CC tissue and normal cervical samples were collected from individuals who underwent surgical resection at Shanghai First Maternity and Infant Hospital between the time span of May 2020 and June 2023. All clinicopathological diagnoses were confirmed by a minimum of two pathologists. None of the patients received chemotherapy or radiotherapy prior to the surgery. Immediately after resection, all tissue samples were promptly preserved in liquid nitrogen and stored in a freezer at a temperature of -80°C until further analysis. The study was granted approval by the Ethics Committee of the Shanghai First Maternity and Infant Hospital, with all participating patients having provided informed consent by signing the necessary forms.

HEK 293T, THP-1, RAW264.7, and CC cell lines (TC-1, HeLa, SiHa, Caski, C33A and ME180) were obtained from the Cell Banks of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), while Caski, C33A, ME180 and TC-1 cell lines were purchased from FuHeng Biology (Shanghai, China). Cells were cultured in DMEM (Gibco, USA) for HEK 293T, RAW264.7, and CC cell lines, and RPMI 1640 medium (Gibco, USA) for THP-1, in a 37°C, 5% CO2 humidified incubator. All media were supplemented with 10% fetal bovine serum (C9050, NCM Biotech, China) and 1% penicillin/streptomycin.

Total RNA was extracted from cells or tissues by using RNAiso Plus reagent (Takara, Japan) in accordance with the instructions. The RNA was reverse-transcribed to cDNA using ABScript II RT Master Mix (RK20429, ABclonal, China). qPCR was performed using Genious 2× SYBR Green Fast qPCR Mix (RK21203, ABclonal, China) according to the manufacturer’s protocols. Target gene expression was standardized to GAPDH expression. The primer sequences are listed in Supplementary Table S1 .

The antibodies and reagents used in this study were shown in Supplementary Table S2 .

In brief, cell or exosome samples were collected using standard RIPA buffer (WB3100, NCM Biotech, China) containing protease and phosphatase inhibitor cocktails (P002, NCM Biotech, China). WB analyses were conducted according to the previously described method (13). The quantification of protein bands was performed using Image J software (USA).

Tissue sections were dewaxed with xylene and dehydrated with different gradient alcohol series. After that, the slices were treated with 3% hydrogen peroxide and pressed boiled to extract the antigens. The slices were then incubated with antibodies overnight at 4°C. Then incubated with the corresponding secondary antibodies at room temperature (25°C) for 1h. Immunostaining is performed using 3’-diaminobenzidine (DAB) according to the manufacturer’s instructions. Finally, the slices were stained with hematoxylin and sealed with neutral gum.

The CMTM6 small interfering RNAs (siRNAs) were provided from Hanbio Biotechnology Co., Ltd (Shanghai, China) and Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher Scientific, USA) was used to transfect the siRNAs into targeted cells according to the manufacturer’s instructions. 12 h after transfection, the cell culture medium was replaced with a fresh medium for subsequent experiments. All sequences used in this study are available in Supplementary Table S3 .

For shRNA work, the designed Oligo (Youbio, Changsha, China) was annealed to form a double-stranded chain, while the carrier pLKO.1 was digested by EcoRI and AgeI enzymes. The annealed product was then connected to the carrier. The synthesized shRNA plasmid, psPAX2, and pMD2.G were all simultaneously transferred into 293T cells to synthesize the virus. The virus was then transfected into the target cells, which were subsequently screened with 4ug/ml puromycin for 48h. The TC-1 cells with stable gene knockout were harvested. Non-targeted shRNA was used as control.

For CRISPR/Cas9 knockout system, lentiCRISPRv2 was utilized as a vector and digested with FastDigest enzyme. The remaining steps followed the same procedure as shRNA work. The sgRNA were designed using the MIT online tool (http://crispr.mit.edu). The effectiveness of gene depletion was assessed through WB. All sequences used in this study are available in Supplementary Table S3 .

