Sulfatase2 affects the polarization of M2 macrophages through the IL-8/JAK2/STAT3 pathway in bladder cancer

doi:10.21203/rs.3.rs-1753488/v1

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Sulfatase2 affects the polarization of M2 macrophages through the IL-8/JAK2/STAT3 pathway in bladder cancer

https://doi.org/10.21203/rs.3.rs-1753488/v1

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Background: Sulfatase 2 (SULF2) can affect the occurrence and development of cancer by regulating HSPG-binding factors. In bladder cancer(BCa), the mechanism of SULF2 is unknown. Here, we clarify the mechanism that SULF2 affects the secretion of HSPG-binding factors (IL-8) and regulates the polarization of M2 macrophages in the BCa microenvironment.

Methods: The RNA sequencing of 90 patients with BCa was analyzed. GSEA analyze the relationship between SULF2 and clinicopathological characteristics and prognosis. SULF2 knockdown or overexpression cell lines, flow cytometry, CCK8, Transwell, Edu assay, Western Blot, qPCR, co-culture systems were used to analyze the effects of SULF2 on the biological functions of BCa in vitro. The mouse orthotopic BCa model, flow cytometry and IHC were used to analyze the changes of SULF2 on the tumor microenvironment of BCa in vivo.

Results: RNA sequencing data analysis showed that the expression of SULF2 is closely related to the prognosis. Knockdown or overexpression of SULF2 revealed that it promotes tumor proliferation and invasion through in vivo and in vitro experiments. Furthermore, SULF2 affected the polarization of macrophages by using the mouse orthotopic BCa and flow cytometric analysis,. Mechanism studies clarify that SULF2 promotes the release of HSPG-binding factors--IL-8 to the microenvironment through β-catenin. Meanwhile, IL-8 activated the JAK2/STAT3 pathway of macrophages to promote the expression of CD163 and CD206, thereby regulating the polarization of macrophages to M2 macrophages.

Conclusions: These data indicate that SULF2 plays an important role in regulating the microenvironment of BCa. SULF2 promotes the polarization of macrophages to M2 by secreting IL-8, which further deepens the malignant progression of BCa.

Bladder cancer

SULF2

Macrophages

Bladder cancer (BCa) is the most common malignant tumor of the urinary system and the fourth most common malignant tumor among men in the world. About 40,000 patients die from BCa every year. 549,393 new cases were reported in 2018[1]. BCa can be divided into non-muscular invasive bladder cancer (NMIBC) and muscular invasive bladder cancer (MIBC). When the patient was first diagnosed, approximately 75% of patients with BCa had NMIBC, and 25% of patients were diagnosed with MIBC. MIBC is characterized by high malignancy, easier metastasis, and poor prognosis. There have been few advances in the research and treatment of BCa in the past thirty years, but with our understanding of the biological functions and immune microenvironment of BCa coupled with large-scale gene sequencing, there have been effective targeted therapy and immunotherapy for BCa[2].

At present, the role of tumor microenvironment (TME) in the progression and metastasis of BCa has attracted great attention. TME includes tumor cells, macrophages, fibroblasts, endothelial cells and non-cellular substances (such as extracellular matrix and soluble signal factors, etc.) [3]. The response of macrophages to microenvironmental signals is usually polarized into two phenotypes-M1 and M2 macrophages. M1 type macrophages are considered to have anti-tumor and immune promotion effects, while M2 type macrophages are mainly used to educate tumor cells to promote tumor immune escape, angiogenesis, tumor growth and metastasis [4, 5]. A variety of factors can affect the polarization of macrophages, such as IL-4, IL-10, and IL-6. So, it is very important to explore the molecular mechanism of the communication between tumor cells and macrophages in the microenvironment of BCa.

Heparin sulfate proteoglycan can bind to a variety of ligands (such as heparin-binding growth factor, fibroblast growth factor, Hepatocyte growth factor and vascular endothelial growth factor) to perform biological functions [6, 7]. In recent years, studies have found that the structure of proteoglycan undergoes profound aberrations, leading to uncontrolled tumor proliferation, immune escape, metastasis and differentiation in different tumors such as rectal cancer [8]. These structural changes are mainly due to abnormal changes in the expression of HS biosynthetic enzymes or catabolic enzymes (such as sulfatase). Sulfatase 2 (SULF2), which belongs to the SULF family, encodes a protein with heparin degrading endosulfatase activity [9]. SULF2 regulates the expression of cell surface signal molecules on tumor cells by regulating the sulfation of the glycosyl part of HSPGs. At present, SULF2 is clearly a potential tumor driver gene for cholangiocarcinoma, glioblastoma [10, 11], but its role in BCa is still unclear.

In this study, we investigated how the overexpression of SULF2 in BCa cells promotes the tumor microenvironmental BCa progression. The results show that SULF2 expression was increased in MIBC and was an adverse prognostic factor in BCa. SULF2 can affect the biological function of BCa, and high expression of SULF2 can promote the polarization of M2 macrophages in vivo and in vitro. SULF2 promoted the secretion of IL-8 through β-catenin, which can secrete IL-8 into TME and promote the polarization of macrophages through JAK2/STAT3, thereby promoting the malignant progression of tumors. Overall, these findings supported the role of SULF2 and macrophages in the malignant progression of BCa, revealing that SULF2 may be a potential therapeutic target for BCa.

