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ZEB1 promotes the immune escape of ovarian cancer through the MCSF-CCL18 axis

Cancer Cell International volume 25, Article number: 95 (2025) Cite this article

Abstract

This study aimed to determine the molecular mechanisms underlying immune escape in ovarian cancer. Samples of ovarian cancer were used to explore the regulatory pathways involved in the malignant phenotype. Tumor cell models with different levels of factor expression were constructed via transfection, and their regulation was determined through investigation of protein expressions. Moreover, our study aimed to investigate the effects of M2 polarization and TAMs aggregation on the apoptosis of CD8 + T-cells, and determine their regulatory axis. Results revealed ZEB1 may promote CCL18 expression via upregulation of MCSF concentration. Notably, high CCL18 expression levels were associated with the aggregation of M2-TAMs and the apoptosis of CD8 + T-cells. In addition, results of the present study demonstrated that the proliferation and invasion of ovarian cancer cells with high expression levels of proteins associated with ZEB1 signal pathway were increased. At the same time the growth rate of tumors in mice was reduced following ZEB1 knockdown, and the volume/weight of tumors were markedly decreased both in vitro and in vivo. Moreover, our results revealed that the aggregation of M2-TAMs and the apoptosis of CD8 + T-cells were significantly decreased in tumor cells following ZEB1 knockdown. Thus, these results verified that ZEB1 may promote the M2 polarization of TAMs via the MCSF axis, leading to the increased secretion of CCL18. Moreover, the MCSF axis may mediate immune escape through the induction of CD8 + T-cell apoptosis, ultimately promoting the malignant phenotype in ovarian cancer cells.

Introduction

Ovarian cancer has the highest mortality rate among gynecological malignant tumors [1], and surgery combined with chemotherapy remains the standard treatment option for this disease. Although recent research has focused on the development of novel treatment methods, the 5-year survival rate of patients with ovarian cancer is < 50% [2]. Notably, platinum resistance may lead to treatment failure or death in patients with ovarian cancer. Patients may present with sensitivity to platinum in the early stages of treatment, or they may develop resistance to platinum following repeated treatments. Thus, the effectiveness of certain chemotherapeutic drugs remains limited in these patients, with a response rate of < 30% (3–4).

In recent years, research has focused on the use of immunotherapy in cancer. Chimeric antigen receptor T-cell (CAR-T) immunotherapy led to a cancer-free survival period of up to 9 years in patients with lymphoblastic leukemia (ALL), and the stagnation of immune checkpoints has also improved the overall survival rate of patients with melanoma, non-small cell lung cancer, urothelial cancer and renal cell carcinoma [5,6,7]. However, drug resistance in patients may limit the use of immunotherapy in clinical practice, and previous studies have demonstrated that this may be associated with the immune escape of ovarian cancer. Thus, further investigations into the mechanisms underlying immune escape are required.

Results of a previous study revealed that zinc finger E box binding homeobox 1 (ZEB1) was involved in the early stages of tumorigenesis via carcinogenesis [8]. Notably, this regulatory factor is located downstream of tumor microenvironment regulatory networks, and directly involved in the microRNA (miRNA/miR)-controlled feedback loop (9–10). Results of a previous study demonstrated that the negative feedback loop between ZEB1 and targeted miRNAs may play a role in the occurrence and development of cisplatin resistance in patients with ovarian cancer [11]. In addition, ZEB1 may promote CD8 + T-cell immunosuppression through alleviating the inhibitory effects of downstream miRNA on tumor cell PD-L1 (Programmed Death Ligand-1) expression [12].

Notably, the formation of tumor associated macrophages (TAMs) is closely associated with the malignancy of ovarian cancer cells. In addition, results of previous studies revealed that ZEB1 induced the transcription of macrophage colony stimulating factor (MCSF) in ovarian cancer cells, ultimately promoting M2 polarization in TAMs (13–14). Chemokine (C-C motif) ligand 18 (CCL18) is a key chemokine secreted by M2-TAMs, and this is closely associated with metastasis and the poor prognosis of patients with breast cancer. Results of a previous study revealed that M2-TAMs may induce the malignant phenotype of ovarian cancer cells via the release of CCL18, and this regulatory factor may also mediate the apoptosis of CD8 + T-cells in the tumor microenvironment [15].

Thus, we hypothesized that ZEB1 may promote ovarian cancer cell proliferation and CD8 + T-cell apoptosis via the MCSF-CCL18 axis, leading to the induction of immune escape in ovarian cancer.

Materials and methods

Clinical samples

Patients who underwent surgical excision for ovarian occupation between May 2022 and April 2024 were considered as potential participants if they met the following criteria: (1) Female adults aged 20–70, (2) Postoperative pathological diagnosis was ovarian cancer, (3) No surgery or other related treatment was performed before this admission. Patients who encountered the following situations should be excluded: (1) Neoplastic cachexia, (2) Liver and kidney dysfunction, (3) Pregnancy and lactation, (4) Major surgery or severe injury within one month prior to admission.

