High expression of formin-2 can promote ovarian cancer chemoresistance via immunosuppressive macrophages

High expression of formin-2 can promote ovarian cancer chemoresistance via immunosuppressive macrophages | 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 High expression of formin-2 can promote ovarian cancer chemoresistance via immunosuppressive macrophages Shuo Feng, Yaping Wang, Ran Ren, Shijia Liu, Xiaotong Wang, Lu Han This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7209240/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Dec, 2025 Read the published version in Functional & Integrative Genomics → Version 1 posted 7 You are reading this latest preprint version Abstract Ovarian cancer (OC) remains a major threat to women’s health, with chemoresistance driven by the immunosuppressive tumor microenvironment. Formin-2 (FMN2), a cytoskeletal regulator, was investigated for its role in OC chemoresistance and macrophage polarization. Bioinformatics analysis identified high FMN2 expression in chemotherapy-resistant OC cell lines, validated experimentally. Stable FMN2 knockdown cell lines were generated via lentiviral transfection. Functional assays revealed that FMN2 overexpression conferred chemoresistance in vitro and in vivo and promoted M2 macrophage polarization via the CCL2/JAK2/STAT3 pathway. Co-culture with M2 macrophages enhanced cisplatin (DDP) resistance in OC cells, mediated by CXCL1 secretion, which activated the epithelial-mesenchymal transition (EMT) pathway. Clinically, FMN2 levels correlated with CCL2 and CD206 (M2 marker) in platinum-resistant patients, and high FMN2, CCL2, or CD206 expression predicted poorer overall and disease-free survival. This study identifies FMN2 as a key mediator of chemoresistance and immune evasion in OC, proposing FMN2-CCL2-CD206 signaling and macrophage-derived CXCL1 as therapeutic targets and prognostic markers for chemotherapy response. FMN2 CCL2 Ovarian cancer Macrophages Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Ovarian cancer (OC) ranks as the second most common gynecological malignancy worldwide. It can affect women of various age groups and can have a significant impact on health and quality of life ( 1 ). Currently, the primary approach for managing OC consists of surgical intervention, which is subsequently accompanied by chemotherapy ( 2 ). Despite the progress made in the management of OC, the 5-year survival rate continues to be disappointingly low ( 3 ). Recurrence, metastasis and drug resistance during chemotherapy significantly threaten patients with OC, especially as resistance to chemotherapy limits the effectiveness of chemotherapy ( 4 ). Approximately 75% of patients exhibit an initial positive response to chemotherapy. However, a significant proportion of women ultimately experience a relapse. Regrettably, they succumb to a form of the disease that shows resistance to chemotherapy ( 5 ). Although the mechanisms of chemotherapy for OC have been extensively studied ( 4 , 6 – 10 ), they are not yet fully elucidated. Studies indicate that the immunosuppressive microenvironment within tumors substantially contributes to the development of resistance to chemotherapy in solid tumors ( 11 ). Improving survival rates and prognostic outcomes for patients with OC requires identifying effective targets to overcome resistance. The present study explored the molecular pathways in the tumor microenvironment that contributed to the development of chemoresistance in patients diagnosed with OC. Multiple elements within the tumor microenvironment, such as immunosuppressive cell populations, various cytokines and chemokines, can play a crucial role in the development of resistance to chemotherapy ( 12 ). Among these factors, tumor-associated macrophages (TAMs) exhibit a notable correlation with resistance to chemotherapy ( 13 ). TAMs present within the OC microenvironment exhibit an immunosuppressive M2 phenotype, a subtype characterized by anti-inflammatory functions that significantly facilitate tumor proliferation, evasion of the immune response and metastasis ( 14 ). Research has indicated that adenosine generated by OC cells plays a significant role in the infiltration of TAMs and their polarization towards an M2-like phenotype, primarily through the upregulation of enzymes such as ectonucleoside triphosphate diphosphohydrolase-1 and cluster of differentiation 73 ( 15 ). Additionally, tumor-derived play ubiquitin protein ligase E3 component n-recognin 5 was shown to facilitate the recruitment and activation of TAMs by secreting cytokines such as CCL2 and colony-stimulating factor-1 ( 16 ). M2 macrophages are present in malignant ascites, and their levels are closely associated with tumor grade and the duration of survival without disease progression in patients ( 17 , 18 ). Therefore, targeting the interactions that promote resistance to chemotherapy and regulate OC-TAMs may highlight strategies to enhance the efficacy of OC therapy. Formin-2 (FMN2) belongs to the formin family of homologous proteins, and the protein is involved in organizing the actin cytoskeleton and maintaining cell polarity ( 19 , 20 ). Cell migration is aided by a variety of actin-based structures (21). In the previous few decades, studies have shown that FMN2 is involved in multiple types of cancer ( 22 – 29 ). The FMN2 gene can function as both an oncogene or a tumor suppressor, depending on the specific type of cancer. Its role varies with changes in its expression profile, meaning that increases or decreases in FMN2 expression can influence cancer development differently ( 30 ). TP53 gene-damaging alterations and co-occurring missense mutations with FMN2 are significantly associated with faster disease relapse following chemotherapy ( 31 ). The FMN2 gene plays a crucial role in the regulation of the immune system ( 32 ). However, the exact role of FMN2 protein in regulating OC chemotherapy resistance remains largely unclear. The present study focused on exploring the mechanisms by which FMN2 reprogrammed the immune microenvironment and contributed to resistance to chemotherapy. Materials and methods Bioinformation analysis. mRNA expression profiles in OC cells exhibiting both high and intermediate levels of resistance were analyzed. Additionally, the mRNA expression profile of intermediate-resistant cells was compared with that of non-resistant cells. Data was sourced from the Gene Expression Omnibus (GEO) database ( 33 ) ( https://www.ncbi.nlm.nih.gov/gds ), specifically the GSE173579 and GSE28739. Datasets were used. Differentially expressed genes (DEGs) in OC cells with different resistance levels were identified using volcano plots and Venn diagrams in R ( 34 ). The online platform Gene Expression Profiling Interactive Analysis (GEPIA) ( http://gepia.cancer-pku.cn/index.html ) was used to verify the DEGs. The OS and DFS rates associated with the differential genes were assessed using data obtained from the Cancer Genome Atlas (TCGA) database. The GSE122287 dataset was used to validate the screened resistance gene FMN2. The relationship between the expression levels of FMN2 and M2 macrophages was assessed using Tumor IMmune Estimation Resource (TIMER) ( http://timer.cistrome.org/ ) in conjunction with data from TCGA. Cell culture. The human OC cell lines SKOV3 and SKOV3-DDP, as well as the mouse OC cell line ID8, were obtained from the Shanghai Bioresource Collection Center. Cells were cultured in either DMEM or RPMI-1640 media, supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were cultured at 37˚C in a humidified incubator supplied with 5% CO 2 air. The growth of SKOV3-DDP required the addition of the drug cisplatin. Additionally, the myeloid cell line THP1 was kindly provided by the laboratory of Dr Shen from Zhengzhou University. THP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; MilliporeSigma) for 24 h to facilitate differentiation. Cell proliferation assays. Cell proliferation was assessed using a CCK-8 assay. For CCK-8 analysis, cells were seeded in 96-well plates (5,000 cells/well). Following a 24h incubation period, 10 µl CCK-8 reagent was added to each well (Meilunbio), and then incubated for 2 h at 37˚C. Absorbance was measured at 450 nm using a microplate reader (BioTek; Agilent Technologies, Inc.). Western blotting. Total protein was extracted from cells using RIPA lysis buffer containing PMSF according to the manufacturer’s protocol. A BCA protein assay kit (Saiwen) was used to quantify protein levels. Total protein was loaded onto a 10% SDS gel, resolved using SDS-PAGE and transferred onto PVDF membranes. The membranes were subsequently blocked with a solution of 5% skim milk 1h at room. Next, the membranes were incubated with a primary antibody overnight at 4˚C, followed by incubation with the secondary antibody at a dilution of 1:20,000 (Abmart Pharmaceutical Technology Co., Ltd.) for 90 min at room temperature. β-actin served as the loading control. Signals were visualized using ECL detection reagent (Meilunbio). All antibodies used are listed in Table SI. Cell transfection. Lentiviral transfection was used to create stable cell lines in which FMN2 expression was knocked down. This was achieved using short hairpin RNA (shRNA) directed against FMN2 (Shanghai GeneChem Co., Ltd.). The SKOV3 and SKOV3-DDP cell lines were transfected using lentiviral delivery of shRNA specifically targeting the FMN2 gene (sh-FMN2), or a corresponding negative control (sh-NC). The shRNA sequences targeting FMN2 are provided in Table SII. Based on initial experimental findings, the multiplicity of infection, defined as the ratio of infectious agents to target cells, was established to be 30. Cells were cultured for 2 weeks in media supplemented with 2 µg/ml puromycin (Sangon Biotech Co., Ltd.). Cells that survived the antibiotic selection were classified as exhibiting stable expression. Reverse transcription-quantitative PCR (RT-qPCR). A total of 20 samples of OC were used. Total RNA was isolated from the cells or tissues using TRIzol® reagent (TransGen Biotech, Co., Ltd.) according to the manufacturer’s protocol. The extracted RNA was reverse transcribed using the PrimeScript RT reagent kit according to the manufacturer’s protocol (Toyobo Co., Ltd.). qPCR was performed using SYBR Green qPCR Master Mix (Jiangsu CoWin Biotech Co., Ltd.). The sequences of the primers are listed in Table SIII. GAPDH served as the housekeeping gene. The relative expression was determined by comparing the 2 −ΔCq value of the samples to the 2 −ΔCq value of the respective GAPDH result ( 35 ). Wound healing assay. The migratory ability of cells was evaluated using a wound healing assay. Cells were cultured to 100% confluence in 6-well plates and serum-starved for 12 h before starting the assay. A standardized wound was created using a 200 µl sterile pipette tip. Cells were maintained in serum-free medium, and wound closure was monitored at 0 and 48 h using an inverted microscope (Olympus Corporation) at x40 magnification. All experiments were performed in triplicate with three independent biological replicates. Transwell assay. Transwell assays were utilized to evaluate the invasive and migratory abilities of OC cells; a 24-well Transwell chamber with 8 µm pores were used (Corning, Inc.). In the invasion assay, the chamber was coated with Matrigel (BD Bioscience). In the migration assay, no coating was applied. A total of 4x10 4 or 6x10 4 cells were re-suspended in 200 µl serum-free media and then added to the upper chamber. The lower chamber was filled with media supplemented with 10% FBS. After a 48 h incubation period, the cells were fixed with methanol 15 min at room, and subsequently stained with a 0.1% solution of crystal violet 15 min at room. The stained cells were observed and counted using an inverted microscope. Chemotaxis assay. Using a 24-well Transwell chamber, cell migration assays were performed to evaluate the migratory potential of THP-1 cells. A total of 6x10 4 cells were suspended in 200 µl serum-free medium and subsequently introduced into the upper chamber. The lower chamber was filled with conditioned media derived from malignant cells or supplemented with CCL2. After a 48 h incubation, the cells were fixed using 4% formaldehyde for 20 min at room. Subsequently, cells were stained with 0.1% crystal violet 15 min at room. The stained cells were visualized using an inverted microscope for further analysis. Flow cytometry . Malignant peritoneal wash cells derived from murine models were stained using antibodies. Additionally, peripheral blood samples were obtained from human participants, along with malignant peritoneal wash cells post-ID8 implantation in mice, and stained using antibodies. In preparation for surface staining, cells were harvested following erythrocyte lysis and then treated with Zombie UV Fixable viability dye (BioLegend, Inc.) for 15 min at ambient temperature. Subsequently, the samples were incubated using fluorochrome-conjugated antibodies for 30 min. After incubation, cells were washed and analyzed using flow cytometry. The quantification of specific immune subsets was determined using a flow cytometer with phenotypic gating criteria. A list of all antibodies used is stated in Table SI. Animal model. First, the successful knockdown of FMN2 in mouse OC ID8 cells was determined. A cohort of 6-week-old female C57BL/6 mice was obtained from the Animal Center of Zhengzhou University. All experimental procedures involving animals were performed