CXCL2 promoted lymphatic metastasis in endometrial cancer by regulating STMN1 related ferroptosis

doi:10.21203/rs.3.rs-2848748/v1

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CXCL2 promoted lymphatic metastasis in endometrial cancer by regulating STMN1 related ferroptosis

https://doi.org/10.21203/rs.3.rs-2848748/v1

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CAFs infiltration increased and ferroptosis decreased in metastatic tissues and lymph nodes compared with non-metastatic endometrial cancer tissues and negative lymph nodes. The ferroptosis-related gene STMN1 was identified by bioinformatics analysis and was closely related to CAFs infiltration. Three STMN1 knockdown endometrial cancer cell lines were constructed to verify the attenuated malignant phenotype and increased ferroptosis. Supernatants of CAFs derived from non-metastatic tissues and metastatic lymphoid tissues were collected for cytokine chip detection. CXCL2 was identified to be closely related to the ferroptosis process of endometrial cancer. Detection of CXCL2 levels in clinical samples showed that CXCL2 levels were increased in tissues, serum and lymphoid tissues of patients with metastatic endometrial cancer. CXCL2 can partially rescue cancer cells from ferroptosis caused by STMN1 knockdown, restore the malignant phenotype, and enhance the tube formation ability of HLEC cells. In vivo experiments showed that CXCL2 promoted cancer cell tumorigenesis and metastasis, while knockdown of STMN1 attenuated this property. In summary, we demonstrated that CXCL2 secreted by CAFs from metastatic tissues regulated STMN1 to inhibit ferroptosis in cancer cells and promote tube formation in HLEC cells. These two synergetic effects promote lymphatic metastasis in endometrial cancer.

Endometrial cancer

Cancer associated fibroblasts

CXCL2

STMN1

Ferroptosis

Lymphatic metastasis

Endometrial cancer (EC) ranks first in the incidence of malignant tumors of the female reproductive system in developed countries. In recent years, its incidence tends to be stable or declining, but its mortality rate increases for decades[1]. In 2020, there were 417,367 new cases of uterine body cancer worldwide, and 97,370 deaths, ranking 6th in female malignant tumor incidence and 13th in mortality. In 2023, 66,200 new cases and 13,030 deaths are expected in the United States[2].The overall 5-year survival rate of patients with EC is as high as 80%, and the 5-year survival rate of low-grade and early-stage patients can even reach 95%. Once tumor recurrence occurs, the patients will rapidly progress to the end-stage[3], and the 5-year survival rate will drop to 17% or even lower[4]. Although a large number of studies have been conducted to reveal the biological characteristics and pathogenesis of EC, due to the fixed treatment mode, the prognosis of EC has remained unchanged for many years, especially for EC with metastasis or recurrence, the mortality rate is still increasing year by year, so it is of great significance to elucidate the mechanism of metastasis and recurrence of EC.

As stromal cells in the tumor microenvironment, CAFs account for the highest proportion, are widely distributed and have complex functions[5, 6]. Their precursors are mainly innate fibroblasts and mesenchymal stem cells in bone marrow and adipose tissue[7]. It can not only release a large number of cytokines to regulate the biological behavior of tumors, but also mediate the proliferation, invasion and metastasis of tumor cells by promoting tumor angiogenesis, interstitial remodeling, drug resistance and immunosuppression. It is one of the hot spots in recent tumor research[8–11].

Ferroptosis is an iron-dependent programmed cell death mode named in 2012. The ultrastructural characteristics of the cells are mainly characterized by cell membrane fragmentation and bleb, mitochondrial shrinkage, and reduction or disappearance of mitochondrial crista[12]. Biochemical levels show glutathione (GSH) depletion, decreased glutathione peroxidase 4 (GPX4) activity, lipid peroxides cannot be reduced to alcohols by GPX4 catalysis, and a large number of reactive oxygen species (ROS) are produced, leading to cell death[13, 14]. In some clinical studies, ferroptosis-induced drugs such as sorafenib and erastin have been used as potential therapeutic strategies for anti-tumor effects by promoting ferroptosis[15, 16]. At present, some drugs may achieve the purpose of adjuvant therapy through ferroptosis pathway of EC[17, 18], but there is no ferroptosis-induced drug for the clinical treatment, and the potentially important role of ferroptosis in lymphatic metastasis in EC has not been fully discussed.