In total, 60 ovarian cancer samples were collected in the Pathology Department of The Affiliated Suzhou Hospital of Nanjing Medical University from May 2022 to April 2024. Notably, complete data was available for all cases. The age of patients ranged from 27 to 63 years, with a median age of 42 years. In total, 17 cases (28.3%) were in stage I, 18 cases (30.0%) were in stage II, 12 cases (20.0%) were in stage III and 13 cases (21.7%) were in stage IV of disease. Histologically, 25 cases were classified in G2 (41.7%) and 35 cases were classified in G3 (58.3%). Moreover, 38 cases (63.3%) presented with lymph node metastasis and 22 cases (36.7%) did not demonstrate lymph node metastasis. Clinical characteristics of these cases were listed in Table 1.

Table 1 Clinical characteristics of patients included in this study
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Tissue collection and protein content detection

Tissue samples were obtained and divided into low-grade (Phase I-II) or high-grade groups (Phase III-IV). All tissues were stored in liquid nitrogen at -80℃ for subsequent protein content detection and immunohistochemical staining.

Cell transfection

Cells and tumor samples were divided into groups as previously described, and lentiviral plasmid transfection was carried out with Lipofectamine-3000 (Thermo Fisher Scientific). During this study, we chose second-generation lentiviral plasmid (Genechem; pLVX: pSPAX2: pMD2.G = 4: 3: 1). Transfection usually performed when cells reached 60-80% (with plasmid concentration ≥ 500 ng/µl). After that a period of resting (4–6 h) was offered to allow plasmids entering cell samples. Then these cells were placed in incubators at 37 °C and 5% CO2 for culture, 48–72 h later cell samples were collected for follow-up experiments.

Following overexpression, samples were divided into OV-negative control (NC), OV-ZEB1, OV-MCSF and OV-CCL18 groups. Following transfection with the respective short-hairpin (Sh) RNA, samples were divided into Sh-NC, Sh-ZEB1, Sh-MCSF and Sh-CCL18 groups.

Cell culture

IOSE-80 (No. bio-106125‌‌) and SKOV3 (bio-105788) cell lines were purchased from the National Experimental Cell Resource Sharing Platform. Cell lines were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Zhejiang Tianhang Biological Company) and at 37℃/5% CO2 for 36 h.

CD8 + T-cell co-culture

The EasySep™ Human CD8 + T-cell Enrichment kit was used to isolate CD8 + T-cells from peripheral blood mononuclear cells (PBMCs), and these cells were treated with the CD3/CD28 activator at a ratio of 1:1. Cells were cultured in DMEM containing 10% FBS, 1% penicillin double antibiotic and 100 U/ml IL-2. Cell density was adjusted to 1 × 106 cells/ml, and SKOV3 cells were subsequently washed with PBS. Following washing, SKOV3 cells were resuspended in cell culture medium at a concentration of 2 × 105 cells/ml, and inoculated in a 96-well U-shaped culture plate with 50 µl/well (10,000 cells). Following culturing, cells were incubated at 37℃ and 5% CO2 overnight. Subsequently, CD8 + T-cells were collected, resuspended in cell culture medium at 1 × 106 cells/ml, and added to the 96-well plate containing SKOV3 cells at a ratio of 1: 1. Following centrifugation (400 g/4℃/10min), cells were incubated at 37℃ and 5% CO2 for 18 h.

Western blot analysis

Both clinical and cell samples were collected, lysed and centrifuged (4℃/12000 g/10min). Supernatants were obtained, and the concentration of pathway proteins was detected through Western Blot. We selected protein extraction buffer (NCM Biotech, Cat No: WB2001) to extract protein from these samples, and used BCA assay kit (Beyotime, P0009) for protein concentration determination. Then we added these samples to electric lanes (Protein: 10 µg/lane, Gel percentage: 12% separated glue/5% concentrated glue), transferred them to the NC membrane after electrophoresis, and added 5% skimmed milk powder to incubated at 4 °C overnight. Primary antibodies were added and incubated at 4 °C overnight, after that diluted secondary antibodies (TBST, 1: 5000) were added and incubated for 30 min at room temperature. Follow-up we carried out luminescence, exposure and photography, finally measured their gray value by AIWBwell™.

The primary and secondary antibody information used in this experiment was listed as follows:

Primary Antibody (abcam): ZEB1:Anti-ZEB1 antibody (Cat No: ab203829) at 1/1000 dilution, Host: Rabbit, Incubated at 4 °C for 24 h. MCSF: Anti-M-CSF antibody (Cat No: ab233387) at 1/1000 dilution, Host: Rabbit, Incubated at 4 °C for 24 h. CCL18: Anti-CCL18 antibody (Cat No: ab300057) at 1/1000 dilution, Host: Rabbit, Incubated at 4 °C for 24 h. Secondary Antibody (abcam): Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/25,000 dilution, Host: Rabbit/Anti-Rabbit Recombinant, Incubated at 4 °C for 6 h.