The cells under investigation were seeded into individual wells of a 6-well plate at a density of 1,000 or 2,000 cells per well. Following a 10-day incubation period, the cells were immobilized using a 4% paraformaldehyde fixative for a duration of 20 min. Subsequently, the cells were stained with a 0.5% crystal violet solution for 20 min. Finally, the number of clonally derived cells was quantified.

Following the digestion with EDTA-free trypsin, the cells intended for analysis were treated with annexin V-FITC and PI fluorescence (40302ES08, Yeason Biotechnology, Shanghai) staining and incubated in 100μL of binding buffer for 30 min in a light-free environment. Flow cytometry was employed to capture fluorescence data, with a total of 1 × 10⁴ cells recorded per sample.

The migratory and invasive capabilities of CC cells were assessed using 24-well transwell plates (Corning, USA) equipped with an 8.0μm pore polycarbonate membrane. For the invasion assay, the upper surface of the membrane was pre-coated with Matrigel mix (D23016-0010, D1 Medical Technology, Hangzhou, China), while for the migration assay, no coating was applied. Tumor cells were seeded in the upper chamber using 200 µL of serum-free DMEM medium, while the bottom chamber was supplemented with 600 µL of medium containing 10% FBS. After a 24-h incubation period, the cells remaining in the upper chamber were gently removed, and the cells that had either migrated through the membrane or invaded it were fixed with paraformaldehyde and stained using a 0.1% crystal violet solution. Images were captured using an inverted microscope, and cell numbers were quantified utilizing the ImageJ software (USA).

The lung tissues were fixed in paraffin and cut into 4 µm sections. Hematoxylin and eosin (H&E) staining was performed using the H&E Staining Kit (SD6146, SIMUWU, China) according to the manufacturer’s instructions.

Specimens were prepared according to the previously described method (3). Labeled cells were analyzed on a BD FACSCalibur or BD FACSCanto™ II flow cytometer and the data were processed using FlowJo software (USA).

Macrophages were transformed from THP-1 cells. Cells were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% FBS at 37°C in a humidified 5% CO₂ atmosphere. To transform the cells into macrophages(M0), THP-1 monocytes were cocultured with 100 ng/mL phorbol 12-myristate 13-acetate (PMA, HY18739, MedChemExpress, China) in RPMI 1640 medium without FBS for 48 h. Macrophages treated with 20 ng/ml interleukin 4 (IL-4) (HY-P70445, MedChemExpress, China) for 24 h were polarized into M2 phenotype (6). To investigate the effects of exosomes on macrophages, 40μg of exosomes were added to the macrophage culture medium and incubated in the cell incubator for another 24 or 48 h prior to harvesting cells for subsequent experiments.

For the in vitro phagocytosis assay, 1×10⁵ macrophages were stained with the CD11b antibody and plated in transparent 96-well plates. Simultaneously, 1×10⁵ tumor cells were labeled with a 5μM green fluorescent dye, CFSE (BD Biosciences). After co-culturing macrophages with tumor cells for 4 h, FCM was employed for result analysis.

Ultracentrifugation methods were applied to isolate exosomes from the supernatants of CC cells according to previously published protocol (14). In brief, the cells were cultured in a complementary medium until reaching approximately 80% confluence, Then the medium was replaced with a defined medium without FBS. After 2 days of culture, the supernatants were harvested and subjected to sequential centrifugations at 800 × g for 15 min, 2,000 × g for 15 min, and 10,000 × g for 60 min. The resulting supernatants were filtrated through a 0.22μm PVDF filter (Millipore, USA). The filtered supernatants were collected to isolate exosomes by ultracentrifugation using 120,000 × g for 2 h (Beckman Coulter). Temporarily unused exosomes were frozen at -80°C with PBS. For exosome identification, 50μL of exosomes resuspended in PBS were adsorbed onto a copper mesh with a polyvinyl methylacetate acetate support membrane, stained with 2% phosphotungstic acid, and observed for size using a transmission electron microscope (TEM). The particle size and concentration of exosomes were observed using nanoparticle tracking analysis (NTA).