in compliance with the Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee at the Third Affiliated Hospital of Zhengzhou University. To establish the syngeneic intraovarian models, mice were first anesthetized using 50 mg/kg pentobarbital sodium. Then, a single dorsal incision was made to access the ovary. Each group was administered injections of 1x10 6 ID8 cells into the left ovarian bursa (Day 7). Each group was administered sh-NC or sh-FMN2 at a dose of 100 nM once a week by tail vein injection. A total of 56 days after the tumors were implanted, they were surgically excised. Tumor sizes were measured using digital calipers. The tumor volumes were determined using the formula (length x width 2 )/2. For chemotherapy treatment, mice received an intraperitoneal injection of 5 mg/kg cisplatin (Beyotime Institute of Biotechnology) three times a week after the tumors were implanted for a total of two doses. Animals were sacrificed using an overdose of isoflurane anesthesia followed by cervical dislocation. The doses of isoflurane used were in accordance with the guidelines provided by the Institutional Animal Care and Use Committee (IACUC). Confirmation of death was based on the absence of respiratory and cardiac activity, as well as a lack of reflexes. The maximum tumor size permitted was 2 cm in diameter. Patient populations and specimens. A total of 26 fresh tumor specimens, 126 cryopreserved tissue samples and 16 blood specimens were obtained from patients with OC who underwent surgical intervention and were subsequently treated with platinum-based chemotherapy at the Third Affiliated Hospital of Zhengzhou University between jan2018 and dec 2024. Prior to enrollment in the study, the participants provided written informed consent. The experimental protocols were approved by the Ethics Committee at the Third Affiliated Hospital of Zhengzhou University (approval no. 2023 − 120). Immunohistochemistry. The tissue sections were first baked in an oven at 75˚C for 4 h, then immediately placed in xylene for dewaxing and incubated with the primary antibody at 4˚C overnight. Subsequently, the tissues were incubated with the secondary antibody at room temperature for 1 h. Finally, the tissue sections were counterstained with DAB and hematoxylin and the display time was observed under the microscope. The assessment of FMN2, CCL2 and CD206 expression was performed by analyzing both the staining intensity and the proportion of positively stained cells. The antibodies used are listed in Table SI. ELISA. The concentrations of CCL2, CCL3, CXCL1 and CCL18 in the supernatants from various conditional OC cell lines were quantified by ELISA using specific kits according to the manufacturer’s protocol (BioLegend, Inc.). Statistical analysis. Bioinformatics analyses were performed using R (version 4.3.1) ( 36 ). For statistical evaluation, GraphPad Prism version 8.0.2 (Dotmatics) was used. Data are presented as the mean ± standard deviation. Standard deviation error bars were included solely for experiments where the sample size was ≥ 3. All data presented are representative of at least two independent experiments yielding consistent results. The comparison of differences between the two groups was performed using an unpaired two-sided Student’s t-test, while comparisons between ≥ 3 groups were performed using a one-way ANOVA. Survival analysis was performed using Kaplan-Meier analysis and a log-rank t-test. P < 0.05 was considered to indicate a statistically significant difference. Results FMN2 expression is correlated with chemoresistance. R was used to examine the mRNA expression profiles to identify genes correlated with chemotherapy resistance in intermediate-resistant and non-resistant OC cell lines (Fig. 1 A and C) and high-resistant and intermediate-resistant cell lines (Fig. 1 B and D) using data from the GEO dataset GSE173579. The analysis revealed that 226 genes were up-regulated and 54 genes were down-regulated in the intermediate-resistant cells compared with the non-resistant cells (Fig. 1 C). When comparing high-resistant cells to intermediate resistant cells, 54 genes were up-regulated and 77 genes were down-regulated (Fig. 1 D). A Venn diagram was constructed based on these two screening methodologies, revealing six DEGs as shown in Fig. 1 . GEPIA was used to assess the clinical relevance of the identified DEGs in OC, which indicated that only the expression levels of FMN2 was significantly associated with both the OS and DFS rates of patients with OC (Fig. 1 F and G). OC cells resistant to cisplatin, specifically SKOV3-DDP cells were obtained. Both the SKOV3 parental cell line and SKOV3-DDP resistant variant were subjected to varying concentrations of cisplatin (0-120 µg/ml) for 24 h. Cell viability was subsequently evaluated using a CCK-8 assay. The findings demonstrated a gradual decrease in the viability of OC cells in response to increasing concentrations of cisplatin (Fig. 1 H). The decrease in viability for the parental OC cells occurred at a significantly faster rate compared to the drug-resistant variants. The IC 50 value of SKOV3-DDP cells, calculated using SPSS version 26.0 (IBM Corp.), was 100 ± 8.43 µg/ml, which was markedly higher than the 25 ± 6.93µg/ml IC 50 value for the parental cells (P < 0.05). Additionally, western blot analysis indicated a significant increase in the expression levels of the chemoresistance-associated protein P-Glycoprotein (P-gp) protein in the drug-resistant cell lines compared to the parental cells (P < 0.05; Fig. 1 I). Collectively, these data confirmed the successful establishment of DDP-resistant ovarian cell lines. The mRNA and protein expression levels of FMN2 were shown to be significantly higher in the chemoresistant OC cells and tissues (both P < 0.05; Fig. 1 J). Wound healing analysis revealed that SKOV3-DDP cells exhibited enhanced cell migration (Fig. 1 K-L). Similarly, Transwell assays showed that SKOV3-DDP cells exhibited increased migratory and invasive capacities (Fig. 1 M-N). These findings suggest that chemoresistance may facilitate proliferation, migration and invasion, and may thus be involved in promoting the progression of OC. FMN2 is upregulated and confers DDP‑resistance in OC cells. To explore the potential involvement of FMN2 in the progression of OC, the expression levels of FMN2 in SKOV3 and SKOV3-DDP cells were examined. The analysis indicated that FMN2 expression was markedly higher in SKOV3-DDP cells compared with SKOV3 cells (Fig. 2 A and B). Stable FMN2 knockdown OC cells were established alongside corresponding negative control cells; successful knockdown was confirmed at both the mRNA and protein level. The data indicated that the knockdown efficiency of sh-FMN2-3 was significantly greater than that of sh-FMN2-1 and sh-FMN2-2 (Fig. 2 C-E), and therefore, this construct was used for further experiments. FMN2 knockdown OC and negative control cells were incubated with 0, 20, 40, 60, 80, 100 or 120 µg/ml DDP for 24 h. CCK-8 assays indicated that FMN2 knockdown significantly reduced DDP-resistance in chemoresistant OC cells (Fig. 2 F and G). Similarly, FMN2 knockdown decreased migration and invasion of chemoresistant OC cells (Fig. 2 H and I). The tumor immunosuppressive microenvironment is an important factor in chemotherapy resistance, and TAMs are a major invasive immune cell subgroup ( 37 ). To investigate the relationship between FMN2 and macrophage infiltration, TIMER2.0 was used. There was a positive correlation between FMN2 expression and the abundance of M2 macrophages (Fig. 2 J). Additionally, GEPIA analysis indicated that the expression levels of FMN2 were significantly associated with the M2 macrophage marker, Mannose receptor C type 1 (MRC1; Fig. 2 K). Next, clinical samples was used to detect the levels of M2 macrophages in the blood of patients with chemotherapy-resistant and chemotherapy-sensitive OC using flow cytometry, and found that the content of M2 macrophages in the blood of patients with chemotherapy resistant OC was higher (Fig. 2 L). The findings suggested that increased M2 macrophage infiltration may be associated with high levels of FMN2 expression. FMN2 regulates macrophage infiltration and M2-like polarization. The quantity and phenotypic variations of TAMs play a pivotal role in the progression of cancer and influence clinical outcomes. To investigate the impact of FMN2 on macrophage behavior, macrophages derived from THP-1 cells were co-cultured with both FMN2 knockdown SKOV3 cells and SKOV3-DDP cells using a Transwell assay for 48 h. The expression levels of the M2 macrophage marker, CD206, were assessed using RT-qPCR. Compared to the negative control cells, co-culturing with FMN2 knockdown SKOV3-DDP cells resulted in a notable reduction in CD206 expression in macrophages (Fig. 2 M and N). Additionally, the proportion of CD206 + macrophages, indicative of M2 macrophage polarization, after co-culture with OC cells was quantified using flow cytometry. Compared to the negative control group, the percentage of CD206 + macrophages decreased following co-culture with FMN2 knockdown chemoresistant OC cells (Fig. 2 O). Therefore, these findings suggest that FMN2 facilitated the polarization of macrophages towards the M2 phenotype. FMN2 induces macrophage polarization towards an M2 phenotype via the CCL2/JAK2/STAT3 pathway. As FMN2 knockdown in non-contact co-cultures with OC cells promoted M2 polarization of macrophages, it was hypothesized that the ability of OC cells to stimulate M2 polarization was associated with differences in the levels of secreted cytokines. A thorough examination of the existing literature led to the identification of 13 cytokines (IL-1α, IL-6, IL-9, CCL2, CCL3, CCL4, CCL5, CXCL1, CXCL2, TNF-α, CSF-1, GM-CSF and LIF) for further analysis via RT-qPCR. As shown in Fig. 2 P and Q, among the 13 cytokines, the most pronounced down-regulation, compared to other cytokines, was observed in the transcriptional levels of CCL2. Using ELISA, the concentration of CCL2 in the culture supernatants of OC cells was evaluated. The levels of CCL2 in the culture supernatants were consistent with the findings obtained from RT-qPCR analysis. Western blotting confirmed that FMN2 knockdown down-regulated the expression of CCL2 (Fig. 2 T). Next, the expression of immune factors in chemotherapy-resistant and sensitive OC tissues was examined in the GSE28739 dataset, and found that most of the immune factors exhibited altered expression, among which the expression of CCL2 was the most significantly up-regulated (Fig. 3 A and B). Studies have shown that CCL2 plays a crucial role in facilitating the chemotaxis of monocytes and macrophages, and in functional suppression ( 38 ). In the clinical specimens, it was found that CCL2 expression gradually increased with the increase of chemotherapy course (Fig. 3 C). As shown in Fig. 3 D-E, a notable reduction in the number of migrating cells was observed when using the conditioned medium from FMN2 knockdown cells. Conversely, treatment with CCL2 increased the number of migrating cells, suggesting that inhibiting FMN2 decreased macrophage migration, and CCL2 promoted it. To further assess functional changes in macrophages, M0 macrophages were treated with conditioned media from sh-NC or sh-FMN2-treated SKOV3 and SKOV3-DDP cells, including media supplemented with CCL2. Numerous studies have confirmed the involvement of the JAK2/STAT3 signaling axis in M2 macrophage polarization. Based on these insights, whether FMN2 promoted M2 macrophage polarization via the JAK2/STAT3 pathway was next assessed. As shown in Fig. 3 F-H, the levels of phosphorylated JAK2 and STAT3 were significantly higher in macrophages treated with CCL2, in macrophages co-cultured with FMN2 knockdown chemoresistant OC cells, and in macrophages co-cultured with FMN-2 knockdown OC cells. Of note, treatment with anti-CCL2R substantially reduced the ability of both FMN2 knockdown chemoresistant and parental OC cells to activate this signaling pathway, potentially by blocking CCL2 receptor interaction. Collectively, these findings indicated that FMN2 enhanced macrophage recruitment and promoted M2-like polarization primarily through a CCL2/JAK2/STAT3 signaling pathway. Coculture with OC cells induces M2 macrophage polarization. THP-1 cells were polarized towards an M0 phenotype by treating cells with PMA. Following this, M0 macrophages were co-cultured with SKOV3 and SKOV3-DDP cells in a Transwell assay for 48 h. RT-qPCR analyses showed that the expression levels of certain markers were significantly diminished in macrophages co-cultured with SKOV3-DDP compared to those co-cultured with SKOV3 cells (Fig. 4 A and B). Conversely, the expression levels of M2-associated genes, including CD163, CD206, IL-10, Arg-1 and VEGF-A, were upregulated following co-culture with ovarian cells, with significantly increased levels observed in macrophage cells co-cultured with SKOV3-DDP cells compared to those co-cultured with SKOV3 cells (Fig. 4 A and B). Additionally, these results highlight the differential impact of drug-resistant OC cells on macrophage polarization. Flow cytometry was employed to examine the surface markers of M0 macrophage cells co-cultured with SKOV3-DDP cells. The results indicated a reduction in the proportion of CD86 + cells, which is representative of M1 macrophages, in the macrophages co-cultured with SKOV3-DDP cells compared to those co-cultured with SKOV3 cells (Fig. 4 C and D). Additionally, the percentages of CD206 + cells (M2 macrophages) were increased in macrophages co-cultured with SKOV3-DDP and SKOV3 cells (Fig. 4 C and E). The findings collectively suggest that OC cells can influence macrophages towards an M2 phenotype, and chemoresistant cells were more proficient in M2 polarization. Co‑culturing OC cells with M2‑polarized macrophages enhances the DDP‑resistance in OC cells. OC cells were co-cultured with macrophages exhibiting distinct phenotypes using a Transwell assay for 48 h. Subsequently, the macrophages were removed, and the OC cells were harvested for further analysis. The CCK-8 assay showed that the presence of macrophages affected resistance to DDP. As shown in Fig. 4 B and C, co-culture with M1 macrophages increased the IC 50 values of SKOV3 cells from 25 ± 6.93 to 55 ± 5.12 µg/ml, while co-culture with M2 macrophages further increased the IC 50 values to 85 ± 6.83 µg/ml. Similarly, the IC 50 values of SKOV3-DDP cells increased from 100 ± 8.43 to 110 ± 7.20 µg/ml after co-culture with M1 macrophages and to 125 ± 7.65 µg/ml after co-culture with M2 macrophages. Furthermore, the expression levels of P-gp were markedly elevated in the drug-resistant cell lines (all P < 0.01, Fig. 4 D and E). Wound healing assays revealed that co-culture with M2 macrophages significantly facilitated the migration of OC cells (Fig. 5 A and B). Collectively, these results suggest that DDP-resistant OC cells effectively induced the polarization of macrophages toward the M2 phenotype, which, in-turn, contributed to enhanced DDP resistance in OC cells. CXCL1 derived from M2polarized macrophages is associated with DDPresistance of OC cells. The varying capacity of macrophages co-cultured with SKOV3 and SKOV3-DDP to enhance DDP resistance in OC cells has been proposed to result from differences in secreted cytokine levels. A comprehensive literature review led to the selection of 11 cytokines (CCL1, CCL2, CCL3, CCL4, CCL5, CCL17, CCL18, CCL22, CXCL1, CXCL2 and CXCL5) for analysis using RT-qPCR. As shown in Fig. 5 A and B, among the 11 cytokines, only CCL18, CCL3 and CXCL1 exhibited transcription levels consistent with the previously observed trends (Fig. 5 C). Notably, transcription levels in macrophages co-cultured with SKOV3-DDP were significantly elevated compared to macrophages co-cultured with SKOV3. Subsequently, the concentrations of CXCL1, CCL3 and CCL18 proteins in the culture supernatants were assessed using ELISA, revealing that only CXCL1 levels were significantly higher than the respective controls (Fig. 5 D-F). In light of these findings, CXCL1 was further studied to elucidate its role in the DDP resistance of OC cells. DDP-sensitive OC cells were cultured either alone or co-cultured with M2 macrophages. These cultures were treated with 10 ng/ml recombinant human CXCL1 (rhCXCL1) or 0.5 µg/ml CXCL1 neutralizing antibody for 48 h. Subsequently, the cells were exposed to various concentrations of 0, 20, 40, 60, 80, 100 and 120 µg/ml DDP for 24 h, and cell survival rates were evaluated. Results from the CCK-8 assay indicated that co-culture with M2 macrophages or the addition of rhCXCL1 induced DDP resistance, whereas administration of the CXCL1 neutralizing antibody diminished the M2 macrophage-mediated DDP resistance in OC cells (Fig. 5 G and H). Crucially, Transwell assays demonstrated that either co-culturing with M2 macrophages or adding rhCXCL1 enhanced the migratory and invasive capacities of OC cells, while CXCL1 neutralizing antibodies mitigated these effects mediated by M2 macrophages (Fig. 5 I and K). Furthermore, western blot analysis revealed that CXCL1 upregulated the mesenchymal markers N-cadherin and vimentin, while downregulating the epithelial marker E-cadherin (Fig. 5 J), indicating epithelial-mesenchymal transition. Collectively, these findings suggest that CXCL1, derived from M2-polarized macrophages, represented a significant chemokine associated with DDP resistance in OC cells. In summary, after the upregulation of FMN2 expression in OC cells, the expression levels of secreted CCL2 increased, recruiting monocytes and promoting their polarization towards M2 macrophages via activation of the JAK2-STAT3 signaling pathway. The secretion of CXCL1 by M2 macrophages, in turn, enhanced the progression of OC cells (Fig. 6 ). Down-regulating FMN2 reduces the presence of TAMs in ovarian masses. To further explore whether chemotherapy, FMN2 and CCL2 affected TAMs, mouse models were used (Fig. 7 A) and tumor volumes were measured to determine whether impaired tumor growth following FMN2 knockdown was the result of reduced recruitment of M2 macrophages. The experimental results showed that tumor growth was inhibited after FMN2 knockdown, but CCL2 and chemotherapy treatment eliminated this difference (Fig. 7 B-D), which may have been related to the increase in M2 macrophages. Survival analysis revealed that mice benefited from the FMN2 knockdown but did not benefit from CCL2 and chemotherapy (Fig. 7 E and F). The expression of the M2 marker CD163 in tumor tissues was detected using RT-qPCR; it was found that the expression levels of CD163 were significantly reduced following sh-FMN2 treatment and the content of CD163 was significantly increased after treatment with CCL2 or chemotherapy (Fig. 7 G and H). The results of the present study indicated that macrophages facilitated tumor progression. Moreover, the impact of sh-FMN2 on tumors was influenced by the immune microenvironment, particularly TAMs. To examine the proportion of macrophages, cells isolated from ascitic fluid were analyzed using flow cytometry (Fig. 7 I). The results of flow cytometry demonstrated a significant reduction in the population of F4/80 + CD11b + TAMs within the ascites of the sh-FMN2 group. Furthermore, the sh-FMN2 group exhibited a lower number of macrophages compared to the sh-NC group; however, treatment with CCL2 and chemotherapy abolished this reduction (Fig. 7 J and K). These findings implied that FMN2 enhanced the recruitment of macrophages and facilitated M2-like polarization of macrophages in vivo . Clinical relevance of FMN2 with CCL2 and CD206. To investigate the relationship between FMN2 and CCL2 and to assess the presence of stromal macrophages, immunostaining of tumor samples for CCL2 and the M2 macrophage marker CD206 was performed. As shown in Fig. 8 A-D, the expression levels of FMN2, CCL2 and CD206 were elevated in tumor tissues from patients with chemotherapy-resistant OC. A notable correlation was identified between the expression levels of FMN2 and CCL2. Additionally, varying degrees of infiltration by CD206 + macrophages were observed. Spearman correlation analysis revealed a statistically significant association between FMN2 and CCL2 expression levels (Fig. 8 E and F). These findings further supported the hypothesis that FMN2 promoted M2 macrophage polarization and this was mediated by CCL2. Expression of FMN2, CCL2 and CD206 was correlated with the prognosis of patients with OC. The OS and DFS rates were evaluated based on the expression levels of FMN2, CCL2 and CD206. As shown in Fig. 8 , patients with elevated expression levels of FMN2, CCL2 and CD206 had a poorer OS (Fig. 8 G-I) and DFS (Fig. 8 J-L) compared to patients with lower expression levels of these markers. These findings suggested a significant correlation between the expression levels of FMN2, CCL2 and CD206, and the prognosis of patients with OC who received DDP-based chemotherapy. Discussion The present study showed that tumor-derived FMN2 modulated the expression of CCL2. This modulation promoted the recruitment of M2-like macrophages in both clinical tumor specimens and murine models. This process contributed to the establishment of an immunosuppressive microenvironment in OC and contributed to resistance to chemotherapy by diminishing the efficacy of chemotherapeutic agents. These results provide valuable insights into the mechanisms by which macrophages infiltrate ovarian tissue. FMN2 is involved in the organization of the actin cytoskeleton, and is pivotal in facilitating cell migration and plays a key role in tumor progression ( 24 ). FMN2 plays different roles in different tumors, both as an oncogene and as a tumor suppressor gene ( 30 ). Among these, its expression is upregulated in melanoma ( 24 ), colorectal cancer ( 22 ), hepatocellular carcinoma ( 28 ) and leukemia ( 23 , 26 ), where it functions as an oncogene, but plays a cancer-inhibiting role in colorectal cancer ( 22 ) and pancreatic cancer ( 25 ). Other researchers used an online database to study the relationship between FMN2 expression and the immune microenvironment in colorectal cancer, and proposed that FMN2 gene mutation could affect the distribution of immune cells and thus influence the immune microenvironment ( 27 ). Nevertheless, there is a paucity of literature addressing the specific function and clinical significance of FMN2 within the immune microenvironment of OC. Through database analysis, it was observed that FMN2 expression was significantly elevated in OC exhibiting resistance to chemotherapy and was negatively associated with the OS and DFS of patients. The expression of FMN2 was also positively correlated with the M2 macrophage marker MRC1 and the abundance of M2 macrophages. The expression of FMN2 increased gradually as the duration of treatment with platinum-based chemotherapy increased. Furthermore, RT-qPCR and western blot analyses revealed that FMN2 levels were markedly upregulated in tissues and cells resistant to OC chemotherapy compared to those sensitive to such treatments. Data analysis from another GEO database showed that CCL2 expression was most significantly upregulated in chemotherapy-resistant OC tissues compared with chemotherapy-sensitive OC tissues. Moreover, analysis of the clinical specimens in the present study showed that CCL2 expression gradually increased as the course of chemotherapy increased. After knocking down FMN2 expression in OC cells, it was found that the expression of CCL2 was significantly decreased at both the mRNA and protein levels. In addition, IHC results showed that the expression of FMN2, CCL2 and CD206 was significantly increased in chemotherapy-resistant OC tissues, and FMN2 was positively correlated with CCL2 and CD206. Of note, M2 macrophages decreased after co-incubation of SKOV3 and SKOV3-DDP of sh-FMN2 with M0, and the levels of M2 macrophages increased after co-incubation with CCL2. The isogenic intraovarian mouse model showed that the tumor volume was significantly reduced and survival time increased in the sh-FMN2 group, which was partially offset by cisplatin or CCL2 treatment. In addition, the levels of M2 macrophages decreased significantly in the sh-FMN2 group and increased again after cisplatin or CCL2 treatment. Therefore, it was speculated that CCL2 may be downstream of FMN2, and as a chemokine, CCL2 promoted the formation of TAMs, thus promoting the progression of tumors and ultimately leading to chemotherapy resistance. Recent investigations have elucidated the significance of the CCL2-CCR2-M2 macrophage axis in the context of OC. One study demonstrated that the administration of CCR2 antagonists in vivo effectively diminished the M2 macrophage population, consequently inhibiting tumor proliferation. This finding underscored the substantial indirect influence exerted by M2 macrophages on tumor growth ( 40 ). CCL2, a crucial mediator of the recruitment, differentiation and functional modulation of TAMs, can be synthesized by both tumor and non-tumor cells ( 41 ). The production of CCL2 within neoplastic environments has been shown to facilitate tumor-induced immunosuppression and the accumulation of TAMs, highlighting it as a pivotal molecular target for the management of cancer ( 42 ). In the present study, FMN2 was observed to increase CCL2 expression, subsequently promoting macrophage recruitment and M2-like polarization; these processes in turn drove OC progression and contributed to the development of cisplatin resistance. Therefore, targeting the FMN2-CCL2 pathway may represent a novel and effective therapeutic approach for managing OC progression and improving sensitivity to cisplatin treatment. Although anti-CCL2 antibodies and CCR2 antagonists have been explored in clinical trials, an animal study revealed that the discontinuation of anti-CCL2 antibody therapy led to a significant increase in lung metastasis and hastened mortality in a mouse isogenic cancer model ( 43 ). Consequently, the efficacy of solely inhibiting the CCL2-CCR2 signaling pathway remains a contentious issue, partly due to the adverse outcomes observed following disruption of therapy ( 44 ). OC is widely recognized for its tendency to develop resistance to chemotherapy. Consequently, numerous investigations have been conducted to enhance the therapeutic response of patients with OC to chemotherapeutic agents. Among these studies, targeting aberrant oncogenic genes in OC cells, such as lysine-specific demethylase 1 and lysine-specific demethylase 1, poly ADP-ribose polymerase, and glutathione S-transferase P1, has demonstrated efficacy in enhancing the therapeutic response to chemotherapy. Furthermore, inhibiting the expression of the FMN2 gene, rather than obstructing the CCL2-CCR2-macrophage signaling pathway, may represent a promising alternative strategy, as it more effectively improves the response to chemotherapy in OC. The present study highlights the critical role of FMN2 in promoting chemoresistance and inducing M2 macrophage polarization in OC. Co-culture with M2-polarized macrophages significantly enhanced the resistance of OC cells to DDP. Moreover, it was confirmed that CXCL1, which originates from M2-polarized macrophages, promoted chemoresistance via the EMT pathway. Taken together, the present study proposed novel targets and strategies for OC treatment and identified potential prognostic indicators for assessing the efficacy of chemotherapy. Declarations Ethics approval and consent to participate The Ethics Committee at the Third Affiliated Hospital of Zhengzhou University approved all the experimental protocols in the present study (approval no. 2023 − 120). Patient consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Clinical trial number Not applicable Funding Statement This study was supported in part by grants from the International Scientific Exchange Foundation of China (no. Z2024LLN005). Author Contribution SF and LH conceived and designed the study. YW and RR performed the experiments. SL and XW analyzed the data. 