In our study, we found that cancer-associated fibroblasts (CAFs) in EC promoted lymphangiogenesis by secreted CXCL2, and promote the high expression of STMN1 in EC cells to induce ferroptosis resistance, which combined to promote lymph node metastasis. These data provide a novel insight into the functional significance of CAFs in lymphatic metastasis of EC.

The infiltration of CAFs in lymph node metastasis of endometrial cancer increased and ferroptosis decreased

In order to detect the expression of CAFs in different clinical samples, 20 cases of primary tumor tissues without metastases and 5 cases of primary tumor tissues with lymph node metastases were selected, the content of CAFs in primary tumor tissues with lymph node metastases was higher than that in primary tumor tissues without metastases (Fig. 1A). In addition, 5 cases of lymph node metastasis of endometrial cancer and their negative lymph nodes of the same patient, and 5 cases of negative lymph nodes without endometrial cancer metastasis were selected for IHC. It was found that CAFs in lymph node metastasis were significantly increased and wrapped around the metastatic endometrial glands, and a small amount of CAFs were seen in the negative lymph nodes of the same patient. There was no CAFs infiltration in the negative lymph nodes without endometrial cancer metastasis (Fig. 1B). It is suggested that CAFs reach the metastatic site before tumor cells, and may form a suitable environment for metastasis, and then induce metastasis and proliferation of EC cells. This view is consistent with previous studies that CAFs regulate the biology of tumor cells via cell-cell contact, releasing a large number of regulatory factors and synthesizing and remodeling the extracellular matrix, and affect cancer initiation and development[19].

Occurrence of ferroptosis was detected by IHC and qRT-PCR in the above 20 cases of primary tumor tissues without metastases and 5 cases of lymph node metastasis. The results showed that the transcription and protein expression levels of GPX4 and SLC7A11 were increased, while ACSL4 were decreased in lymph node metastasis tissues (Fig. 1C and D).That is, the degree of ferroptosis is decreased in lymph node metastasis. Summary statistics are shown in Supplementary Table S3.

STMN1 is a ferroptosis-related core gene in endometrial carcinoma and is associated with CAFs infiltration

In order to find the core genes related to ferroptosis in EC, we downloaded the genes involved in ferroptosis from FerrDb ferroptosis database (Fig. 2A). 552 endometrial cancer samples and 23 normal endometrial samples were obtained from TCGA database for correlation analysis. The total of 259 ferroptosis-related genes were introduced, and 99 genes were found to be differentially expressed. Univariate analysis showed that 20 differentially expressed genes were related to survival time and survival outcome (Fig. 2B). Seven key genes were identified by multivariate analysis: PROM2, GPX2, TSC22D3, IL6, HIC1, STMN1 and AURKA. Analysis of the above genes through the UALCAN database revealed that STMN1 had a high transcript level and protein expression level in EC (Fig. 2C and D). Stathmin1 (STMN1), also known as Oncoprotein 18, is a microtubule-binding protein that binds to α/β-Tubulin heterodimers, leading to inhibition of microtubule assembly, or promoting microtubule dissociation[20]. Recent studies have shown that high levels of STMN1 can be detected in a variety of malignant tumor cells, and it is related to the promotion of tumor tumorigenicity and tumor metastasis[21, 22]. The transcription and expression level of STMN1 is higher in TP53 mutant EC than that in non-TP53 mutant EC (Fig. 2E and F), suggesting that STMN1 has a greater significance in TP53 mutant EC. The expression level of STMN1 is increased in pan-cancer (Fig. 2G). The expression level of STMN1 increased gradually in G1, G2 and G3 of EC, and the differences were statistically significant (Fig. 2H). The expression of STMN1 tends to be increased in advanced stage of EC (Fig. 2I), suggesting that STMN1 is more valuable in high-grade and advanced stage of EC.

TIMER2.0 database was used to analyze the correlation between STMN1 expression and CAFs infiltration in EC. EPIC, MCP-counter and TIDE all suggested that the expression of STMN1 in EC was positively correlated with CAFs infiltration (Fig. 2J). This further validates the importance of CAFs in the mechanism of ferroptosis in EC.