To monitor exosomal trafficking, exosomes isolated from the culture medium were labeled with a Dil fluorescent cell linker kit (Thermo Fisher Scientific, USA). After Dil staining, the exosomes were washed in PBS and resuspended in PBS. Plus, the Dil-labeled exosomes were incubated with macrophages for 24 h. Nuclei and the cytoskeleton were stained with DAPI and FITC-phalloidin (40735ES75, Yeason Biotechnology, Shanghai), respectively. Finally, the uptake of exosomes was examined by confocal fluorescence microscopy.

Secretion of CCL2 into the culture supernatant from macrophages were detected with the ELISA kits (YPG0179, UpingBioHANGZHOU, China) according to the manufacturer’s instructions.

All animal care and experiments were conducted in accordance with the guidelines stipulated by the National Institutes of Health and approved by the Animal Care Committee of Tongji University. C57BL/6 and Balb/c mice (6–8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Company and settled in a specific pathogen-free environment. Mice were randomly allocated to groups and received a subcutaneous injection of 2 × 10⁶ mouse tumor cells (150μL) in the animal’s right flank.

In tumor metastasis assay in vivo, Balb/c mice were intravenously injected with tumor cells (2 × 10⁶ cells per mouse) via their tail veins. After an 8-week period, the mice were humanely sacrificed, and the number of metastatic foci was computed.

For the effects of exosomes on macrophages in vivo, the macrophages were pretreated with either PBS or exosomes derived from TC-1. Next, a mixture of tumor cells and macrophages in a 10-to-1 ratio was injected subcutaneously into C57BL/6 mice. A total of 4 intratumoral injections of either PBS or exosomes were administered before the mice were euthanized.

Tumor volumes were measured at fixed intervals and calculated as volume = width² × length × 0.5. The tumor and lung tissues were fixed with paraformaldehyde for HE and IHC staining.

Statistical analyses were performed using R (v4.2.2) or GraphPad Prism (v.9) software. Cell counts were analyzed using the ImageJ software, and FCM data were quantified using the FlowJo (v10) software. Details are provided in the figure legends. A p-value of <0.05 was considered statistically significant.

To investigate the potential role of CMTM family in CC, we initially analyzed the copy number variations (CNV) of CMTM1–8 in The Cancer Genome Atlas (TCGA) dataset as shown in Figure 1A . It was found that CMTM2/6/8 are typically amplified in CC and the amplification of these genes is correlated with increased mRNA expression. Further research showed a significant upregulation of CMTM6 compared to CMTM2 and CMTM8 ( Figure 1B ). Following that, we found that CMTM6 genetic alteration was linked with lymph node HE staining positive and advanced stage via cbioportal (https://www.cbioportal.org/) database ( Figure 1C ; Supplementary Figure S1A-C ). In addition, the data from TCGA showed that CMTM6 expression in CC tissues was higher than that in normal tissues ( Figure 1D ). This is further confirmed by the cohort (30 CC tissues and 20 normal tissues) of Shanghai First Maternal and Infant Health Hospital and Gene Expression Omnibus (GEO: GSE7410, GSE6791, GSE63514) database ( Figures 1E–H ). The relationship between CMTM6 expression and a range of clinicopathological parameters was systematically evaluated and found that CMTM6 expression exhibited a significant positive correlation with both tumor size and stromal invasion. In contrast, no statistically significant associations were observed between CMTM6 expression and age, pathologic types, FIGO stage, lymph node metastasis, lymphovascular invasion, and vaginal involvement ( Table 1 ). Then, WB and IHC analysis both revealed that CMTM6 expression was higher in tumor tissues than matched non-tumor tissues ( Figures 1I, J ). Finally, we analyzed the relationship between CMTM6 expression levels and patient prognosis using the TCGA cohort and/found that higher CMTM6 expression was positively associated with poorer overall survival (OS) and progression-free interval (PFI) ( Figure 1K ). To assess the diagnostic value of CMTM6 in CC, a receiver operating characteristic (ROC) curve was generated, and the area under the curve (AUC) for CMTM6 was calculated to be 0.721 ( Figure 1L ), suggesting that CMTM6 mRNA expression may serve as a predictive factor for CC progression. Collectively, CMTM6 probably serve as a promising diagnostic biomarker for CC patients and its upregulation linked with inferior survival.