3D-proliferating ball formation

Matrigel matrix glue was incubated at 4℃ for melting into a liquid state. Cells in the logarithmic growth phase were digested with trypsin for counting, and the concentration was adjusted to 2.0 × 105 cells/ml. Subsequently, 300 µl matrix glue was added (2.5%, v/v) to cells, and the cell suspension was mixed in 10 ml culture medium. In total, 600 µl of the cell suspension was mixed to reach a cell concentration of 10,000 cells/ml. Subsequently, cell suspensions containing Matrigel matrix glue were added to a sampling tank, and 200 µl of this solution was added to a 96-well plate coated with agarose. All samples were centrifuged at 1,000 g and 4℃ for 10 min, sealed and cultured at 37℃/5% CO2 for 7 days.

We observed these samples through an inverted microscope, and took pictures of all spheroids. Then we used image software (Image J) to analyze these images, thus detect the change in the area of these proliferating cell sphere. Based on this we evaluated the formation and growth of these 3D spheroids (Global), and finally reflected the proliferating ability in these cell samples.

Cell invasion

Following 48 h of transfection, 30 µg of matrix adhesive (BD Biosciences) was used to detect cell invasion (MilliporeSigma). Cells were cultured at 37℃ to form the reconstituted matrix film. Subsequently, 200 µl of serum-free medium containing 1 × 105 cells was placed in the upper chamber, and 600 µl serum-containing medium was placed in the lower chamber. Cells were fixed at 4℃for 15 min with 4% paraformaldehyde, and stained with hematoxylin at 35℃ for 15 min. Non-migrating cells were removed, and cell invasion was determined using a microscope (Olympus Corporation/Inverted/100x).

Immunohistochemical analysis

Samples were fixed with 4% paraformaldehyde and stored at 4℃ overnight. All samples were cut into slices (5 μm) and incubated with 0.3% hydrogen peroxide methanol at 4℃ for 30 min. Subsequently, samples were incubated with 0.3%Triton™ X-100 at 37℃ for 30 min, and anti-rabbit monoclonal antibody (Abcam) diluted with serum (Constituents: 59% PBS, 40% Glycerol, 0.05% BSA; ZEB1: ab303480, MCSF: ab233387, CCL18: ab233099) was added. Following overnight incubation at 4℃, samples were washed in TBS-tween 20 (TBS-T, 0.05%) and incubated with the goat anti-rabbit IgG secondary antibody (proteintech, Constituents: 0.01 M Sodiumphosphate, 0.25 M NaCl, 50% glycerol; No. SA00001-2). Following incubation at room temperature for 2 h, color solution was added for immunohistochemical analysis. Sections were dehydrated using gradient ethanol, sealed and imaged with Image J software (20x).

Immunofluorescence

Cells were digested, dispersed and added to a six-well plate at 5 × 105 cells/well. Following 24-h incubation, cells were fixed using 4% paraformaldehyde at 4℃ and incubated at room temperature for 15 min. Subsequently, cells were incubated with 0.5% Triton™ X-100 at room temperature for 20 min, and sealed using goat serum for 30 min. Cells were incubated with the anti-rabbit monoclonal antibody (Abcam, Constituents: 59% PBS, 40% Glycerol, 0.05% BSA; ZEB1: ab303480, MCSF: ab233387, CCL18: ab233099) overnight at 4℃ overnight. Following primary incubation, cells were incubated with the fluorescent goat anti-rabbit IgG secondary antibody (proteintech, Constituents: 0.01 M Sodiumphosphate, 0.25 M NaCl, 50% glycerol; No. SA00001-2) for 1 h. Hoechst stain was added, and cells were incubated in the dark for 5min. Subsequently, cells were sealed using an anti-fluorescence quenching agent, and images were obtained using a fluorescence microscope (Olympus Corporation).

Flow cytometry

Cell samples were collected and resuspended in PBS. In total, 1 × 106 cells were centrifuged (4℃/500 g/5min) and stained with 10 µl Annexin V-FITC and 5 µl PI (Thermo Fisher Scientific/room temperature/10min). Subsequently, CD206 and CD11b (Abcam, No. ab270647/ab8878) were added, and cells were analyzed.

We selected FACS-Calibur system for flow cytometry analysis, while in the detection of M2-TAMs cells, we added CD206 and CD11b as specific antibodies. In the process of CD8+-T cell apoptosis detection, we used cell sorting kit to screen CD8+-T, then double-stained these cells with AnnexinV-FITC (FL1 channel) and PI (FL2 channel). When proceed apoptosis testing on the system we selected FITC channel for Annexin V-FITC and PerCP/Cy5.5 channel for PI.