The 99 differentially expressed ferroptosis related genes between EC and normal endometrial tissues in TCGA database were introduced into KEGG database, and the results suggested that the mechanism of ferroptosis in EC was closely related to cell cycle, DNA replication signaling pathways (Fig. 2K). KEGG analysis showed that STMN1 was involved in the MAPK signaling pathway, which was related to cell proliferation and differentiation, suggesting that STMN1 may be regulated by the upstream MAPK signaling pathway in the mechanism of ferroptosis in EC.

Knockdown of STMN1 reduced the malignant phenotype of endometrial cancer in vitro

To verify the role of STMN1 in the progression of EC, we used three endometrial cancer cell lines: ISK, HEC-1-A and KLE, which represent the well, moderately and poorly differentiated types of EC, respectively (Fig. 3A-C).

The changes in cell proliferation, migration and invasion ability of the three cells after knockdown of STMN1 were further detected. After 72 hours of adherent culture, the proliferation ability of the three STMN1 knockdown cells was significantly decreased (Fig. 3D). The migration and invasion ability of the three cells was significantly decreased after shSTMN1 knockdown (Fig. 3E and 3F).

Knockdown of STMN1 promoted ferroptosis of endometrial cancer in vitro

KLE cell line was selected to detect the ferroptosis of EC cells after knockdown of STMN1. The mRNA and protein expression levels of GPX4 and SLC7A11 in the shSTMN1 group were significantly decreased, while ACSL4 showed an increasing trend (Fig. 4A and B). ROS accumulation and inhibition of GSH synthesis can induce ferroptosis[23, 24]. The concentration of GSH in the shSTMN1 group was decreased (Fig. 4C), and the concentrations of MDA (Fig. 4D) and ROS (Fig. 4E) were increased, suggesting that ferroptosis occurred strongly in KLE cells after knockdown of STMN1.

In order to further verify that STMN1 knockdown could induce ferroptosis in KLE cells, the KLE cells were treated with ferroptosis inducer RSL3 at 0.2 µM concentration and the KLE cells after knockdown of STMN1 were treated with ferroptosis inhibitor Ferrostatin-1 at 1 µM concentration. The levels of ferroptosis related metabolites were detected again. The results showed that ferroptosis induced by RSL3 was similar to that induced by STMN1 knockdown, and ferrostatin-1 could reverse the effect of ferroptosis induced by STMN1 knockdown (Fig. 4F-J). TEM was used to observe the morphology of cells and internal mitochondria. In KLE cells with shSTMN1 knockdown and KLE cells with RSL3 treatment, mitochondrial shrinkage, mitochondrial cristae reduction and outer membrane fragmentation were observed (Fig. 4K). These results further confirmed that knockdown of STMN1 could produce a similar ferroptosis effect as ferroptosis inducers.

CAFs in lymphatic metastasis could secrete more CXCL2 to inhibit ferroptosis

Five cases of primary EC without metastasis and five cases of lymph node metastasis were selected (Fig. 5A). Immunofluorescence staining showed that the four surface markers of Vimentin, α-SMA, FSP-1 and FAP were all positive in CAFs (Fig. 5B). The expressions of Vimentin and FSP-1 of lymphatic metastasis were significantly higher than those in primary tumor. The expressions of α-SMA and FAP were slightly increased, but there were no statistical differences (Fig. 5C). The supernatant of CAFs from 2 types of specimens were collected and mixed each for Cytokine Antibody Arrays. The five cytokines with the highest fold difference were obtained (Fig. 5D). After 24 hours of incubation with 5 exogenous cytokines, the transcription levels of GPX4, SLC7A11 and STMN1 were significantly increased in KLE cells treated with CXCL2, suggesting that CXCL2 plays a key role in the process of ferroptosis in EC (Fig. 5E). Serum and metastatic tissue samples from 2 patients with ovarian metastasis, 1 patient with vaginal metastasis, 4 patients with pelvic lymph node metastasis, and the serum, primary EC tissue samples and negative lymph node samples from 8 patients without metastasis were collected. The CXCL2 level of serum and tissues in patients with EC metastasis was significantly higher than that in patients without EC metastasis. CXCL2 level in metastatic lymph nodes was higher than that in negative lymph nodes, but the difference was not statistically significant probably due to the number of samples (Fig. 5F), suggesting that CXCL2 plays a key role in EC metastasis.