To explore the biological effects of CMTM6 in CC, we firstly detected CMTM6 expression in CC cell lines ( Supplementary Figure S2A ). Therefore, we transfected CMTM6 siRNA (defined as siCMTM6#1 and siCMTM6#2, respectively) into HeLa and Caski cell lines. WB analysis exhibited that siRNAs obviously suppress CMTM6 protein expression ( Figure 2A ). We also constructed CMTM6 knockdown TC-1 cell—a murine-derived cell line expressing HPV16 E6 and E7—with two shRNAs (named as sh-CMTM6–1 and sh-CMTM6-2), with sh-NC as the control ( Supplementary Figure S2B ). Compared to the control, silencing CMTM6 impaired the clonogenicity of CC cells ( Figures 2B, C ; Supplementary Figure S2C ). Consistently, Transwell assay revealed that downregulation of CMTM6 expression remarkedly impaired the migration and invasion behavior in CC cells ( Figure 2D, E ; Supplementary Figures S2D, E ). As expected, the down-regulation of CMTM6 enhanced the apoptosis ability of CC cells ( Figures 2F, G ; Supplementary Figure S2F ). Given the aforementioned findings, we examined the influence of CMTM6 of CC in vivo employing TC-1 tumor- bearing mice and lung metastasis model. The results disclosed that CMTM6 knockdown undermined tumor proliferation ( Figures 2H, I ). Afterwards, the WB analysis indicated CMTM6 low-expression in tumor tissues and IHC showed that CMTM6 knockdown inhibited the Ki67 expression ( Figures 2J, K ). In the lung metastasis model, CMTM6 knockdown markedly reduced both the number and size of metastatic nodules. Metastatic lung tissues showed severe disruption of alveolar architecture, enlarged pleomorphic tumor cell nuclei, and abundant neovascularization ( Figure 2L ). Generally speaking, these discoveries indicate that CMTM6 exerts an oncogenic role in CC.

To further investigate the oncogenic role of CMTM6 in CC, we firstly queried the STRING database to identify 50 interactors of CMTM6 ( Figure 3A ). Then we performed enrichment analysis on these interactors, and GO results showed they are significantly linked to immune regulation, including cytokine activity and immune response signaling. ( Figure 3B ; Supplementary Figures S3A, B ). Building on these results, we speculated that CMTM6 might play a role in shaping the tumor immune microenvironment in CC. Hence, we first analyzed the relationship between CMTM6 expression and tumor-infiltrating immune cells using the GEO dataset GSE63514 (n = 104). The results showed that CMTM6 expression was significantly correlated with increased infiltration of CD68-positive macrophages, whereas the correlations with markers of CD8⁺ T cells (CD8A), B cells (CD19), regulatory T cells (Tregs, CD25), dendritic cells (DCs, CD11c), and neutrophils (LY6G) were relatively weak and not statistically significant. Similarly, the correlation with markers of myeloid-derived suppressor cells (MDSCs) was also limited ( Figure 3C ; Supplementary Figure S3C ). Collectively, the data suggest that CMTM6 may be specifically associated with the infiltration of TAMs in the CC microenvironment. Moreover, IHC demonstrated that heightened CMTM6 in human tissues was closely linked to a marked augmentation in macrophage infiltration, a trend that was also observed in cases with TC-1 mouse tumors ( Figures 3D, E ). These results indicate that CMTM6 is associated with enhanced macrophage infiltration, suggesting its potential role in modulating macrophage-mediated immune responses in CC.