Detection of TAMs in co-culture

THP-1 cells were digested and counted when 80–90% confluence was reached, and subsequently added to PRMI-1640 complete medium containing 5 × 105/ml tumor cells. In total, 100 ng/ml PMA (Propylene Glycol Methyl Ether Acetate) was added, and cells were incubated in six-well plates with 5% CO2 at 37℃ for 24 h. Following incubation, cell supernatants were discarded, and cells were washed three times using PBS. PRMI-1640 complete medium was added and cells were incubated at 37℃ for 24 h.

In addition, ovarian cancer cells were washed with PBS and mixed with PRMI-1640 basal medium with 1% double antibodies without serum. Following incubation for 24 h, cell supernatants were extracted and centrifuged at 1,000 g and 4℃ for 20 min. Subsequently, 10% FBS was added, and cells were incubated for 24 h. Cells were washed with PBS and the conditioned co-culture medium was obtained. Samples were added into six-well plates supplemented with PRMI-1640 complete medium (2 ml/ well), and co-cultured for 24 h.

The ratio of M2 polarization was detected using flow cytometry, and aggregation of M2-TAMs was detected using cellular immunofluorescence as previously described.

Xenograft assay

SKOV3 cells in the logarithmic growth phase were adjusted to a density of 80–90% (2 × 107 cells/ml) and digested using trypsin. Cells were incubated on ice to reduce cell metabolism, and the left axilla of 12 BALB/c nude mice (weight, 18–20 g) was shaved. Animals were injected with 200 µl (2 × 106 cells/100 µl) of cell suspension, using SKOV3 cells for the NC group and SKOV3 cells with ZEB1 knockdown for the experimental group. All animals were initially anesthetized via inhalation of 4–5% + 0.8-1 l/min halothane, and anesthesia was maintained at a concentration of 1–2% + 0.8-1 l/min (Approved by the Ethics Committee of Suzhou Hospital Affiliated to Nanjing Medical University; nos. SUDA202306080A).

Tumor volumes were measured on Days 7, 14, 21, 28, 35, 42 and 49. All tumor-bearing mice were euthanized via an intraperitoneal injection of pentobarbital sodium (200 mg/kg) on the 50th day. Tumors were removed and weighed, and tumor tissues were obtained for immunohistochemical staining to detect the number of M2-TAMs and CD8 + T-cells. In the present study, tumors did not exceed 2,000 mm3 in diameter.

Statistical analysis

Data are expressed as the mean ± standard deviation (x ± s). All data were analyzed using GraphPad Prism 10 (GraphPad Software, Inc.), and Student’s t-tests (unpaired) were used for comparisons between two groups. P < 0.05 was considered to indicate a statistically significant difference.

Results

Expression of the ZEB1 signaling pathway in ovarian cancer samples

Results of the western blot analysis demonstrated that the expression levels of ZEB1 were markedly increased in ovarian cancer cells, compared with healthy ovarian cells (Fig. 1A). Detection of protein concentration in clinical tissue samples revealed that ZEB1 protein levels significantly increased in high-grade ovarian cancer samples, compared with low-grade samples (Fig. 1B). Results of the immunehistochemical analysis revealed that ZEB1 was mainly expressed in the tumor nuclei, and the positive expression rate of ZEB1 was 44 ± 2% in high-grade ovarian cancer samples, compared with 17 ± 1% in low-grade ovarian cancer samples (Fig. 1C). Notably, the difference between these samples was statistically significant. These results suggested that ZEB1 may mediate the malignant phenotype in ovarian cancer cells.

Fig. 1
figure 1

Expression of the ZEB1 signaling pathway in ovarian cancer samples. (A) Total protein was extracted from ovarian cancer cells and healthy ovarian cells, Western Blot was used to detect the protein level of ZEB1, statistical analysis of ZEB1 expression was offered. (B) Total protein was extracted from low- and high-grade clinical ovarian cancer tissue samples, Western Blot was used to detect the protein level of ZEB1, statistical analysis of ZEB1 expression was offered. (C) HE staining was proceeded and immunohistochemical was used to analyze ZEB1 expression in low- and high-grade clinical ovarian cancer tissue samples, statistical analysis of ZEB1 component was offered. (D) Total protein was extracted from ovarian cancer cells and healthy ovarian cells, Western Blot was used to detect the proteins associated with the ZEB1 signal pathway (MCSF-CCL18), statistical analysis of pathway expression (MCSF-CCL18) was offered. (E) Total protein was extracted from low- and high-grade clinical ovarian cancer tissue samples, Western Blot was used to detect the proteins associated with the ZEB1 signal pathway (MCSF-CCL18), statistical analysis of pathway expression (MCSF-CCL18) was offered. (F) HE staining was proceeded and immunohistochemical was used to analyze proteins associated with the ZEB1 signal pathway (MCSF-CCL18) in low- and high-grade clinical ovarian cancer tissue samples, statistical analysis of ZEB1 component was offered. *P < 0.05, **P < 0.01. ZEB1, zinc finger E box binding homeobox 1

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Results of the western blot analysis also demonstrated that the expression levels of factors associated with the ZEB1 signaling pathway; for example, MCSF and CCL18, were markedly increased in ovarian cancer cells, compared with healthy ovarian cells (Fig. 1D). Results of the western blot analysis using clinical tissue samples also revealed that both MCSF and CCL18 expression levels were significantly increased in high-grade ovarian cancer samples, compared with low-grade ovarian cancer samples (Fig. 1E).