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Supplementary Files Wb.zip SupplementaryTables.docx Cite Share Download PDF Status: Published Journal Publication published 20 Dec, 2025 Read the published version in Functional & Integrative Genomics → Version 1 posted Editorial decision: Revision requested 25 Sep, 2025 Reviews received at journal 14 Sep, 2025 Reviewers agreed at journal 13 Aug, 2025 Reviewers invited by journal 04 Aug, 2025 Editor assigned by journal 31 Jul, 2025 Submission checks completed at journal 31 Jul, 2025 First submitted to journal 24 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-7209240","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":495389791,"identity":"451bdd27-1a37-47cb-86fb-8353b4b2e492","order_by":0,"name":"Shuo Feng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYBAC9gYgwQgk2JgZGw58gAga4NXCcwCqhY+9ufHgDJK0yPEcbz7MQ5QW9t7DL3/uOCzHJpHYcNjmT11iA3vzNgmGmju4tfCcS7PmPXPYGKwlh4ctsYHnWJkEw7FnOLXYS+SYGTO2HU5sA2uR4ElsAIpIMDYcxm2L/Bszw58wLRYGQBIogl+LBI/xA16QFp6DQGUJBkBbeAho4ckxY+ZtSzdmY29sONhzIMG4jSet2CLhGB4t7GeMP/5ss5aTb2Z//OHHnzrZfvbDG298qMGtBQjYJFC5ICIBnwYGBuYP+OVHwSgYBaNgxAMAwXZU1i3c8l0AAAAASUVORK5CYII=","orcid":"","institution":"Dalian Medical University","correspondingAuthor":true,"prefix":"","firstName":"Shuo","middleName":"","lastName":"Feng","suffix":""},{"id":495389794,"identity":"49bb5e09-e790-49b3-a3e3-8d8430b5a4f0","order_by":1,"name":"Yaping Wang","email":"","orcid":"","institution":"the Third Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yaping","middleName":"","lastName":"Wang","suffix":""},{"id":495389796,"identity":"306fc1ed-c2a4-41e3-8290-e35e9ceeb2ea","order_by":2,"name":"Ran Ren","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ran","middleName":"","lastName":"Ren","suffix":""},{"id":495389797,"identity":"a50d98df-7076-4755-a254-621955b387f7","order_by":3,"name":"Shijia Liu","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shijia","middleName":"","lastName":"Liu","suffix":""},{"id":495389799,"identity":"7d02c560-eda1-4d45-b855-e95570f7c1c3","order_by":4,"name":"Xiaotong Wang","email":"","orcid":"","institution":"the Third Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xiaotong","middleName":"","lastName":"Wang","suffix":""},{"id":495389800,"identity":"d80a2f03-da98-416a-a614-7278263eeba9","order_by":5,"name":"Lu Han","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Han","suffix":""}],"badges":[],"createdAt":"2025-07-25 01:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7209240/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7209240/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10142-025-01766-z","type":"published","date":"2025-12-20T15:58:41+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88500482,"identity":"63306c89-f15a-4308-99f0-627482b68c52","added_by":"auto","created_at":"2025-08-07 06:51:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3701595,"visible":true,"origin":"","legend":"\u003cp\u003eFMN2 promotes ovarian cancer chemotherapy resistance. (A) Volcano map showing the different genes of moderately resistant OC and non-resistant OC cell lines. The volcano map shows the mRNA levels based on data obtained from GEO; the x-axis shows the log2 transformed fold change rate and the Y-axis represents the log10 adjusted P-value. The red dots represent DEGs based on the fold changes. (B) The 226 upregulated and 39 downregulated genes based on the volcano plot. (C) The 54 upregulated and 77 downregulated genes based on the volcano plot. (D) Volcano maps of mRNA levels of different genes in highly resistant OC cell lines and moderately resistant ovarian cancer cell lines.\u003cstrong\u003e \u003c/strong\u003e(E) Venn diagram representing the distribution of DEGs in the different groups. (F) Prognostic value of FMN2 expression in patients with OC evaluated using Kaplan-Meier analysis and (G) DFS in\u003cstrong\u003e \u003c/strong\u003ehigh vs. low FMN2 expression.\u003cstrong\u003e \u003c/strong\u003e(H)\u003cstrong\u003e \u003c/strong\u003eSKOV3 and SKOV3-DDP cells were cultured in the presence of DDP for 24 h, cell viability was assessed using a CCK-8 assay. (I) Western blot analysis showed P-gp was significantly increased in SKOV3-DDP cells compared with parental cells.\u003cstrong\u003e \u003c/strong\u003e(J) Western blot analysis showed that the expression of FMN2 was significantly increased in chemotherapy-resistant tissues compared with chemotherapy-sensitive tissues.\u003cstrong\u003e \u003c/strong\u003e(K and L) Wound healing assays were performed to assess the migratory abilities of SKOV3 and SKOV3-DDP cells. (M and N) Transwell assays were performed to determine the migratory and invasive abilities of SKOV3 and SKOV3-DDP cells. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01 and \u003csup\u003e****\u003c/sup\u003eP\u0026lt;0.0001. FMN2, Formin-2; OC, ovarian cancer; DDP, cisplatin; DEG, differentially expressed gene; P-gp, P-Glycoprotein.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7209240/v1/7dbd2a47711a91f25accedac.png"},{"id":88500479,"identity":"9137168a-02ca-4380-8a92-818896a6eda5","added_by":"auto","created_at":"2025-08-07 06:51:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3331504,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of FMN2 on DDP-resistance and macrophage abundance of OC cells \u003cem\u003ein vitro\u003c/em\u003e. (A) Expression of FMN2 in ovarian cancer cells (SKOV3 and SKOV3-DDP) by RT-qPCR.\u003cstrong\u003e \u003c/strong\u003e(B) FMN2 expression in SKOV3, SKOV3-DDP cells and OC tissues following treatment with platinum-based chemotherapy. (C and D) FMN2 protein and mRNA expression in OC cells following FMN2 knockdown. (F and G)\u003cstrong\u003e \u003c/strong\u003eFMN2 knockdown OC cells were incubated with different concentrations of cisplatin (0, 20, 40, 60, 80, 100 and 120 μg/ml) for 24 h, and cell viability was detected using a CCK-8 assay.\u003cstrong\u003e \u003c/strong\u003e(H) Wound healing assays were performed to determine the migratory abilities of SKOV3 and SKOV3-DDP cells following FMN2 knockdown. (I) Transwell assays were performed to determine the migratory and invasive abilities of SKOV3 and SKOV3-DDP cells following FMN2 knockdown.\u003cstrong\u003e \u003c/strong\u003e(J) Association between the abundance of M2 macrophages and mRNA expression of FMN2 in OC in the TIMER2.0 database.\u003cstrong\u003e \u003c/strong\u003e(K) Association between the mRNA expression levels of MRC1 and mRNA expression of FMN2 in OC based on data obtained from GEPIA.\u003cstrong\u003e \u003c/strong\u003e(L) Percentage of CD4+CD206+cells in the peripheral blood of eight OC patients were analyzed by flow cytometry. (M and N) mRNA expression of CD206 in macrophages using the supernatant of SKOV3 and SKOV3-DDP cells with FMN2 knockdown by RT-qPCR. (O) Flow cytometry analysis of CD206+ cells in PMA-stimulated THP-1 cells treated with the supernatant of SKOV3 and SKOV3-DDP cells with FMN2 expression knocked down for 48 h.\u003cstrong\u003e \u003c/strong\u003e(P and Q) RT-qPCR was used to detect the mRNA levels of 13 cytokines in OC cells, the change in CCL2 expression was statistically significant. (R and S) ELISA was used to detect the CCL2 levels in culture supernatants of SKOV3 and SKOV3-DDP cells.\u003cstrong\u003e \u003c/strong\u003e(T) Western blot analysis indicated the expression of CCL2 was significantly decreased in SKOV3 and SKOV3-DDP with FMN2 expression was knocked down. DDP, cisplatin-resistant. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003eP\u0026lt;0.0001. FMN2, Formin-2; OC, ovarian cancer; DDP, cisplatin; RT-qPCR, reverse transcription-quantitative PCR; TIMER, Tumor IMmune Estimation Resource; MRC1, Mannose Receptor C-Type 1; GEPIA, Gene Expression Profiling Interactive Analysis.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7209240/v1/367b6eaec62d402287d20ee8.png"},{"id":88500478,"identity":"3f67cd82-d1ef-4e37-899b-df7ca285dbb4","added_by":"auto","created_at":"2025-08-07 06:51:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1675388,"visible":true,"origin":"","legend":"\u003cp\u003eFMN2 induced polarization of macrophages towards an M2 phenotype via a CCL2/JAK2/STAT3 pathway.\u003cstrong\u003e \u003c/strong\u003emRNA expression levels of (A)\u003cstrong\u003e \u003c/strong\u003eimmune factors and (B) CCL2 in chemotherapy-sensitive and resistant OC tissues based on data obtained from GEO.\u003cstrong\u003e \u003c/strong\u003e(C) Concentration of CCL2 (pg/ml) in the serum obtained from chemosensitive and chemoresistant patients with OC at different stages in their chemotherapy, as measured by ELISA. (D and E)\u003cstrong\u003e \u003c/strong\u003eChemotactic migration assays of macrophages using the supernatant of SKOV3 and SKOV3-DDP cells with FMN2 expression knocked down or in cells treated with CCL2.\u003cstrong\u003e \u003c/strong\u003e(F-H) Macrophages were incubated with CCL2, co-cultured with parental OC cells with FMN2 expression knocked down or the respective negative control cells in the presence or absence of anti-CCL2-1R, then the expression of members of the JAK2/STAT3 pathway in these macrophages were detected by western blot. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003eP\u0026lt;0.0001. FMN2, Formin-2; OC, ovarian cancer; DDP, cisplatin.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7209240/v1/2de5f63c4e818a4142197f57.png"},{"id":88500485,"identity":"5c9f829f-4d4a-436b-a481-aea074dfc58d","added_by":"auto","created_at":"2025-08-07 06:51:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1469011,"visible":true,"origin":"","legend":"\u003cp\u003eCo-culturing OC cells induced polarization of macrophages towards an M2 phenotype.\u003cstrong\u003e \u003c/strong\u003e(A and B) mRNA expression levels of M1-related genes, CD86, TNF-α and IL-12, as well as M2-related genes, CD163, CD206, IL-10, Arg-1, VEGF-A and VEGF-C in M0, M1 and M2 cells. (C-E)\u003cstrong\u003e \u003c/strong\u003ePercentages of CD86+ (M1 macrophages), CD163+ cells (M2 macrophages) and CD206+ cells (M2 macrophages) were measured using flow cytometry.\u003cstrong\u003e \u003c/strong\u003e(F)\u003cstrong\u003e \u003c/strong\u003eCCK-8 analysis indicated that culturing with M1 and M2 macrophages induced DDP-resistance and enhanced the IC\u003csub\u003e50\u003c/sub\u003e values of SKOV3 and SKOV3-DDP cells.\u003cstrong\u003e \u003c/strong\u003e(H and I)\u003cstrong\u003e \u003c/strong\u003eWestern blot analysis indicated that the expression of P-gp was significantly increased in OC cells co-cultured with M2 macrophages. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001. ns, not significant; OC, ovarian cancer; DDP, cisplatin; P-gp, P-Glycoprotein.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7209240/v1/8ef5c7fc4d9bc6d48ecba5e2.png"},{"id":88500491,"identity":"fd08457a-ea9c-4187-b824-f12ef4f42946","added_by":"auto","created_at":"2025-08-07 06:51:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3896586,"visible":true,"origin":"","legend":"\u003cp\u003eCXCL1 derived from M2-polarized macrophages was associated with DDP-resistance of OC cells.\u003cstrong\u003e \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003eWound healing assays were performed to determine the migratory abilities of SKOV3-DDP and SKOV3-DDP cells co-cultured with M2 macrophages. (B) Transwell assays were performed to determine the migratory and invasive abilities of SKOV3-DDP and SKOV3-DDP cells co-cultured with M2 macrophages.\u003cstrong\u003e \u003c/strong\u003e(C) mRNA expression levels of 11 cytokines in M0, MS and MR macrophages. ELISA was used to detect the levels of (D) CCL3, (E) CXCL1 and \u003cstrong\u003e(F)\u003c/strong\u003e CCL18 in culture supernatants. (G and H) CCK-8 assays were performed to assess the role of CXCL1 in DDP-resistant OC cells. (I-K) Transwell assays showed that M2 macrophages promoted OC cell DDP-induced migration and invasion via CXCL1. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003eP\u0026lt;0.0001. OC, ovarian cancer; DDP, cisplatin.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7209240/v1/3102b7c57102a8a9bb814066.png"},{"id":88502670,"identity":"980967e9-a328-4412-8aa4-8549ae263454","added_by":"auto","created_at":"2025-08-07 06:59:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":327417,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical representation showing how macrophages interact with OC cells to promote the development of a chemoresistant microenvironment and how M2 macrophages promote chemotherapy resistance in OC. OC, ovarian cancer.