To further validate our findings, we stratified the TCGA-CESC cohort into four groups based on CMTM6 expression and macrophage infiltration levels. Survival analysis revealed that patients with high CMTM6 expression and increased macrophage infiltration had the poorest prognosis ( Figure 3F ). Macrophages are key to phagocytosis, a vital defense mechanism. Tumor cells exploit immune checkpoint molecules (ICMs), like PD-L1 and CD47, to escape macrophage-mediated surveillance (15). To verify our hypothesis, we conducted in vitro macrophage phagocytosis assays. The results proved that macrophages engulfed more CMTM6-KO cells compared to the control cells ( Figure 3G , Supplementary Figures S3D–F ). FCM results exposed a significant downregulation of CD47 expression in CMTM6-KO CC cells, but PD-L1 expression was not affected ( Figures 3H, I ; Supplementary Figure S3G, H ). Likewise, the findings from the TCGA-CESC reinforce this ( Figures 3J ). To assess whether the suppression of tumor growth following CMTM6 knockdown is attributable to a reduction in macrophage-mediated effects, we employed anti-F4/80 (α-F4/80) antibody to deplete macrophages in the TC-1 tumor model. Macrophage depletion alone produced a tumor-inhibitory effect comparable to that observed with CMTM6 knockdown. Notably, the most pronounced attenuation of tumor progression occurred when CMTM6 knockdown was combined with macrophage depletion ( Figures 3K–M ). WB results also showed down-regulated protein expression of CD68 in the tumor-bearing mice ( Figure 3N ). These findings suggest that the absence of CMTM6 in the tumor impairs the infiltration of TAMs and reduces oncogenesis.

Subsequently, we expounded which phenotype of macrophages the CMTM6 molecule is associated with. Through the analysis of online databases, we identified a negative correlation between CMTM6 expression and M1 macrophage ( Figure 4A ). In contrast, M2 macrophages are now recognized to have four phenotypes (M2a, M2b, M2c, M2d), and we found that CMTM6 is positively correlated with CD206, a classical marker of M2a ( Figure 4B ). Enrichment analysis of 50 genes positively correlated with CMTM6 expression in TCGA-CESC cohort showed that CMTM6 might play a role in cytosolic transport, which was confirm by GEPIA database ( Figure 4C ; Supplementary Figures S4A, B ), suggesting that CMTM6 may be packaged in exosomes. Therefore, we investigated the potential presence of CMTM6 in exosomes, after characterizing the exosomes via TEM and NTA, WB analysis revealed significantly higher CMTM6 protein expression in exosomes compared to whole cell lysates (WCL) ( Figure 4D ; Supplementary Figures S4C, D ). Consistently, cellular changes in CMTM6 also impact its levels in exosomes. Notably, when CC cells are treated with exosomal pharmacological inhibitors (GW4869), the CMTM6 expression in exosomes is altered, while its cellular levels remain unaffected ( Supplementary Figures S4E–G ). This suggests the presence of a compensatory intracellular degradation mechanism, likely mediated by both the proteasome and autophagy pathways, which prevents accumulation of CMTM6 upon inhibition of its exosomal secretion. The effect of GW4869 on exosomal CMTM6 mainly relies on these compensatory degradation processes rather than intracellular accumulation, which still needs further investigation to fully elucidate the underlying molecular mechanisms.