Results of the immunohistochemical analysis demonstrated that MCSF and CCL18 were mainly expressed in the tumor nuclei, and the positive expression rate of MCSF was 44 ± 2% in high-grade ovarian cancer samples, compared with 16 ± 1% in low-grade ovarian cancer samples. In addition, the positive expression rate of CCL18 was 32 ± 1% in high-grade ovarian cancer samples, compared with 13 ± 1% in low-grade ovarian cancer samples (Fig. 1F) Notably, differences between these groups were statistically significant. These results suggested that ZEB1 related signal pathway (MCSF-CCL18) may mediate the malignant phenotype in ovarian cancer cells. Meanwhile, this axis may be closely related to the malignancy of ovarian cancer patients.

Targeted regulation in the ZEB1-MCSF-CCL18 axis

In the present study, SKOV3 cell models with different ZEB1, MCSF and CCL18 expression levels were constructed via transfection, and the concentration of these pathway proteins was detected using western blot analysis. Results of the present study revealed that the expression levels of MCSF and CCL18 were significantly increased in all OV-ZEB1 groups, compared with the corresponding NC group. Moreover, the expression levels of MCSF and CCL18 were markedly decreased in the OV-ZEB1 + Sh-MCSF group, compared with the NC group (Fig. 2A, B).

Fig. 2
figure 2

Targeted regulation between the ZEB1 axis and proliferation in ovarian cancer cells. (A) Cells were transfected with ZEB1 and MCSF plasmids, total cell protein was extracted, and the expression of pathway proteins ZEB1, MCSF and CCL18 was detected by Western Blot (OV Group). (B) Statistical analysis of pathway expression (ZEB1-MCSF-CCL18) was offered to analyze the regulation of these signal factors in Over-Expressing Group. (C) Cells were transfected with ZEB1 and MCSF plasmids, total cell protein was extracted, and the expression of pathway proteins ZEB1, MCSF and CCL18 was detected by Western Blot (Sh Group). (D) Statistical analysis of pathway expression (ZEB1-MCSF-CCL18) was offered to analyze the regulation of these signal factors in Sh-Group. (E) After transfection, these cells were centrifuged and observed through an inverted microscope, all spheroids were captured with Image J. (F) Statistical analysis of sphere projected area was used to analyze the changes in the volume of these proliferating cell sphere. *P < 0.05, **P < 0.01. ZEB1, zinc finger E box binding homeobox 1; OV, overexpression; Sh, short hairpin RNA

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Results of the present study also revealed that the expression levels of MCSF and CCL18 in the Sh-ZEB1 group were markedly decreased, compared with the NC group. By contrast, the expression levels of MCSF and CCL18 in the Sh-ZEB1 + OV-MCSF group were increased compared with the NC group, and these results were statistically significant (Fig. 2C, D). Collectively, these results revealed that ZEB1 may promote MCSF expression via targeted binding, leading to the increased expression of downstream CCL18.

Effects of ZEB1 and the regulatory axis on the proliferation and invasion of ovarian cancer cells

Results of the present study revealed that the proliferation and invasion of SKOV3 cells in the OV-ZEB1 group were significantly increased, while the proliferation and invasion of SKOV3 cells in the OV-ZEB1 + Sh-MCSF and OV-ZEB1 + Sh-CCL18 groups decreased, compared with the corresponding NC groups.

In addition, results of the present study revealed that the proliferation and invasion of SKOV3 cells were markedly decreased in the Sh-ZEB1 group, while the proliferation and invasion of SKOV3 cells in the Sh-ZEB1 + OV-MCSF and Sh-ZEB1 + OV-CCL18 groups were increased, compared with the corresponding NC groups (Figs. 2E and F and 3A and B). Notably, these results were statistically significant. The above experimental results showed that ZEB1 could promote the proliferation and invasion abilities through MCSF-CCL18 axis, thereby mediating the malignant phenotypes in ovarian cancer cells.