CXCL2 could rescue STMN1-downregulation-induced ferroptosis in endometrial cancer cells

Previous study showed that ferroptosis was increased in KLE cells with knockdown of STMN1, and exogenous CXCL2 could promote the transcription levels of GPX4, SLC7A11 and STMN1 in KLE cells. To further verify whether CXCL2 could rescue the increased ferroptosis caused by knockdown of STMN1. The cells were divided into three groups: CXCL2-treated KLE cells, CXCL2-treated KLE cells with knockdown of STMN1, and KLE cells with knockdown of STMN1. After cultured for 24 hours with the exogenous CXCL2 concentration of 30 ng/ml, the proliferation (Fig. 6A), invasion (Fig. 6B, 6E) and migration (Fig. 6C, 6D) abilities of KLE cells with STMN1 knockdown were significantly enhanced under the addition of CXCL2. The occurrence of ferroptosis was confirmed by qRT-PCR and WB detection of ferroptosis related gene expression, and the levels of ferroptosis related factors GSH, MDA and ROS. The results suggested that compared with the KLE cells with knockdown of STMN1, the CXCL2-treated KLE cells with knockdown of STMN1 had increased GPX4 and SLC7A11 transcription (Fig. 6G) and protein expression (Fig. 6J) levels, increased GSH level (Fig. 6H), and decreased ROS levels (Fig. 6K). All these results suggest that exogenous CXCL2 may play a role in the rescue of ferroptosis caused by knockdown of STMN1.

Bioinformatics analysis suggests that STMN1 is related to cell cycle, cell proliferation and differentiation, and may be regulated by the MAPK pathway, leading to ferroptosis resistance in EC. Exogenous addition of CXCL2 increased the expression of p-ERK protein and STMN1. Exogenous addition of MEK inhibitor PD98059 decreased the expression of p-ERK protein and STMN1. CXCL2 involved in the MAPK signaling pathway and activating p-ERK and up-regulating STMN1 in KLE cells were preliminarily identified (Fig. 6F).

CXCL2 promoted lymphangiogenesis

In the mainstream view, lymphatic vessels serve as rapid pathways for metastasis and spread. The denser the lymphatic vessels within or near the tumor, the more potential lymphatic protective niches that tumor cells can enter, thus lymphatic vessel density is significantly correlated with lymph node and organ metastasis[25, 26]. The proliferation (Fig. 7A), invasion (Fig. 7B) and migration (Fig. 7C) abilities of HLECs were significantly enhanced under the addition of CXCL2. Previous study showed that CXCL2 can up-regulate STMN1 and inhibit ferroptosis in EC by activating p-ERK, leading to cancer progression and metastasis. Therefore, to further verify whether CXCL2 is involved in the MAPK pathway to regulate the role of HLECs, HLECs were divided into the experimental group after CXCL2 treatment and the control group without intervention, and the expression of ERK protein in the MAPK pathway was detected. The results showed that exogenous CXCL2 increased the expression of p-ERK protein in HLECs. The expression of p-ERK protein in HLECs was decreased after the addition of MEK inhibitor PD98059 (Fig. 7D). Therefore, CXCL2 is involved in the MAPK signaling pathway and plays a role in the activation of p-ERK in HLEC cells.

In order to detect the effect of CXCL2 on the tube formation ability of HLEC cells, HLEC cells were divided into experimental group treated with CXCL2 and control group only cultured with Matrigel and ECM medium. The relative length of tube formation and the relative number of branch nodes of HLEC cells were measured at 2 h, 6 h and 12 h after incubation. The results showed that the relative length of tube formation and the relative number of branch nodes of HLEC cells treated with CXCL2 were significantly increased compared with the control group (Fig. 7E). It was preliminarily verified that CXCL2 may promotes the metastasis and invasion of EC cells by promoting lymphangiogenesis.