Recent studies have highlighted the crucial role of exosomes in regulating the interaction between tumor cells and TAMs (14, 16). We hypothesize that CMTM6 is transferred to TAMs via exosomes, promoting their differentiation into M2a. Confocal and WB analyses confirmed that macrophages can internalize exosomes ( Figures 4E, F ). Macrophages educated with CMTM6-altered exosomes (Exos^{CMTM6 KO} and Exos^TC1-sh-CMTM6) had fewer M2a macrophages compared to those educated with control (WT-Exos and Exos^TC1-sh-nc) ( Figures 4G, H ). To rule out the possibility that other changes in exosomal content caused this effect, we measured mRNA levels of several M2-related factors (TGF-β, IL-10, ARG1) in exosomes from CMTM6-knockdown cells, and found no significant differences compared to controls. This suggests the effect is mainly due to CMTM6 loss ( Supplementary Figure S5 ). Due to the fact that macrophage polarization is usually accompanied by activation of intracellular signaling pathways, we firstly conducted gene set enrichment analysis (GSEA) found that CMTM6 in the TCGA-CESC cohort has a regulatory effect on mTOR signaling ( Figure 4I ). To support this, we further showed that CMTM6 knockdown in HeLa and Caski cells led to a significant decrease in phosphorylated mTOR levels, suggesting that CMTM6 may help sustain mTOR pathway activation in CC cells ( Supplementary Figure S6 ). Building on this observation, we performed WB and found that macrophages (THP-1 and RAW264.7) treated with exosomes derived from CC cells exhibited activation of the mTOR signaling pathway. However, this phenomenon was attenuated when CMTM6 was absent in the exosomes ( Figure 4J ). Furthermore, mTOR inhibition also reversed the exosome-induced M2a macrophage polarization ( Figures 4K, L ). To further validate the regulatory role of CMTM6 in M2a polarization, we transfected IL-4-treated macrophages with CMTM6 siRNA. As expected, CMTM6 knockdown in macrophages reduced their tendency toward M2a activation, accompanied by decreased phosphorylation of the mTOR (p-mTOR) pathway ( Figures 4M, N ). These outcomes imply that CMTM6 was encapsulate into exosomes exerts an influence on M2a macrophage polarization by modulating the mTOR pathway.

M2a is a malignant macrophage phenotype known to foster tumor progression (17, 18). We constructed a coculture system to elucidate the function of tumor exosomal CMTM6 internalized by TAMs (TAMs/WT−Exos or TAMs/Exos-CMTM6 KO). Subsequently, tumor exosome-induced TAMs were cocultured with tumor cells for 48 h, and then these tumor cells were collected for Transwell assays ( Figure 5A ). As expected, the migrated and invasive ability of CC cells were notably increased after coculturing with exosome-pulsed TAMs, especially coculturing with TAMs^WT−Exos ( Figures 5B, C ). To validate the above results in vitro, we conducted in vivo experiments. We first verified whether exosomes alone can directly promote tumor growth in vivo, TC–1 cells were implanted subcutaneously into C57BL/6 mice, followed by three intratumoural injections of TC–1–derived exosomes or PBS without macrophage supplementation. Tumor volumes did not differ significantly between the exosome–treated and control groups ( Supplementary Figure S7 ), indicating that exosomes by themselves are insufficient to drive tumor outgrowth in the absence of macrophages. We then validated the macrophage−dependent effect in vivo. TC-1 and RAW264.7 cells, pretreated with PBS or exosomes, were mixed and subcutaneously injected into the right abdomen of mice. Subsequently, PBS or exosomes from TC-1/sh-NC or TC-1/sh-CMTM6 cells were intratumorally injected into the tumor-bearing mice. Notably, the sh-NC-Exos group displayed larger tumors with faster growth rates compared to other ( Figures 5D–F ). In addition, FCM showed more m2a macrophage infiltration in tumor tissue of sh-NC-Exos group ( Figure 5G ). IHC analysis further confirmed that exosomal CMTM6 accelerates CC progression and induces a higher proportion of M2a, accompanied by the activation of the mTOR pathway ( Figure 5H ). Similar findings were obtained in WB and quantification analysis ( Figures 5I, J ). Together, exosomal CMTM6 can drive the M2a macrophages and promote the progression of CC.