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7209240/v1/13e79bade97dba4277cdd386.png"},{"id":88500498,"identity":"d81c3f83-8121-4c63-8c9e-75604f0fc316","added_by":"auto","created_at":"2025-08-07 06:51:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1921790,"visible":true,"origin":"","legend":"\u003cp\u003eMacrophage recruitment and tumor growth \u003cem\u003ein vivo\u003c/em\u003e are impaired following treatment with shFMN2 in the mouse model of OC.\u003cstrong\u003e \u003c/strong\u003e(A) Workflow of the experiments with or without CCL2 and cisplatin treatment.\u003cstrong\u003e \u003c/strong\u003e(B) Tumor volumes in the orthotopic syngenetic model mice.\u003cstrong\u003e \u003c/strong\u003e(C) Tumors from orthotopic syngeneic models with or without CCL2 treatment. (D) Tumors from orthotopic syngeneic models with or without cisplatin treatment. (E and F) Overall survival curves were obtained using Kaplan-Meier and log-rank analysis. (G and H) mRNA expression levels of CD206 in tumor tissues with or without CCL2 and cisplatin treatment. (I-K) The percentage of CD11b+ cells in the orthotopic syngeneic model mice with or without CCL2 and cisplatin treatment was analyzed by flow cytometry. \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003eP\u0026lt;0.0001. OC, ovarian cancer; DDP, cisplatin-resistant;\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7209240/v1/20cc5d30a1af34c17fa56df6.png"},{"id":88504430,"identity":"c100b7bb-d497-4b77-b1fe-c4808df57fff","added_by":"auto","created_at":"2025-08-07 07:08:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2485299,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of FMN2 with CCL2 and CD206 and with the prognosis of patients with OC. (A-D) Representative images of immunohistochemical staining for FMN2, CCL2 and CD206 in human OC tissues. Magnification, x400. (E and F)\u003cstrong\u003e \u003c/strong\u003eThe correlation between FMN2 with CCL2 and CD206 in OC tissues was analyzed using Spearman’s rank-order correlation. (G-I) Overall survival analysis based on FMN2, CCL2 and CD206 expression. (J-L)DFS analysis based on FMN2, CCL2 and CD206 expression. FMN2, Formin-2; OC, ovarian cancer; DFS, disease-free survival.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7209240/v1/a9c59282eb6a8a2147a46ffc.png"},{"id":98814061,"identity":"6d6111b9-2a0f-474c-ad1c-b530c1202637","added_by":"auto","created_at":"2025-12-22 16:10:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19203787,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7209240/v1/8676ac58-db9c-4693-aa0c-c5abd4f1788e.pdf"},{"id":88500514,"identity":"007bb418-47fe-4b74-8e6e-9ac9aac769a8","added_by":"auto","created_at":"2025-08-07 06:52:01","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":70612922,"visible":true,"origin":"","legend":"","description":"","filename":"Wb.zip","url":"https://assets-eu.researchsquare.com/files/rs-7209240/v1/3bb4d686ea130cdbe90959e4.zip"},{"id":88502665,"identity":"7b7efe74-3804-4eb9-80c9-88a5b67b1cf8","added_by":"auto","created_at":"2025-08-07 06:59:58","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21887,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7209240/v1/ee7dd72342db099e62da90c1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"High expression of formin-2 can promote ovarian cancer chemoresistance via immunosuppressive macrophages","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOvarian cancer (OC) ranks as the second most common gynecological malignancy worldwide. It can affect women of various age groups and can have a significant impact on health and quality of life (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Currently, the primary approach for managing OC consists of surgical intervention, which is subsequently accompanied by chemotherapy (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Despite the progress made in the management of OC, the 5-year survival rate continues to be disappointingly low (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Recurrence, metastasis and drug resistance during chemotherapy significantly threaten patients with OC, especially as resistance to chemotherapy limits the effectiveness of chemotherapy (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Approximately 75% of patients exhibit an initial positive response to chemotherapy. However, a significant proportion of women ultimately experience a relapse. Regrettably, they succumb to a form of the disease that shows resistance to chemotherapy (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Although the mechanisms of chemotherapy for OC have been extensively studied (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), they are not yet fully elucidated. Studies indicate that the immunosuppressive microenvironment within tumors substantially contributes to the development of resistance to chemotherapy in solid tumors (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Improving survival rates and prognostic outcomes for patients with OC requires identifying effective targets to overcome resistance.\u003c/p\u003e\u003cp\u003eThe present study explored the molecular pathways in the tumor microenvironment that contributed to the development of chemoresistance in patients diagnosed with OC. Multiple elements within the tumor microenvironment, such as immunosuppressive cell populations, various cytokines and chemokines, can play a crucial role in the development of resistance to chemotherapy (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Among these factors, tumor-associated macrophages (TAMs) exhibit a notable correlation with resistance to chemotherapy (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). TAMs present within the OC microenvironment exhibit an immunosuppressive M2 phenotype, a subtype characterized by anti-inflammatory functions that significantly facilitate tumor proliferation, evasion of the immune response and metastasis (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Research has indicated that adenosine generated by OC cells plays a significant role in the infiltration of TAMs and their polarization towards an M2-like phenotype, primarily through the upregulation of enzymes such as ectonucleoside triphosphate diphosphohydrolase-1 and cluster of differentiation 73 (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Additionally, tumor-derived play ubiquitin protein ligase E3 component n-recognin 5 was shown to facilitate the recruitment and activation of TAMs by secreting cytokines such as CCL2 and colony-stimulating factor-1 (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). M2 macrophages are present in malignant ascites, and their levels are closely associated with tumor grade and the duration of survival without disease progression in patients (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Therefore, targeting the interactions that promote resistance to chemotherapy and regulate OC-TAMs may highlight strategies to enhance the efficacy of OC therapy.\u003c/p\u003e\u003cp\u003eFormin-2 (FMN2) belongs to the formin family of homologous proteins, and the protein is involved in organizing the actin cytoskeleton and maintaining cell polarity (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Cell migration is aided by a variety of actin-based structures (21). In the previous few decades, studies have shown that FMN2 is involved in multiple types of cancer (\u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26 CR27 CR28\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The FMN2 gene can function as both an oncogene or a tumor suppressor, depending on the specific type of cancer. Its role varies with changes in its expression profile, meaning that increases or decreases in FMN2 expression can influence cancer development differently (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). TP53 gene-damaging alterations and co-occurring missense mutations with FMN2 are significantly associated with faster disease relapse following chemotherapy (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). The FMN2 gene plays a crucial role in the regulation of the immune system (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). However, the exact role of FMN2 protein in regulating OC chemotherapy resistance remains largely unclear. The present study focused on exploring the mechanisms by which FMN2 reprogrammed the immune microenvironment and contributed to resistance to chemotherapy.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003eBioinformation analysis.\u003c/em\u003e mRNA expression profiles in OC cells exhibiting both high and intermediate levels of resistance were analyzed. Additionally, the mRNA expression profile of intermediate-resistant cells was compared with that of non-resistant cells. Data was sourced from the Gene Expression Omnibus (GEO) database (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/gds\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/gds\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), specifically the GSE173579 and GSE28739. Datasets were used. Differentially expressed genes (DEGs) in OC cells with different resistance levels were identified using volcano plots and Venn diagrams in R (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). The online platform Gene Expression Profiling Interactive Analysis (GEPIA) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/index.html\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to verify the DEGs. The OS and DFS rates associated with the differential genes were assessed using data obtained from the Cancer Genome Atlas (TCGA) database. The GSE122287 dataset was used to validate the screened resistance gene FMN2. The relationship between the expression levels of FMN2 and M2 macrophages was assessed using Tumor IMmune Estimation Resource (TIMER) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://timer.cistrome.org/\u003c/span\u003e\u003cspan address=\"http://timer.cistrome.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) in conjunction with data from TCGA.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCell culture.\u003c/em\u003e The human OC cell lines SKOV3 and SKOV3-DDP, as well as the mouse OC cell line ID8, were obtained from the Shanghai Bioresource Collection Center. Cells were cultured in either DMEM or RPMI-1640 media, supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were cultured at 37˚C in a humidified incubator supplied with 5% CO\u003csub\u003e2\u003c/sub\u003e air. The growth of SKOV3-DDP required the addition of the drug cisplatin. Additionally, the myeloid cell line THP1 was kindly provided by the laboratory of Dr Shen from Zhengzhou University. THP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; MilliporeSigma) for 24 h to facilitate differentiation.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCell proliferation assays.\u003c/em\u003e Cell proliferation was assessed using a CCK-8 assay. For CCK-8 analysis, cells were seeded in 96-well plates (5,000 cells/well). Following a 24h incubation period, 10 \u0026micro;l CCK-8 reagent was added to each well (Meilunbio), and then incubated for 2 h at 37˚C. Absorbance was measured at 450 nm using a microplate reader (BioTek; Agilent Technologies, Inc.).\u003c/p\u003e\u003cp\u003e\u003cem\u003eWestern blotting.\u003c/em\u003e Total protein was extracted from cells using RIPA lysis buffer containing PMSF according to the manufacturer\u0026rsquo;s protocol. A BCA protein assay kit (Saiwen) was used to quantify protein levels. Total protein was loaded onto a 10% SDS gel, resolved using SDS-PAGE and transferred onto PVDF membranes. The membranes were subsequently blocked with a solution of 5% skim milk 1h at room. Next, the membranes were incubated with a primary antibody overnight at 4˚C, followed by incubation with the secondary antibody at a dilution of 1:20,000 (Abmart Pharmaceutical Technology Co., Ltd.) for 90 min at room temperature. β-actin served as the loading control. Signals were visualized using ECL detection reagent (Meilunbio). All antibodies used are listed in Table SI.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCell transfection.\u003c/em\u003e Lentiviral transfection was used to create stable cell lines in which FMN2 expression was knocked down. This was achieved using short hairpin RNA (shRNA) directed against FMN2 (Shanghai GeneChem Co., Ltd.). The SKOV3 and SKOV3-DDP cell lines were transfected using lentiviral delivery of shRNA specifically targeting the FMN2 gene (sh-FMN2), or a corresponding negative control (sh-NC). The shRNA sequences targeting FMN2 are provided in Table SII. Based on initial experimental findings, the multiplicity of infection, defined as the ratio of infectious agents to target cells, was established to be 30. Cells were cultured for 2 weeks in media supplemented with 2 \u0026micro;g/ml puromycin (Sangon Biotech Co., Ltd.). Cells that survived the antibiotic selection were classified as exhibiting stable expression.\u003c/p\u003e\u003cp\u003e\u003cem\u003eReverse transcription-quantitative PCR (RT-qPCR).