CXCL2 and STMN1 promoted endometrial cancer metastasis by suppressing ferroptosis in vivo

The subcutaneous tumor tissue was completely removed and minced, and mixed with homologous cell samples for in situ EC formation experiment in BALB/c nude mice. They were divided into three groups, CXCL2-treated KLE cells (CXCL2), CXCL2-treated KLE cells with knockdown of STMN1 (shSTMN1), and KLE cells with knockdown of STMN1 (CXCL2 + shSTMN1), with 10 mice in each group. In vivo imaging was performed every week after implantation. The results showed that the tumor load in group CXCL2 was significantly higher than that in group shSTMN1 and group CXCL2 + shSTMN1 (Fig. 8A). When the luminescent tumor was about 1 cm in diameter, the mice were sacrificed at about 3–4 weeks. The primary tumors and metastatic tumors were removed completely, and the total tumor weight of each mouse was weighed. Histogram analysis showed a significant difference in tumor weight between the CXCL2 group with the shSTMN1 group and CXCL2 + shSTMN1 group (Fig. 8B). Dissection of the tumor-bearing mice revealed in situ uterine tumors (Fig. 8C) and enlargement of the iliac lymph nodes suspected of metastasis (Fig. 8D).

Paraffin-embedded sections of in situ tumors and lymph nodes were subjected to HE staining and ferroptosis-related gene IHC. The results showed that GPX4, SLC7A11, and STMN1 were up-regulated and ACSL4 was down-regulated in lymph node metastases (Fig. 8E). The mRNA levels of above genes were basically consistent with the IHC results (Fig. 8I). These results suggest that ferroptosis is decreased in metastatic lymph nodes. The above two tissues were also used for IHC of CAFs related markers, and it was found that lymph node metastases had more CAFs content, further verifying that CAFs played a role in promoting metastasis in EC (Fig. 8F). Summary statistics are shown in Supplementary Table S4. The level of GSH (Fig. 8G) was higher and the level of MDA (Fig. 8H) was lower in lymphatic metastasis, indicating that ferroptosis was decreased in lymphatic metastasis.

In this study, we demonstrated that CXCL2 could be secreted by CAFs from metastatic tissues to regulate the core genes of ferroptosis in endometrial cancer cells, inhibit the ferroptosis, and also promote the tube formation of HLEC cells. The two synergies promote lymphatic metastasis of endometrial cancer (Fig. 7F).

A large number of previous studies have shown that there are different secretory functions between normal endometrium and EC derived fibroblasts, and all suggest that the medium supernatant of EC derived CAFs can increase the proliferation, invasion and metastasis of primary EC cells and EC cell lines in a dose-dependent manner[27–29]. A study had shown that CAFs promote the proliferation of EC cells in an autocrine manner through SDF-α/CXCR4 axis, involving PI3K/AKT and MAPK/ERK pathways[30]. However, for cytokine screening, only five candidate cytokines, SDF-1α, MCP-1, MIF, CSF-1 and IL-1, were examined in concentrations in both samples, resulting in the neglect of other potential factors. Another study, which selected fibroblasts supernatants from normal endometrium and endometrial cancer for the screening of 10 cytokines, also had limitations[31]. In this study, cytokine microarray was used to screen the CAFs supernatant from non-metastatic endometrial cancer tissues and lymph node metastasis tissues, and 80 cytokines were selected to identify the key cytokines involved in the ferroptosis process of endometrial cancer more widely and accurately. The PI3K/Akt and MAPK/ERK signaling pathways may be the key regulators of the response of endometrial cancer cells to their microenvironment. Our findings are consistent with those of these previous studies.

CXCL2, also known as GRO-β and macrophage inflammatory protein (MIP)-2α, is a member of the CXC chemokine family. CXCL2 is a chemotactic factor for neutrophils and is also produced by immune cells such as macrophages in response to infection or injury[32, 33]. A previous in vivo study showed that CXCL2 induced neutrophil-dependent angiogenesis and promoted liver metastasis of colon cancer through the release of VEGFA from neutrophils[34]. Another study demonstrated that CXCL2 secretion by omental adipocytes has also been shown to promote gastric cancer progression, angiogenesis and peritoneal dissemination of gastric cancer cells[35]. The results of our study of lymph node metastasis in EC were also consistent with these previous studies. In gynecologic tumors, it has been reported that CXCL2 expression is increased in endometrial cancer, ovarian cancer, and breast cancer, and silencing CXCL2 can inhibit the invasion ability of endometrial cancer cells and reduce the expression of metastasis-related proteins[36]. However, the potential role of CXCL2 in EC remains unclear. Therefore, our study first demonstrated that CXCL2 secreted by CAFs can promote the malignant phenotype of EC cells and inhibit ferroptosis, leading to the occurrence of metastasis. The serum level of CXCL2 in patients with metastasis was significantly higher than that in patients without metastasis. Serum level of CXCL2 may be a potential therapeutic target and predictive biomarker for lymph node metastasis of EC.