Fig. 3
figure 3

Cell invasion and regulation of TAMs in ovarian cancer cells. (A) ZEB1/MCSF/CCL18 overexpression and inhibition cells were constructed, Transwell Assay was used to detect the migration of these ovarian cancer cells. (B) Statistical analysis of invasive cells was offered to analyze the migration of these cell models in OV and Sh Group. (C) Assessment of M2-TAMs cells in ovarian cancer cell models was proceeded by flow cell sorting (CD11b and CD206). (D) Statistical analysis of M2-TAMs cell numbers was offered to analyze the changes of M2 polarization in TAMs cells. (E) Assessment of M2-TAMs aggregation in ovarian cancer cell models was detected through immunofluorescence staining. (F) Statistical analysis of M2-TAMs cell aggregation was offered to analyze the changes of M2 polarization in TAMs cells. *P < 0.05, **P < 0.01. TAM, tumor associated macrophage

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Effects of ZEB1 on CD8 + T-cell apoptosis in the tumor microenvironment of ovarian cancer cells

Results of the present study revealed that the M2 polarization ratio and aggregation of M2-TAMs was markedly increased in the OV-ZEB1 group, compared with the NC group. By contrast, the M2 polarization ratio and aggregation of M2-TAMs was decreased in the OV-ZEB1 + Sh-MCSF group, compared with the control group. In addition, results of the present study revealed that the M2 polarization ratio and the aggregation of M2-TAMs was significantly reduced in the Sh-ZEB1 group compared with NC Group, while the M2 polarization ratio and aggregation of M2-TAMs in the Sh-ZEB1 + OV-MCSF group markedly increased compared with Sh-ZEB1 group (Fig. 3C, D, E, F). Notably, these results were statistically significant.

Results of the present study also revealed that the percentage of apoptotic CD8 + T-cells in the OV-ZEB1 group was significantly increased compared with the NC group. By contrast, the percentage of apoptotic CD8 + T-cells in the OV-ZEB1 + Sh-MCSF and OV-ZEB1 + Sh-CCL18 groups significantly decreased, compared with the NC group. Results of the present study also demonstrated that the percentage of apoptotic CD8 + T-cells was markedly decreased in the Sh-ZEB1 group, while the percentage of apoptotic CD8 + T-cells was significantly increased in the Sh-ZEB1 + OV-MCSF and Sh-ZEB1 + OV-CCL18 groups (Fig. 4A, B). Elisa test showed that factor secretion (IL-10 and TNF-α) in CD8 + T-cells (OV-ZEB1 group) obviously reduced, while these factors correspondingly increased in OV-ZEB1 + Sh-MCSF and OV-ZEB1 + Sh-CCL18 groups (Fig. 4C).

Fig. 4
figure 4

Regulation of CD8 + T-cell apoptosis and tumorigenic ability in ovarian cancer cells. (A) ZEB1/MCSF/CCL18 overexpression and inhibition cells were constructed, flow cytometry was used to detect CD8 + T-cell apoptosis in ovarian cancer cell models. (B) Statistical analysis was offered to analyze the changes of apoptosis rate in CD8 + T-cells. (C) After transfection, ELISA Assay was used to detect the cytokine release in CD8 + T-cells, statistical analysis was offered to analyze these changes. (D) Ovarian cancer cells (SKOV3) were cultured in vitro, these cells were implanted into the left axilla of BALB/c nude mice via subcutaneous injection. The volumes of these subcutaneously transplanted tumors (V = 1/2 * L * W * W) were measured. (E) Tumor volume of samples from tumor-bearing mice in vitro. (F) Tumor weight analysis of samples from tumor-bearing mice in different groups (NC and Sh-ZEB1) in vitro. (G) HE staining was used to detect the aggregation of M2-TAMs and CD8 + T-cells in tissue samples obtained from tumor-bearing mice. (H) Statistical analysis was offered to analyze these data. *P < 0.05, **P < 0.01. TAM, tumor associated macrophage

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The above experimental results showed that ZEB1 could promote M2-polarization of TAMs cells and M2-TAMs aggregation through MCSF axis in ovarian cancer cells. Meanwhile this axis could also induce the apoptosis of CD8 + T cells via promoting CCL18 secretion in ovarian cancer tumor microenvironment. Finally this regulatory pathway could mediate immune escape, thus enhance proliferation and invasion in ovarian cancer cells.

In this section we found that when M2-TAM aggregation and their M2 polarization increased (reduced), the CD8 + T-cell apoptosis correspondingly up-regulated (down-regulated). Meanwhile the proliferation and invasion abilities in ovarian cancer cells obviously enhanced (inhibited). Their range and trend of changes were consistent. So we concluded that the levels of M2 polarization and M2-TAM aggregation were significantly associated with the levels of CD8 + T-cell apoptosis. Moreover, results of the present study demonstrated that cell proliferation and invasion were positively associated with M2 polarization, TAM aggregation and CD8 + T-cell apoptosis in ovarian cancer cells.