\u003c/em\u003e A total of 20 samples of OC were used. Total RNA was isolated from the cells or tissues using TRIzol\u0026reg; reagent (TransGen Biotech, Co., Ltd.) according to the manufacturer\u0026rsquo;s protocol. The extracted RNA was reverse transcribed using the PrimeScript RT reagent kit according to the manufacturer\u0026rsquo;s protocol (Toyobo Co., Ltd.). qPCR was performed using SYBR Green qPCR Master Mix (Jiangsu CoWin Biotech Co., Ltd.). The sequences of the primers are listed in Table SIII. GAPDH served as the housekeeping gene. The relative expression was determined by comparing the 2\u003csup\u003e\u0026minus;ΔCq\u003c/sup\u003e value of the samples to the 2\u003csup\u003e\u0026minus;ΔCq\u003c/sup\u003e value of the respective GAPDH result (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eWound healing assay.\u003c/em\u003e The migratory ability of cells was evaluated using a wound healing assay. Cells were cultured to 100% confluence in 6-well plates and serum-starved for 12 h before starting the assay. A standardized wound was created using a 200 \u0026micro;l sterile pipette tip. Cells were maintained in serum-free medium, and wound closure was monitored at 0 and 48 h using an inverted microscope (Olympus Corporation) at x40 magnification. All experiments were performed in triplicate with three independent biological replicates.\u003c/p\u003e\u003cp\u003e\u003cem\u003eTranswell assay.\u003c/em\u003e Transwell assays were utilized to evaluate the invasive and migratory abilities of OC cells; a 24-well Transwell chamber with 8 \u0026micro;m pores were used (Corning, Inc.). In the invasion assay, the chamber was coated with Matrigel (BD Bioscience). In the migration assay, no coating was applied. A total of 4x10\u003csup\u003e4\u003c/sup\u003e or 6x10\u003csup\u003e4\u003c/sup\u003e cells were re-suspended in 200 \u0026micro;l serum-free media and then added to the upper chamber. The lower chamber was filled with media supplemented with 10% FBS. After a 48 h incubation period, the cells were fixed with methanol 15 min at room, and subsequently stained with a 0.1% solution of crystal violet 15 min at room. The stained cells were observed and counted using an inverted microscope.\u003c/p\u003e\u003cp\u003e\u003cem\u003eChemotaxis assay.\u003c/em\u003e Using a 24-well Transwell chamber, cell migration assays were performed to evaluate the migratory potential of THP-1 cells. A total of 6x10\u003csup\u003e4\u003c/sup\u003e cells were suspended in 200 \u0026micro;l serum-free medium and subsequently introduced into the upper chamber. The lower chamber was filled with conditioned media derived from malignant cells or supplemented with CCL2. After a 48 h incubation, the cells were fixed using 4% formaldehyde for 20 min at room. Subsequently, cells were stained with 0.1% crystal violet 15 min at room. The stained cells were visualized using an inverted microscope for further analysis.\u003c/p\u003e\u003cp\u003e\u003cem\u003eFlow cytometry\u003c/em\u003e. Malignant peritoneal wash cells derived from murine models were stained using antibodies. Additionally, peripheral blood samples were obtained from human participants, along with malignant peritoneal wash cells post-ID8 implantation in mice, and stained using antibodies. In preparation for surface staining, cells were harvested following erythrocyte lysis and then treated with Zombie UV Fixable viability dye (BioLegend, Inc.) for 15 min at ambient temperature. Subsequently, the samples were incubated using fluorochrome-conjugated antibodies for 30 min. After incubation, cells were washed and analyzed using flow cytometry. The quantification of specific immune subsets was determined using a flow cytometer with phenotypic gating criteria. A list of all antibodies used is stated in Table SI.\u003c/p\u003e\u003cp\u003e\u003cem\u003eAnimal model.\u003c/em\u003e First, the successful knockdown of FMN2 in mouse OC ID8 cells was determined. A cohort of 6-week-old female C57BL/6 mice was obtained from the Animal Center of Zhengzhou University. All experimental procedures involving animals were performed in compliance with the Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee at the Third Affiliated Hospital of Zhengzhou University. To establish the syngeneic intraovarian models, mice were first anesthetized using 50 mg/kg pentobarbital sodium. Then, a single dorsal incision was made to access the ovary. Each group was administered injections of 1x10\u003csup\u003e6\u003c/sup\u003e ID8 cells into the left ovarian bursa (Day 7). Each group was administered sh-NC or sh-FMN2 at a dose of 100 nM once a week by tail vein injection. A total of 56 days after the tumors were implanted, they were surgically excised. Tumor sizes were measured using digital calipers. The tumor volumes were determined using the formula (length x width\u003csup\u003e2\u003c/sup\u003e)/2. For chemotherapy treatment, mice received an intraperitoneal injection of 5 mg/kg cisplatin (Beyotime Institute of Biotechnology) three times a week after the tumors were implanted for a total of two doses. Animals were sacrificed using an overdose of isoflurane anesthesia followed by cervical dislocation. The doses of isoflurane used were in accordance with the guidelines provided by the Institutional Animal Care and Use Committee (IACUC). Confirmation of death was based on the absence of respiratory and cardiac activity, as well as a lack of reflexes. The maximum tumor size permitted was 2 cm in diameter.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePatient populations and specimens.\u003c/em\u003e A total of 26 fresh tumor specimens, 126 cryopreserved tissue samples and 16 blood specimens were obtained from patients with OC who underwent surgical intervention and were subsequently treated with platinum-based chemotherapy at the Third Affiliated Hospital of Zhengzhou University between jan2018 and dec 2024. Prior to enrollment in the study, the participants provided written informed consent. The experimental protocols were approved by the Ethics Committee at the Third Affiliated Hospital of Zhengzhou University (approval no. 2023\u0026thinsp;\u0026minus;\u0026thinsp;120).\u003c/p\u003e\u003cp\u003e\u003cem\u003eImmunohistochemistry.\u003c/em\u003e The tissue sections were first baked in an oven at 75˚C for 4 h, then immediately placed in xylene for dewaxing and incubated with the primary antibody at 4˚C overnight. Subsequently, the tissues were incubated with the secondary antibody at room temperature for 1 h. Finally, the tissue sections were counterstained with DAB and hematoxylin and the display time was observed under the microscope. The assessment of FMN2, CCL2 and CD206 expression was performed by analyzing both the staining intensity and the proportion of positively stained cells. The antibodies used are listed in Table SI.\u003c/p\u003e\u003cp\u003e\u003cem\u003eELISA.\u003c/em\u003e The concentrations of CCL2, CCL3, CXCL1 and CCL18 in the supernatants from various conditional OC cell lines were quantified by ELISA using specific kits according to the manufacturer\u0026rsquo;s protocol (BioLegend, Inc.).\u003c/p\u003e\u003cp\u003e\u003cem\u003eStatistical analysis.\u003c/em\u003e Bioinformatics analyses were performed using R (version 4.3.1) (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). For statistical evaluation, GraphPad Prism version 8.0.2 (Dotmatics) was used. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Standard deviation error bars were included solely for experiments where the sample size was \u0026ge;\u0026thinsp;3. All data presented are representative of at least two independent experiments yielding consistent results. The comparison of differences between the two groups was performed using an unpaired two-sided Student\u0026rsquo;s t-test, while comparisons between \u0026ge;\u0026thinsp;3 groups were performed using a one-way ANOVA. Survival analysis was performed using Kaplan-Meier analysis and a log-rank t-test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eFMN2 expression is correlated with chemoresistance.\u003c/em\u003e R was used to examine the mRNA expression profiles to identify genes correlated with chemotherapy resistance in intermediate-resistant and non-resistant OC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and C) and high-resistant and intermediate-resistant cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and D) using data from the GEO dataset GSE173579. The analysis revealed that 226 genes were up-regulated and 54 genes were down-regulated in the intermediate-resistant cells compared with the non-resistant cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). When comparing high-resistant cells to intermediate resistant cells, 54 genes were up-regulated and 77 genes were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). A Venn diagram was constructed based on these two screening methodologies, revealing six DEGs as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. GEPIA was used to assess the clinical relevance of the identified DEGs in OC, which indicated that only the expression levels of FMN2 was significantly associated with both the OS and DFS rates of patients with OC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and G).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOC cells resistant to cisplatin, specifically SKOV3-DDP cells were obtained. Both the SKOV3 parental cell line and SKOV3-DDP resistant variant were subjected to varying concentrations of cisplatin (0-120 \u0026micro;g/ml) for 24 h. Cell viability was subsequently evaluated using a CCK-8 assay. The findings demonstrated a gradual decrease in the viability of OC cells in response to increasing concentrations of cisplatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). The decrease in viability for the parental OC cells occurred at a significantly faster rate compared to the drug-resistant variants. The IC\u003csub\u003e50\u003c/sub\u003e value of SKOV3-DDP cells, calculated using SPSS version 26.0 (IBM Corp.), was 100\u0026thinsp;\u0026plusmn;\u0026thinsp;8.43 \u0026micro;g/ml, which was markedly higher than the 25\u0026thinsp;\u0026plusmn;\u0026thinsp;6.93\u0026micro;g/ml IC\u003csub\u003e50\u003c/sub\u003e value for the parental cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, western blot analysis indicated a significant increase in the expression levels of the chemoresistance-associated protein P-Glycoprotein (P-gp) protein in the drug-resistant cell lines compared to the parental cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Collectively, these data confirmed the successful establishment of DDP-resistant ovarian cell lines. The mRNA and protein expression levels of FMN2 were shown to be significantly higher in the chemoresistant OC cells and tissues (both P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). Wound healing analysis revealed that SKOV3-DDP cells exhibited enhanced cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK-L). Similarly, Transwell assays showed that SKOV3-DDP cells exhibited increased migratory and invasive capacities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM-N). These findings suggest that chemoresistance may facilitate proliferation, migration and invasion, and may thus be involved in promoting the progression of OC.\u003c/p\u003e\u003cp\u003e\u003cem\u003eFMN2 is upregulated and confers DDP‑resistance in OC cells.\u003c/em\u003e To explore the potential involvement of FMN2 in the progression of OC, the expression levels of FMN2 in SKOV3 and SKOV3-DDP cells were examined. The analysis indicated that FMN2 expression was markedly higher in SKOV3-DDP cells compared with SKOV3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). Stable FMN2 knockdown OC cells were established alongside corresponding negative control cells; successful knockdown was confirmed at both the mRNA and protein level. The data indicated that the knockdown efficiency of sh-FMN2-3 was significantly greater than that of sh-FMN2-1 and sh-FMN2-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E), and therefore, this construct was used for further experiments. FMN2 knockdown OC and negative control cells were incubated with 0, 20, 40, 60, 80, 100 or 120 \u0026micro;g/ml DDP for 24 h. CCK-8 assays indicated that FMN2 knockdown significantly reduced DDP-resistance in chemoresistant OC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and G). Similarly, FMN2 knockdown decreased migration and invasion of chemoresistant OC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH and I).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe tumor immunosuppressive microenvironment is an important factor in chemotherapy resistance, and TAMs are a major invasive immune cell subgroup (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). To investigate the relationship between FMN2 and macrophage infiltration, TIMER2.0 was used. There was a positive correlation between FMN2 expression and the abundance of M2 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Additionally, GEPIA analysis indicated that the expression levels of FMN2 were significantly associated with the M2 macrophage marker, Mannose receptor C type 1 (MRC1; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). Next, clinical samples was used to detect the levels of M2 macrophages in the blood of patients with chemotherapy-resistant and chemotherapy-sensitive OC using flow cytometry, and found that the content of M2 macrophages in the blood of patients with chemotherapy resistant OC was higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL). The findings suggested that increased M2 macrophage infiltration may be associated with high levels of FMN2 expression.\u003c/p\u003e\u003cp\u003e\u003cem\u003eFMN2 regulates macrophage infiltration and M2-like polarization.