The clinical attitude towards suspicious lymph node metastasis of EC is inclined to complete dissection in the initial operation, but some people believe that lymph node resection may overtreat and no significant improvement in the prognosis[37]. According to a large number of convincing clinical studies[38–40], lymph node removal has a diagnostic and therapeutic role in patients at risk for metastasis. For intermediate or high-risk patients, the number and extent of resected lymph nodes are positively correlated with good survival prognosis[41, 42]. These are consistent with the view of this study. Mild CAFs infiltration was also detected in negative lymph nodes of EC patients who developed metastasis, suggesting that CAFs may reach distant sites before EC cells and provide a suitable protective niche and nourish soil for EC cells to metastasize[43]. We also found that CXCL2 level was increased in metastatic lymph nodes tissues compared with negative lymph nodes tissues, and verified that CXCL2 promoted the malignant phenotype progression and tube formation ability of HLEC by activating p-ERK, which provided a rapid pathway for lymphatic metastasis of EC cells. Therefore, in the case of suspected lymphatic metastasis, we believe that complete resection is beneficial.

We also have identified STMN1 as a core gene in EC ferroptosis. STMN1 is first characterized as a microtubule-destabilizing phosphoprotein[44]. STMN1 is highly expressed in a variety of tumors and is associated with poor prognosis[45, 46]. Although the link between STMN1 and ferroptosis in endometrial cancer has not yet been determined, previous studies have reported that STMN1 is involved in the regulation of GSH levels in liver cancer cells, thereby interfering with the ferroptosis process. However, in the present study, we found that STMN1 regulated the expression of ferroptosis-related genes and GSH level. After STMN1 knockdown, GSH level decreased, MDA and ROS levels increased, and ferroptosis occurred (Fig. 7G).

The cellular components of metastases in vivo are complex, involving many different cell types in the tumor microenvironment[47, 48], and we also selected BALB/c mice for in vivo experiments. Knockdown of STMN1 can attenuate the tumor-promoting properties of CXCL2, which supports the role of CXCL2 in metastasis of endometrial cancer. Targeting STMN1 may be a promising approach to reduce metastatic dissemination of endometrial cancer.

In conclusion, CAFs promote metastasis of EC by secreting CXCL2 to form a microenvironment suitable for metastasis, promote lymphangiogenesis and ferroptosis resistance of cancer cells. CXCL2 may be a potential therapeutic target to regulate or prevent metastasis of EC and an indicator to monitor cancer progression.

Patient information and tissue specimens

The clinical samples used in this study were from the Department of Gynecology, the First Affiliated Hospital of Jinan University. From September 2019 to March 2023, 80 cases of EC tissues and 28 cases of lymphatic tissues were collected, including 7 cases of metastatic lymph nodes and 21 cases of negative lymph nodes.

Written informed consent was obtained from each patient before collection of clinical data and specimens. This study was performed in accordance with the principles of the Declaration of Helsinki. The Ethics Committee of the First Affiliated Hospital of Jinan University approved this study (approval number: 20210303).

Bioinformatic analysis

TCGA clinical data was downloaded from The Cancer Genome Atlas official website (as of September 2021), including 552 endometrial carcinoma samples and 23 normal endometrial samples. Ferroptosis related genes were obtained from FerrDb database. The online database UALCAN was used to analyze the transcription and expression levels of target genes, the TIMER2.0 database was used to analyze the infiltration of tumor immune cells, and the KEGG and GO databases were used to locate the signal pathways involved in core genes.

Cell lines and cell culture

Cell line sources and culture conditions are described in the supplementary material. Primary human endometrial CAFs isolated from fresh EC tissue or lymphoid metastatic tissue using 0.2% collagenase I and collagenase II for digestion, filtered with 70 µm cell strainer, and identified by means of morphology and immunofluorescence staining for Vimentin、α-SMA、FSP-1、FAP, were cultured in DMEM medium with 10% FBS.

Detection of cytokine microarray

Semi-quantitative detection of 80 human proteins with RayBio ® C-Series Human Cytokine Antibody Array C5 (AAH-CYT-5-2, RayBiotech, USA) according to the manufacturer’s instructions. The specific steps are described in the supplementary material.