Cytokine secretion, a key indicator of macrophage function, was investigated to uncover how exosomal CMTM6 enhances macrophage-driven migration and invasion of CC cells. Employing qPCR, we conducted a comprehensive analysis of the expression profiles of ten inflammatory mediators and chemokines implicated in mTOR pathway activation in macrophages. Intriguingly, the data unveiled a marked elevation of CCL2 expression in macrophages stimulated with normal exosomes ( Figure 6A ). ELISA results further confirmed the increased secretion of CCL2 ( Figure 6B ). Simultaneously, the CCL2 baseline level in macrophages greatly surpassed that found in HeLa cells, while co-culturing exosomes with macrophages notably enhanced the CCL2 expression of macrophages ( Figure 6C ). Moreover, GEPIA database indicated higher CCL2 expression in M2 compared to M1 and M0 macrophages and a strong correlation between CMTM6 and CCL2 expression was confirmed by our cohort data and TCGA datasets ( Figures 6D–F ). Thus, we suggest that CMTM6 enhances CCL2 secretion by driving M2a polarization. As expected, CCL2 was highly expressed in CC tissues, linked to poor prognosis ( Figures 6G, H ). Next, we sought to determine whether CCL2 levels contribute to tumorigenic processes in CC. Using Transwell assays, we first verified that CCL2 markedly enhanced the migratory and invasive potential of CC cells ( Figures 6I, J ). Finally, we explored whether the elevated CCL2 contribute to the tumor-promoting effects mediated by M2a macrophages induced by CC-derived exosomal CMTM6. To address this, CC cells co-cultured with exosome-treated macrophages were subjected to RS504393 (CCL2 receptor antagonist) intervention. Transwell assays revealed that RS504393 effectively suppressed tumor cell migration and invasion ( Figures 6K, L ). To further evaluate this regulatory axis in vivo aiming to dissect the individual and combined effects of CMTM6 depletion and CCL2 inhibition. Tumor-bearing mice treated with sh-CMTM6-Exos or RS504383 alone exhibited moderate but comparable reductions in tumor growth, indicating that both interventions can restrain tumor progression to a certain extent. Notably, the combination of sh-CMTM6-Exos and RS504383 resulted in the most significant tumor suppression ( Figure 6M ), suggesting a potential additive or synergistic interaction between exosomal CMTM6 and CCL2 signaling in promoting CC progression. Together, these findings strongly suggest that macrophage-derived CCL2 may be a key cytokine mediating the development of CC. In summary, CMTM6-mediated exosomes can promote the polarization of M2a. These macrophages then release CCL2 to increase tumorigenicity.

To thoroughly investigate the clinical significance of the CMTM6/M2a polarization/CCL2 axis, we performed an extensive evaluation of CMTM6 expression in CC specimens and analyzed its relationship with the corresponding levels of CD206 and CCL2. Data derived from the GEO databases (GSE9750, GSE6791) revealed a strong correlation between elevated CMTM6 mRNA expression and increased M2a macrophage infiltration (CD206) as well as higher CCL2 mRNA levels, and this trend also existed in samples with low CMTM6 expression ( Figures 7A, B ). Furthermore, survival analyses based on TCGA-CESC indicated that CC patients exhibiting higher mRNA expression levels of CMTM6, CD206, or CCL2 had markedly worse OS and DSS outcomes compared to their counterparts with lower expression levels ( Figures 7C–F ). These observations highlight the substantial clinical relevance of CMTM6, CCL2, and M2a macrophage infiltration as powerful prognostic biomarkers in CC. Specifically, patients characterized by elevated CMTM6 and CCL2 mRNA expression, coupled with a high proportion of CD206-positive macrophages, demonstrated the most unfavorable OS and DSS outcomes ( Figures 7G, H ). Building on these prognostic indicators, we constructed a detailed nomogram to quantitatively predict the OS and DSS of CC patients with high accuracy ( Figures 7I, J ). In summary, our findings shed light on the prognostic significance of CMTM6/M2a polarization/CCL2 axis in CC. Additionally, Figure 7K presents a comprehensive and systematic overview of the pathways, processes, and mechanisms explored in this study.