Effects of ZEB1 on the tumorigenesis of ovarian cancer cells in vivo

Results of the present study indicated that the growth rate in the Sh-ZEB1 group was markedly lower than that of the NC group, and this difference was apparent from Week 3 (Fig. 4D). Results of the in vitro analysis revealed the weight of tumors in the Sh-ZEB1 group was significantly lower than those in the NC group (Fig. 4E, F).

Results of the immunohistochemical staining assay revealed that the number of M2-TAMs in tumor samples obtained from the Sh-ZEB1 group was markedly decreased, compared with the control group. By contrast, the number of CD8 + T-cells in tumor samples obtained from the Sh-ZEB1 group significantly increased compared with those in NC Group (Fig. 4G, H).

The above experimental results indicated that ZEB1 may induce the immune escape in ovarian cancer by mediating M2-polarization of TAMs cells and apoptosis of CD8 + T-cells, finally promoting the tumorigenesis of ovarian cancer cells in vivo condition.

Discussion

Results of a previous study revealed that the generation of drug resistance in malignant tumors was closely associated with the immune escape of cancer cells [16]. At present, immunotherapy exhibits potential in the treatment of cancer, using the body’s immune system to induce tumor immune surveillance or the reversal of tumor immune escape. Thus, inhibiting the immune escape of cancer cells may be crucial in reducing the generation of drug resistance and improving the effectiveness of tumor immunotherapy.

Research has focused on developments in surgery and chemotherapy for the treatment of ovarian cancer; however, the overall efficacy in patients remains low. Results of previous studies revealed that ovarian cancer cells may secrete a variety of immunosuppressive molecules, leading to an anti-tumor immune response in a low-energy state, and the escape of ovarian cancer cells from immune surveillance.

ZEB1 is an important member of the zinc finger transcription factor family, and abnormal expression of this factor has been reported in numerous malignant tumors. Thus, ZEB1 may promote cancer cell invasion and metastasis [17]. Results of a previous study revealed that ZEB1 was highly expressed in cancer infiltrating cells, highlighting its potential as a regulatory factor that may activate or inhibit gene expression through targeting downstream regulatory regions. Moreover, ZEB1 may act as a key regulatory factor, leading to the promotion of the protoplasm function of TAMs. Notably, CCL18 is the main chemokine secreted by M2-TAMs, and this is closely associated with cell metastasis and the poor prognosis of patients with breast cancer [18]. In addition, the expression levels of MCSF are closely associated with TAM-mediated secretion of CCL18.

Results of a previous study revealed that ZEB1 contains four MCSF binding sites. In a previous study, chromatin immunoprecipitation experiments were performed using SKOV3 cells, and the results demonstrated that the ZEB1 protein accumulated rapidly in the promoter region of MCSF. In addition, luciferase detection confirmed that MCSF activity was significantly increased in ovarian cancer cells with ZEB1 overexpression [19]. Thus, ZEB1 may activate the promoter region of MCSF, while the Siglec-15/sialic acid glyco-immune checkpoint axis may exhibit potential as a downstream target pathway in the tumor microenvironment [20].

Results of the present study revealed that expression levels of ZEB1, MCSF and CCL18 were markedly increased in ovarian cancer cells, compared with healthy ovarian cells. The change trend revealed that expression levels of the aforementioned proteins were closely associated with malignant phenotype of ovarian cells. Results of the present study also highlighted that ZEB1 may increase the levels of CCL18 through the promotion of MSCF expression. Collectively, these results suggested that MCSF-CCL18 was the downstream regulatory axis of ZEB1, and this regulatory pathway was closely associated with the malignant phenotype of ovarian cells.

At present, research is focused on TAMs, which act as key immunosuppressive cells that suppress immunity in the tumor microenvironment. Results of a previous study revealed that TAMs expressed PD-1 (Programmed Death-1) ligands, PD-L1 and PD-L2; thus, inhibiting T-cell function [21]. Results of a previous study also revealed that TAMs secrete immunosuppressive factors, such as IL-10 and TGF-β. In addition, TAMs inhibit CD4+/CD8 + T-cells, leading to increased expansion of regulatory T-cells. A further previous study used the peritoneal effusion of patients with ovarian cancer, and the results demonstrated that TAMs express immune-suppressive chemokines; namely, CCL18/CCL22. Collectively, these results suggested that this regulatory factor may participate in mediating the apoptosis of CD8 + T-cells [22].

Results of a previous study revealed that inhibition of MCSF expression may prevent the M2 polarization of TAMs in the tumor microenvironment of ovarian cancer cells [23]. Moreover, MCSF promoted cell differentiation and proliferation in macrophages, leading to the increased expression of the surface Siglec, which directly binds and inhibits CD8 + T-cells in cancer [24].