ELISA of CXCL2

Quantitative determination of Human GROβ/CXCL2 concentrations in serum and other biological fluids with an ELISA kit (E-EL-H1904c, Elabscience, China). The specific steps are described in the supplementary material.

Transmission electron microscopy (TEM)

The collected cells were immersed in 2.5% neutral glutaraldehyde and fixed at room temperature for 30 min. The images were collected and analyzed under a transmission electron microscope (HT7800, Hitachi, Japan), focusing on the mitochondrial morphology of tumor cells.

ROS, MDA and GSH assay

ROS levels were measured with a Reactive oxygen species assay kit (E004-1-1, Nanjing Jiancheng, China) according to the manufacturer’s instructions. Cells were seeded in six-well plates and treated with RSL3 (0.2 µM) or Ferrostatin-1 (1 µM). Then, cells were incubated with DCFH-DA at a final concentration of 10 µM in serum-free medium at 37°C for 30 min and washed three times with PBS. The level of ROS was determined by flow cytometer. The results were analyzed with FlowJo V10 software.

MDA levels were measured with a Lipid Peroxidation MDA Assay Kit (BC0025, Solarbio, China) according to the manufacturer’s instructions.

GSH levels were measured with a Micro-reduced glutathione assay kit (A006-2-1, Nanjing Jiancheng, China) according to the manufacturer’s instructions.

Tube-formation assay

HLEC less than 10 generations were used for tube-formation assay. Incubated HLECs were washed with PBS and added to serum-free medium. After 2h of serum starvation, HLEC cells were suspended in ECM complete medium and the cell concentration was adjusted to 3*10⁵ cells/ml. CXCL2 was added to the medium of the experimental group with an additional 30 ng/ml. 100 µl cell suspension was added to each well which pre-coated with Matrigel. The chambers were incubated for 6h, photographed under a microscope and branching numbers were counted. The length of pipe and the number of branch nodes were measured by Image J software.

Animal experiments

BALB/c mice were purchased from Guangdong Vital River Laboratory Animal Technology Company Limited. Treated luciferase tumor cells were injected subcutaneously into the right shoulder of female mice aged 4 to 5 weeks. When the tumor volume was about 1 cm³, the tumor was removed and cut up, and the tissue homogenates were mixed with the cells of the same treatment group, each of which contained about 50 mg tissue homogenates and 10⁷ cells. After abdominal exposure to the uterus, the tumor samples with Matrigel were injected into both sides of the uterus. The tumor growth was observed by IVIS® Lumina III. All animal experiments were performed in accordance with the guidelines of the Experimental Animal Ethics Committee of the First Affiliated Hospital of Jinan University (approval number: 20220905-09).

Other methods are described in the Supplementary Information.

Data availability

The raw data is available without undue reservation.

Acknowledgments

The work was partly supported by Guangdong Basic and Applied Basic Research Foundation (2019A1515012091).

Author statement

YM and GL performed the experiments and wrote the manuscript. HF contributed to the data analysis. JL prepared the figures and designed the layout. XJ and QT conceived the experiments and participated in the revision of the manuscript. All authors read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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There is NO conflict of interest to disclose.

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CXCL2 promoted lymphatic metastasis in endometrial cancer by regulating STMN1 related ferroptosis

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Abstract

Figures

Introduction

Results

The infiltration of CAFs in lymph node metastasis of endometrial cancer increased and ferroptosis decreased

STMN1 is a ferroptosis-related core gene in endometrial carcinoma and is associated with CAFs infiltration

Knockdown of STMN1 reduced the malignant phenotype of endometrial cancer in vitro

Knockdown of STMN1 promoted ferroptosis of endometrial cancer in vitro

CAFs in lymphatic metastasis could secrete more CXCL2 to inhibit ferroptosis

CXCL2 could rescue STMN1-downregulation-induced ferroptosis in endometrial cancer cells

CXCL2 promoted lymphangiogenesis

CXCL2 and STMN1 promoted endometrial cancer metastasis by suppressing ferroptosis in vivo

Discussion

Materials and methods

Patient information and tissue specimens

Bioinformatic analysis

Cell lines and cell culture

Detection of cytokine microarray

ELISA of CXCL2

Transmission electron microscopy (TEM)

ROS, MDA and GSH assay

Tube-formation assay

Animal experiments

Declarations

References

Additional Declarations

Supplementary Files

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