\u003c/em\u003e The quantity and phenotypic variations of TAMs play a pivotal role in the progression of cancer and influence clinical outcomes. To investigate the impact of FMN2 on macrophage behavior, macrophages derived from THP-1 cells were co-cultured with both FMN2 knockdown SKOV3 cells and SKOV3-DDP cells using a Transwell assay for 48 h. The expression levels of the M2 macrophage marker, CD206, were assessed using RT-qPCR. Compared to the negative control cells, co-culturing with FMN2 knockdown SKOV3-DDP cells resulted in a notable reduction in CD206 expression in macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM and N). Additionally, the proportion of CD206\u0026thinsp;+\u0026thinsp;macrophages, indicative of M2 macrophage polarization, after co-culture with OC cells was quantified using flow cytometry. Compared to the negative control group, the percentage of CD206\u0026thinsp;+\u0026thinsp;macrophages decreased following co-culture with FMN2 knockdown chemoresistant OC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO). Therefore, these findings suggest that FMN2 facilitated the polarization of macrophages towards the M2 phenotype.\u003c/p\u003e\u003cp\u003e\u003cem\u003eFMN2 induces macrophage polarization towards an M2 phenotype via the CCL2/JAK2/STAT3 pathway.\u003c/em\u003e As FMN2 knockdown in non-contact co-cultures with OC cells promoted M2 polarization of macrophages, it was hypothesized that the ability of OC cells to stimulate M2 polarization was associated with differences in the levels of secreted cytokines. A thorough examination of the existing literature led to the identification of 13 cytokines (IL-1α, IL-6, IL-9, CCL2, CCL3, CCL4, CCL5, CXCL1, CXCL2, TNF-α, CSF-1, GM-CSF and LIF) for further analysis via RT-qPCR. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eP and Q, among the 13 cytokines, the most pronounced down-regulation, compared to other cytokines, was observed in the transcriptional levels of CCL2. Using ELISA, the concentration of CCL2 in the culture supernatants of OC cells was evaluated. The levels of CCL2 in the culture supernatants were consistent with the findings obtained from RT-qPCR analysis. Western blotting confirmed that FMN2 knockdown down-regulated the expression of CCL2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eT).\u003c/p\u003e\u003cp\u003eNext, the expression of immune factors in chemotherapy-resistant and sensitive OC tissues was examined in the GSE28739 dataset, and found that most of the immune factors exhibited altered expression, among which the expression of CCL2 was the most significantly up-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). Studies have shown that CCL2 plays a crucial role in facilitating the chemotaxis of monocytes and macrophages, and in functional suppression (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In the clinical specimens, it was found that CCL2 expression gradually increased with the increase of chemotherapy course (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E, a notable reduction in the number of migrating cells was observed when using the conditioned medium from FMN2 knockdown cells. Conversely, treatment with CCL2 increased the number of migrating cells, suggesting that inhibiting FMN2 decreased macrophage migration, and CCL2 promoted it. To further assess functional changes in macrophages, M0 macrophages were treated with conditioned media from sh-NC or sh-FMN2-treated SKOV3 and SKOV3-DDP cells, including media supplemented with CCL2. Numerous studies have confirmed the involvement of the JAK2/STAT3 signaling axis in M2 macrophage polarization. Based on these insights, whether FMN2 promoted M2 macrophage polarization via the JAK2/STAT3 pathway was next assessed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-H, the levels of phosphorylated JAK2 and STAT3 were significantly higher in macrophages treated with CCL2, in macrophages co-cultured with FMN2 knockdown chemoresistant OC cells, and in macrophages co-cultured with FMN-2 knockdown OC cells. Of note, treatment with anti-CCL2R substantially reduced the ability of both FMN2 knockdown chemoresistant and parental OC cells to activate this signaling pathway, potentially by blocking CCL2 receptor interaction. Collectively, these findings indicated that FMN2 enhanced macrophage recruitment and promoted M2-like polarization primarily through a CCL2/JAK2/STAT3 signaling pathway.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCoculture with OC cells induces M2 macrophage polarization.\u003c/em\u003e THP-1 cells were polarized towards an M0 phenotype by treating cells with PMA. Following this, M0 macrophages were co-cultured with SKOV3 and SKOV3-DDP cells in a Transwell assay for 48 h. RT-qPCR analyses showed that the expression levels of certain markers were significantly diminished in macrophages co-cultured with SKOV3-DDP compared to those co-cultured with SKOV3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). Conversely, the expression levels of M2-associated genes, including CD163, CD206, IL-10, Arg-1 and VEGF-A, were upregulated following co-culture with ovarian cells, with significantly increased levels observed in macrophage cells co-cultured with SKOV3-DDP cells compared to those co-cultured with SKOV3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). Additionally, these results highlight the differential impact of drug-resistant OC cells on macrophage polarization. Flow cytometry was employed to examine the surface markers of M0 macrophage cells co-cultured with SKOV3-DDP cells. The results indicated a reduction in the proportion of CD86\u0026thinsp;+\u0026thinsp;cells, which is representative of M1 macrophages, in the macrophages co-cultured with SKOV3-DDP cells compared to those co-cultured with SKOV3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). Additionally, the percentages of CD206\u0026thinsp;+\u0026thinsp;cells (M2 macrophages) were increased in macrophages co-cultured with SKOV3-DDP and SKOV3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe findings collectively suggest that OC cells can influence macrophages towards an M2 phenotype, and chemoresistant cells were more proficient in M2 polarization.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCo‑culturing OC cells with M2‑polarized macrophages enhances the DDP‑resistance in OC cells.\u003c/em\u003e OC cells were co-cultured with macrophages exhibiting distinct phenotypes using a Transwell assay for 48 h. Subsequently, the macrophages were removed, and the OC cells were harvested for further analysis. The CCK-8 assay showed that the presence of macrophages affected resistance to DDP. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and C, co-culture with M1 macrophages increased the IC\u003csub\u003e50\u003c/sub\u003e values of SKOV3 cells from 25\u0026thinsp;\u0026plusmn;\u0026thinsp;6.93 to 55\u0026thinsp;\u0026plusmn;\u0026thinsp;5.12 \u0026micro;g/ml, while co-culture with M2 macrophages further increased the IC\u003csub\u003e50\u003c/sub\u003e values to 85\u0026thinsp;\u0026plusmn;\u0026thinsp;6.83 \u0026micro;g/ml. Similarly, the IC\u003csub\u003e50\u003c/sub\u003e values of SKOV3-DDP cells increased from 100\u0026thinsp;\u0026plusmn;\u0026thinsp;8.43 to 110\u0026thinsp;\u0026plusmn;\u0026thinsp;7.20 \u0026micro;g/ml after co-culture with M1 macrophages and to 125\u0026thinsp;\u0026plusmn;\u0026thinsp;7.65 \u0026micro;g/ml after co-culture with M2 macrophages. Furthermore, the expression levels of P-gp were markedly elevated in the drug-resistant cell lines (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and E). Wound healing assays revealed that co-culture with M2 macrophages significantly facilitated the migration of OC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). Collectively, these results suggest that DDP-resistant OC cells effectively induced the polarization of macrophages toward the M2 phenotype, which, in-turn, contributed to enhanced DDP resistance in OC cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eCXCL1 derived from M2polarized macrophages is associated with DDPresistance of OC cells.\u003c/em\u003e The varying capacity of macrophages co-cultured with SKOV3 and SKOV3-DDP to enhance DDP resistance in OC cells has been proposed to result from differences in secreted cytokine levels. A comprehensive literature review led to the selection of 11 cytokines (CCL1, CCL2, CCL3, CCL4, CCL5, CCL17, CCL18, CCL22, CXCL1, CXCL2 and CXCL5) for analysis using RT-qPCR. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B, among the 11 cytokines, only CCL18, CCL3 and CXCL1 exhibited transcription levels consistent with the previously observed trends (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Notably, transcription levels in macrophages co-cultured with SKOV3-DDP were significantly elevated compared to macrophages co-cultured with SKOV3. Subsequently, the concentrations of CXCL1, CCL3 and CCL18 proteins in the culture supernatants were assessed using ELISA, revealing that only CXCL1 levels were significantly higher than the respective controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F).\u003c/p\u003e\u003cp\u003eIn light of these findings, CXCL1 was further studied to elucidate its role in the DDP resistance of OC cells. DDP-sensitive OC cells were cultured either alone or co-cultured with M2 macrophages. These cultures were treated with 10 ng/ml recombinant human CXCL1 (rhCXCL1) or 0.5 \u0026micro;g/ml CXCL1 neutralizing antibody for 48 h. Subsequently, the cells were exposed to various concentrations of 0, 20, 40, 60, 80, 100 and 120 \u0026micro;g/ml DDP for 24 h, and cell survival rates were evaluated. Results from the CCK-8 assay indicated that co-culture with M2 macrophages or the addition of rhCXCL1 induced DDP resistance, whereas administration of the CXCL1 neutralizing antibody diminished the M2 macrophage-mediated DDP resistance in OC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and H). Crucially, Transwell assays demonstrated that either co-culturing with M2 macrophages or adding rhCXCL1 enhanced the migratory and invasive capacities of OC cells, while CXCL1 neutralizing antibodies mitigated these effects mediated by M2 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI and K). Furthermore, western blot analysis revealed that CXCL1 upregulated the mesenchymal markers N-cadherin and vimentin, while downregulating the epithelial marker E-cadherin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ), indicating epithelial-mesenchymal transition. Collectively, these findings suggest that CXCL1, derived from M2-polarized macrophages, represented a significant chemokine associated with DDP resistance in OC cells.\u003c/p\u003e\u003cp\u003eIn summary, after the upregulation of FMN2 expression in OC cells, the expression levels of secreted CCL2 increased, recruiting monocytes and promoting their polarization towards M2 macrophages via activation of the JAK2-STAT3 signaling pathway. The secretion of CXCL1 by M2 macrophages, in turn, enhanced the progression of OC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eDown-regulating FMN2 reduces the presence of TAMs in ovarian masses.\u003c/em\u003e To further explore whether chemotherapy, FMN2 and CCL2 affected TAMs, mouse models were used (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) and tumor volumes were measured to determine whether impaired tumor growth following FMN2 knockdown was the result of reduced recruitment of M2 macrophages. The experimental results showed that tumor growth was inhibited after FMN2 knockdown, but CCL2 and chemotherapy treatment eliminated this difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-D), which may have been related to the increase in M2 macrophages. Survival analysis revealed that mice benefited from the FMN2 knockdown but did not benefit from CCL2 and chemotherapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and F). The expression of the M2 marker CD163 in tumor tissues was detected using RT-qPCR; it was found that the expression levels of CD163 were significantly reduced following sh-FMN2 treatment and the content of CD163 was significantly increased after treatment with CCL2 or chemotherapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG and H). The results of the present study indicated that macrophages facilitated tumor progression. Moreover, the impact of sh-FMN2 on tumors was influenced by the immune microenvironment, particularly TAMs. To examine the proportion of macrophages, cells isolated from ascitic fluid were analyzed using flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). The results of flow cytometry demonstrated a significant reduction in the population of F4/80\u0026thinsp;+\u0026thinsp;CD11b\u0026thinsp;+\u0026thinsp;TAMs within the ascites of the sh-FMN2 group. Furthermore, the sh-FMN2 group exhibited a lower number of macrophages compared to the sh-NC group; however, treatment with CCL2 and chemotherapy abolished this reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ and K). These findings implied that FMN2 enhanced the recruitment of macrophages and facilitated M2-like polarization of macrophages \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eClinical relevance of FMN2 with CCL2 and CD206.