The ratio of M1/M2 polarization of TAMs is associated with the survival rate of patients with ovarian cancer. Notably, an increase in M2-TAMs cells may be indicative of a poor prognosis of patients with ovarian cancer [25]. The present study aimed to determine a therapeutic target for the inhibition of the M2 phenotype in macrophages. This inhibition may reduce the number of M2-TAMs in ovarian cancer, leading to the reversal of immunosuppressive effects and improvements in the efficacy of immunotherapy in patients with ovarian cancer. Collectively, results of the present study revealed that ZEB1 may regulate M2 polarization in TAMs via the MCSF axis; thus, impacting the corresponding secretion of CCL18 in ovarian cancer cells.

CD8 + T-cells exhibit anti-tumor properties, highlighting the potential of these cells in promoting an anti-tumor immune response. Notably, the cytotoxicity of T-cells may exhibit potential in immunotherapeutic regimens. Results of previous studies demonstrated the effectiveness of CAR-T treatment in numerous malignant tumors (such as leukemia, lymphoma, glioma and neuroblastoma). Results of a previous study demonstrated that the concentration of immuno-suppressive factors, such as regulatory T-cells, plasma cell-like dendritic cells and cytokines, including IL-10, IL-6, TNF-α and TGF-β, were notably increased in the tumor microenvironment [26]. Notably, malignant tumors are susceptible to targeted by T-cells, leading to immune escape. Results of a previous study highlighted the role of antigen presentation, IFN-γ signaling [27,28,29], chromatin remodeling, TNF-α and the autophagy pathway in immune escape [30].

Therefore, further research is required to clarify the genetic heterogeneity of malignant tumors and understand the molecular mechanisms underlying immune escape in the dynamic immune microenvironment. At present, research is focused on the inhibition of pathways associated with killer T-cells.

Conclusion and prospect

In our research the molecular mechanism of interactions between these key factors in this axis, and their target regulating in TAMs cell M2 polarization/CD8+-T apoptosis needs to be further studied in subsequent cellular and molecular research. Meanwhile, the effect of this regulatory axis on the malignant progression, recurrence, chemotherapy tolerance and immunotherapy resistance in ovarian cancer patients has not been elaborated. This area needs to be clarified through subsequent cellular/ molecular/animal/clinical trials.

In conclusion, results of the present study revealed that the ZEB1 axis promoted M2 polarization in ovarian cancer cells, and TAM-mediated CCL18 secretion induced the apoptosis of CD8 + T-cells, leading to immune escape. In addition, this regulatory axis may promote the proliferation, invasion and tumorigenesis of ovarian cancer cells in vivo, through CD8 + T-cell suppression-mediated immune escape. In subsequent applied research we could use small molecule compounds or monoclonal antibodies to inhibit pivotal genes and proteins in this axis (ZEB1-MCSF-CCL18). To interfere with target cell delivery mechanisms, and down-regulate the release of CCL18, which could inhibit CD8+-T cells. Based on these we could combine this strategy with CAR-T therapy in ovarian cancer, to improve the clinical effect of these patients. Collectively, results of the present study provided a novel theoretical basis for improved treatment of patients with ovarian cancer.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

My deepest gratitude goes first and foremost to Guo-Wei Li, for his constant encouragement and guidance. Without his consistent and illuminating instruction, this thesis could not have reached its present form. Second, my thanks go to my beloved family for their support and great confidence in me throughout all of these years. I also owe my sincere gratitude to my friends and my fellow workmates who gave me their help and time in listening to me and helping me to work out my problems during hard times.

Funding

The present study was supported by the Scientific Research of Jiangsu Health Vocational College (grant no. JKCY202401).

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Authors and Affiliations

  1. School of Nursing, Jiangsu Health Vocational College, Nanjing, 211899, Jiangsu, China

    Yan-Ping Jin

  2. School of Rehabilitation Science, Nanjing Normal University of Special Education, Nanjing, 210038, Jiangsu, China

    Guo-Wei Li

  3. Department of Obstetrics and Gynecology, The Affiliated Suzhou Hospital of Nanjing Medical University (Suzhou Municipal Hospital North), Suzhou, 215008, China

    Qian-Qian Xu

  4. Department of Obstetrics and Gynecology, Zhongda Hospital Jiangbei Branch, School of Medicine, Southeast University, Nanjing, 210048, Jiangsu, China

    Xiao-Lan Wang

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Contributions

Y-PJ conducted this experiment and analyzed the results. G-WL designed the experiment and reviewed the manuscript. Q-QX and X-LW analyzed the data.

Corresponding author

Correspondence to Guo-Wei Li.

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The present study was approved by the Ethics Committee of Suzhou Hospital Affiliated to Nanjing Medical University. The present study was performed in accordance with the Declaration of Helsinki.

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Jin, YP., Li, GW., Xu, QQ. et al. ZEB1 promotes the immune escape of ovarian cancer through the MCSF-CCL18 axis. Cancer Cell Int 25, 95 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12935-025-03724-y

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Cancer Cell International

ISSN: 1475-2867

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