B in the poor prognosis of cervical cancer patients. We speculate that SEMA6B can serve as a new immunotherapy target in cervical cancer, providing new ideas for in-depth research on the effectiveness and safety of immunotherapy. However, the mechanism of SEMA6B in the hypoxic tumor immune microenvironment of cervical cancer and its potential target for targeting TAMs still need further exploration. In addition, these findings require additional preclinical models, clinical patient cohorts, and clinical trials for further validation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (UHCT-IEC-SOP-016-03-01). Written informed consent was obtained from all patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;No fundings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLufang Wang:\u003c/strong\u003e Conceptualization, Methodology, Software. \u003cstrong\u003eShuyan Yi:\u003c/strong\u003e Data curation, Writing- Original draft preparation. \u003cstrong\u003eSha Hu:\u003c/strong\u003e Visualization, Investigation. \u003cstrong\u003eWenhan Li:\u003c/strong\u003eSupervision.\u003cstrong\u003e\u0026nbsp;Jing Cai:\u003c/strong\u003e Software, Validation. \u003cstrong\u003eLiqiong Cai:\u003c/strong\u003e Writing- Reviewing and Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analyzed in this study are available at TCGA (http://www.cancer.gov/tcga), GEO (https://www.ncbi.nlm.nih.gov/gds/) and TIMER database (http://timer.comp-genomics.org/ ).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSingh D, Vignat J, Lorenzoni V, Eslahi M, Ginsburg O, Lauby-Secretan B, Arbyn M, Basu P, Bray F, Vaccarella S. 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Intensive Care Med Exp 3:56, 2015.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hypoxia, Semaphorin 6B, cervical cancer, macrophage polarization","lastPublishedDoi":"10.21203/rs.3.rs-5304636/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5304636/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe progression of cervical cancer is closely related to the tumor microenvironment (TME) (which includes hypoxia and macrophages). However, the impact of hypoxia on macrophages remains to be determined. In the present study, mRNA sequencing was used to detect differential gene expression in macrophages induced from peripheral blood mononuclear cells of cervical cancer patients under hypoxic and normoxic conditions, and 236 genes were upregulated in macrophages exposed to hypoxia; these genes were mainly enriched in response to chemokines and the actin cytoskeleton. The expression of semaphorin 6B (SEMA6B) significantly increased after hypoxia treatment, and high expression of SEMA6B was related to poorer survival in cervical cancer patients. Multicolor immunofluorescence revealed that abundant CD206\u0026thinsp;+\u0026thinsp;SEMA6B\u0026thinsp;+\u0026thinsp;TAMs were associated with poor prognosis, late clinical stage, lymph node metastasis, poor differentiation, and lymphovascular space invasion in cervical cancer patients. TIMER database analysis revealed that SEMA6B expression was positively correlated with the infiltration of M2 macrophages and Tregs and negatively correlated with the infiltration of CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells. In vitro, knocking down SEMA6B in TAMs inhibited macrophage M2 polarization and the migration of macrophages. Furthermore, after coculture of macrophages with SEMA6B knockdown and cervical cancer cells, the proliferation, migration and invasion of SiHa and HeLa cells was significantly reduced. In conclusion, SEMA6B is a promoting factor for the development of cervical cancer. Targeting SEMA6B may be a potential immunotherapy approach for treating cervical cancer.\u003c/p\u003e","manuscriptTitle":"Hypoxia-induced Semaphorin 6B promotes the development of cervical cancer through regulating macrophage polarization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-10 05:40:31","doi":"10.21203/rs.3.rs-5304636/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2024-12-25T14:11:33+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-11-05T22:46:51+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-05T22:38:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-01T04:43:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2024-10-21T08:55:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7299cf92-5e21-4d8b-a965-9dcf16980c91","owner":[],"postedDate":"December 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-14T16:09:35+00:00","versionOfRecord":{"articleIdentity":"rs-5304636","link":"https://doi.org/10.1186/s12967-025-06692-z","journal":{"identity":"journal-of-translational-medicine","isVorOnly":false,"title":"Journal of Translational Medicine"},"publishedOn":"2025-07-10 15:57:46","publishedOnDateReadable":"July 10th, 2025"},"versionCreatedAt":"2024-12-10 05:40:31","video":"","vorDoi":"10.1186/s12967-025-06692-z","vorDoiUrl":"https://doi.org/10.1186/s12967-025-06692-z","workflowStages":[]},"version":"v1","identity":"rs-5304636","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5304636","identity":"rs-5304636","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}