\u003c/em\u003e To investigate the relationship between FMN2 and CCL2 and to assess the presence of stromal macrophages, immunostaining of tumor samples for CCL2 and the M2 macrophage marker CD206 was performed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-D, the expression levels of FMN2, CCL2 and CD206 were elevated in tumor tissues from patients with chemotherapy-resistant OC. A notable correlation was identified between the expression levels of FMN2 and CCL2. Additionally, varying degrees of infiltration by CD206\u0026thinsp;+\u0026thinsp;macrophages were observed. Spearman correlation analysis revealed a statistically significant association between FMN2 and CCL2 expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE and F). These findings further supported the hypothesis that FMN2 promoted M2 macrophage polarization and this was mediated by CCL2.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eExpression of FMN2, CCL2 and CD206 was correlated with the prognosis of patients with OC.\u003c/em\u003e The OS and DFS rates were evaluated based on the expression levels of FMN2, CCL2 and CD206. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, patients with elevated expression levels of FMN2, CCL2 and CD206 had a poorer OS (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG-I) and DFS (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ-L) compared to patients with lower expression levels of these markers. These findings suggested a significant correlation between the expression levels of FMN2, CCL2 and CD206, and the prognosis of patients with OC who received DDP-based chemotherapy.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study showed that tumor-derived FMN2 modulated the expression of CCL2. This modulation promoted the recruitment of M2-like macrophages in both clinical tumor specimens and murine models. This process contributed to the establishment of an immunosuppressive microenvironment in OC and contributed to resistance to chemotherapy by diminishing the efficacy of chemotherapeutic agents. These results provide valuable insights into the mechanisms by which macrophages infiltrate ovarian tissue.\u003c/p\u003e\u003cp\u003eFMN2 is involved in the organization of the actin cytoskeleton, and is pivotal in facilitating cell migration and plays a key role in tumor progression (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). FMN2 plays different roles in different tumors, both as an oncogene and as a tumor suppressor gene (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Among these, its expression is upregulated in melanoma (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), colorectal cancer (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), hepatocellular carcinoma (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) and leukemia (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), where it functions as an oncogene, but plays a cancer-inhibiting role in colorectal cancer (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and pancreatic cancer (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Other researchers used an online database to study the relationship between FMN2 expression and the immune microenvironment in colorectal cancer, and proposed that FMN2 gene mutation could affect the distribution of immune cells and thus influence the immune microenvironment (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Nevertheless, there is a paucity of literature addressing the specific function and clinical significance of FMN2 within the immune microenvironment of OC. Through database analysis, it was observed that FMN2 expression was significantly elevated in OC exhibiting resistance to chemotherapy and was negatively associated with the OS and DFS of patients. The expression of FMN2 was also positively correlated with the M2 macrophage marker MRC1 and the abundance of M2 macrophages. The expression of FMN2 increased gradually as the duration of treatment with platinum-based chemotherapy increased. Furthermore, RT-qPCR and western blot analyses revealed that FMN2 levels were markedly upregulated in tissues and cells resistant to OC chemotherapy compared to those sensitive to such treatments.\u003c/p\u003e\u003cp\u003eData analysis from another GEO database showed that CCL2 expression was most significantly upregulated in chemotherapy-resistant OC tissues compared with chemotherapy-sensitive OC tissues. Moreover, analysis of the clinical specimens in the present study showed that CCL2 expression gradually increased as the course of chemotherapy increased. After knocking down FMN2 expression in OC cells, it was found that the expression of CCL2 was significantly decreased at both the mRNA and protein levels. In addition, IHC results showed that the expression of FMN2, CCL2 and CD206 was significantly increased in chemotherapy-resistant OC tissues, and FMN2 was positively correlated with CCL2 and CD206. Of note, M2 macrophages decreased after co-incubation of SKOV3 and SKOV3-DDP of sh-FMN2 with M0, and the levels of M2 macrophages increased after co-incubation with CCL2. The isogenic intraovarian mouse model showed that the tumor volume was significantly reduced and survival time increased in the sh-FMN2 group, which was partially offset by cisplatin or CCL2 treatment. In addition, the levels of M2 macrophages decreased significantly in the sh-FMN2 group and increased again after cisplatin or CCL2 treatment. Therefore, it was speculated that CCL2 may be downstream of FMN2, and as a chemokine, CCL2 promoted the formation of TAMs, thus promoting the progression of tumors and ultimately leading to chemotherapy resistance.\u003c/p\u003e\u003cp\u003eRecent investigations have elucidated the significance of the CCL2-CCR2-M2 macrophage axis in the context of OC. One study demonstrated that the administration of CCR2 antagonists \u003cem\u003ein vivo\u003c/em\u003e effectively diminished the M2 macrophage population, consequently inhibiting tumor proliferation. This finding underscored the substantial indirect influence exerted by M2 macrophages on tumor growth (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). CCL2, a crucial mediator of the recruitment, differentiation and functional modulation of TAMs, can be synthesized by both tumor and non-tumor cells (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). The production of CCL2 within neoplastic environments has been shown to facilitate tumor-induced immunosuppression and the accumulation of TAMs, highlighting it as a pivotal molecular target for the management of cancer (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). In the present study, FMN2 was observed to increase CCL2 expression, subsequently promoting macrophage recruitment and M2-like polarization; these processes in turn drove OC progression and contributed to the development of cisplatin resistance. Therefore, targeting the FMN2-CCL2 pathway may represent a novel and effective therapeutic approach for managing OC progression and improving sensitivity to cisplatin treatment. Although anti-CCL2 antibodies and CCR2 antagonists have been explored in clinical trials, an animal study revealed that the discontinuation of anti-CCL2 antibody therapy led to a significant increase in lung metastasis and hastened mortality in a mouse isogenic cancer model (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Consequently, the efficacy of solely inhibiting the CCL2-CCR2 signaling pathway remains a contentious issue, partly due to the adverse outcomes observed following disruption of therapy (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOC is widely recognized for its tendency to develop resistance to chemotherapy. Consequently, numerous investigations have been conducted to enhance the therapeutic response of patients with OC to chemotherapeutic agents. Among these studies, targeting aberrant oncogenic genes in OC cells, such as lysine-specific demethylase 1 and lysine-specific demethylase 1, poly ADP-ribose polymerase, and glutathione S-transferase P1, has demonstrated efficacy in enhancing the therapeutic response to chemotherapy. Furthermore, inhibiting the expression of the FMN2 gene, rather than obstructing the CCL2-CCR2-macrophage signaling pathway, may represent a promising alternative strategy, as it more effectively improves the response to chemotherapy in OC.\u003c/p\u003e\u003cp\u003eThe present study highlights the critical role of FMN2 in promoting chemoresistance and inducing M2 macrophage polarization in OC. Co-culture with M2-polarized macrophages significantly enhanced the resistance of OC cells to DDP. Moreover, it was confirmed that CXCL1, which originates from M2-polarized macrophages, promoted chemoresistance via the EMT pathway. Taken together, the present study proposed novel targets and strategies for OC treatment and identified potential prognostic indicators for assessing the efficacy of chemotherapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\u003cp\u003eThe Ethics Committee at the Third Affiliated Hospital of Zhengzhou University approved all the experimental protocols in the present study (approval no. 2023\u0026thinsp;\u0026minus;\u0026thinsp;120).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003ePatient consent for publication\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eClinical trial number\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding Statement\u003c/h2\u003e\u003cp\u003eThis study was supported in part by grants from the International Scientific Exchange Foundation of China (no. Z2024LLN005).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSF and LH conceived and designed the study. YW and RR performed the experiments. SL and XW analyzed the data. YW and SF wrote the manuscript. All authors read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArmstrong DK, Alvarez RD, Backes FJ, Bakkum-Gamez JN, Barroilhet L, Behbakht K et al (2022) NCCN Guidelines\u0026reg; Insights: Ovarian Cancer, Version 3.2022. 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Nature 515(7525). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature13862\u003c/span\u003e\u003cspan address=\"10.1038/nature13862\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNawghare BR, Joshi RR, Lokhande PD, CHEMOSELECTIVE METAL FREE, DEALLYLATION OF α-ALLYL-PHENYLCARBOXYLIC ESTERS UNDER REDUCTION CONDITION (2025) B Chem Soc Ethiopia 39(1):131\u0026ndash;139. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dx.doi.org/10.4314/bcse.v39i1.11\u003c/span\u003e\u003cspan address=\"10.4314/bcse.v39i1.11\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"FMN2, CCL2, Ovarian cancer, Macrophages","lastPublishedDoi":"10.21203/rs.3.rs-7209240/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7209240/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOvarian cancer (OC) remains a major threat to women\u0026rsquo;s health, with chemoresistance driven by the immunosuppressive tumor microenvironment. Formin-2 (FMN2), a cytoskeletal regulator, was investigated for its role in OC chemoresistance and macrophage polarization. Bioinformatics analysis identified high FMN2 expression in chemotherapy-resistant OC cell lines, validated experimentally. Stable FMN2 knockdown cell lines were generated via lentiviral transfection. Functional assays revealed that FMN2 overexpression conferred chemoresistance \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e and promoted M2 macrophage polarization via the CCL2/JAK2/STAT3 pathway. Co-culture with M2 macrophages enhanced cisplatin (DDP) resistance in OC cells, mediated by CXCL1 secretion, which activated the epithelial-mesenchymal transition (EMT) pathway. Clinically, FMN2 levels correlated with CCL2 and CD206 (M2 marker) in platinum-resistant patients, and high FMN2, CCL2, or CD206 expression predicted poorer overall and disease-free survival. This study identifies FMN2 as a key mediator of chemoresistance and immune evasion in OC, proposing FMN2-CCL2-CD206 signaling and macrophage-derived CXCL1 as therapeutic targets and prognostic markers for chemotherapy response.\u003c/p\u003e","manuscriptTitle":"High expression of formin-2 can promote ovarian cancer chemoresistance via immunosuppressive macrophages","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-07 06:51:53","doi":"10.21203/rs.3.rs-7209240/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-25T18:27:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-15T01:42:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99609418260241278267404562343248762117","date":"2025-08-13T18:54:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-04T11:30:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-31T11:01:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-31T11:01:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Functional \u0026 Integrative Genomics","date":"2025-07-25T01:04:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5249a33e-0852-4fe8-b5bb-53443ff87f61","owner":[],"postedDate":"August 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T16:03:50+00:00","versionOfRecord":{"articleIdentity":"rs-7209240","link":"https://doi.org/10.1007/s10142-025-01766-z","journal":{"identity":"functional-and-integrative-genomics","isVorOnly":false,"title":"Functional \u0026 Integrative Genomics"},"publishedOn":"2025-12-20 15:58:41","publishedOnDateReadable":"December 20th, 2025"},"versionCreatedAt":"2025-08-07 06:51:53","video":"","vorDoi":"10.1007/s10142-025-01766-z","vorDoiUrl":"https://doi.org/10.1007/s10142-025-01766-z","workflowStages":[]},"version":"v1","identity":"rs-7209240","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7209240","identity":"rs-7209240","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}