Data processing

Ninety BCa tissues were collected from patients that underwent either TURBT or radical cystectomy in Shanghai Tenth People’s Hospital (STPH) between November 2019 and September 2020. Informed consent was prior obtained from patients. The protocol of total RNA extraction, paired-end libraries synthetization and RNA-sequence were available in our previous methods. The mRNA (RNA-sequencing) Fragments Per Kilobase of transcripts per Million mapped reads (FPKM) standardized values of genes were calculated and selected for further analyses. Monthly telephone follow-up was done to obtain the disease-free survival (DFS) of these patients. This study was reviewed and approved by the Ethics Committee of Shanghai Tenth People’s Hospital.

The FPKM standardized mRNA expression data and clinical information of BLCA patients were downloaded from TCGA (https://gdc-portal.nci.nih. gov/). The processed gene expression data of GSE13507, GSE48277, GSE32894 and GSE31684 with the prognostic information of BLCA patients were downloaded from the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/). The processed gene expression data and clinical information of IMvigor210 trial that included patients with metastatic urothelial cancer treated with atezolizumab (PD-L1 inhibitor) were obtained from the website http://research‐pub.gene.com/IMvigor210CoreBiologies.

Meta-analysis

Meta-analysis was performed to investigate the prognostic value of in SULF2 using ‘meta’ R package v4.16 and ‘metafor’ R package v2.4. Univariate Cox analysis was used to calculate the hazard ratio (HR) and 95% confidence interval (CI) of SULF2 in each data set. Then, the HR and 95% CI from each data set were selected to calculate the combined value. The χ² -based Q test and I² statistical test were used to investigate the heterogeneity. When P<0.05 or I²>50%, the random effects model was used to calculate the combined value; otherwise, the fixed effects model (P>0.05 or I²<50%) was used. Egger’s funnel plot was used to evaluate the publication bias. P>0.05 was considered as no publication bias.

Gene Set Enrichment Analysis (GSEA)

In STPH, BCa patients were divided into high and low expression groups based on the median expression of SULF2. GSEA (http://www.broadinstitute.org/gsea/index.jsp) was performed to investigate the BLCA-related signaling pathways affected by high expression of SULF2. A nominal P<0.05 and a false discovery rate (FDR)<0.25 were considered significant.

Cell lines and Reagents

Human BCa cell lines T24(Cat.SCSP-536), UMUC3(Cat.CRL-1749), 5637(Cat.TCHu 1), RT4(Cat.TCHu226) and J82(Cat.TCHu218), normal urothelial cell line SV-HUC-1(Cat.TCHu169), mouse BCa cell MB49(Cat.AC-340699), and human acute monocyte THP-1 leukemia cells(Cat.TCHu 57) were purchased from the Chinese Academy of Sciences, Institute of Chemistry and Cell Biology (Shanghai, China). The above cells are all in DMEM or RPMI-1640 medium with 10% fetal bovine serum (Gibco)and 1% penicillin/streptomycin (Hyclone; GE Healthcare Life Sciences, Logan, UT). McCoy's 5A medium containing 10% FBS and 1% penicillin/streptomycin used for RT4. All cells were cultured in a 37°C incubator with 5% CO₂.

In order to obtain macrophage-like differentiated THP-1 cells, THP-1 cells were seeded at a density of 3×10⁶ cells/flask and exposed to 100 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) 48 hours. After that, replace the medium containing PMA with fresh medium to obtain the resting state of macrophages (M0), and then add 20 ng/ml IFNγ and 1 mg/ml LPS (Sigma-Aldrich) to differentiate into M1 Phenotype, or adding 20 ng/ml IL-4 to differentiate into M2 phenotype.

Cell proliferation assay

The effect of SULF2 on cell proliferation was tested by CCK-8 assay and 5-Ethynyl-20-Deoxyuridine (EDU) incorporation experiment. For the EDU assay, the experiment was carried out according to the manufacturer’s instructions of the kit (Beyotime Biotechnology, Shanghai, China). For CCK 8 determination, Cells were seeded into 96-well plates at a density of 2×10 3 cells/well, each well added 10μl CCK-8 reagent (Yeasen Biotechnology, Shanghai, China) and the cells were Maintained at 37°C for 1.5 hours. The absorbance at 450 nm was measured using a microplate spectrophotometer (BioTek, Winooski, VT). The experiment was repeated three times.

RNA extraction and real-time polymerase chain reaction (qPCR)

According to the manufacturer's instructions, Trizol reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA from human tissues or cells, and then a reverse transcription system kit (Vazyme Biotech Co., Ltd, China) was used to generate the first strand cDNA. Use ChamQ universal SYBR qPCR master mix (Vazyme Biotechnology Co., Ltd, China) and ABI Prism 7500 sequence detection system (Applied Biosystems, CA) for qRT-PCR. The qPCR parameters are as follows: 40 cycles of 30 seconds at 95°C, then 10 seconds at 95°C and 30 seconds at 60°C. GAPDH was used as an endogenous control(Supplementary Table3). The relative fold change is analyzed by the 2-ΔΔC(t) method. Repeat the measurement 3 times.

Western Blot

Use RIPA buffer to extract total protein from cells or tissues. After quantifying the lysate by the bicinchoninic acid protein assay (Beyotime, China), 30μg protein sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membrane (Sigma-Aldrich; Merck KGaA) The blot was blocked with 5% skim milk at room temperature for 1 hour, and immunoblotting was performed with primary antibody at 4°C overnight. Subsequently, the blot was incubated with the appropriate HRP-labeled secondary antibody. Use Super ECL Detection (Yeasen Biotech Co. Ltd, China) to visualized and quantified the immunoreactive bands. GAPDH was used as an internal reference. Supplementary Table 1 lists the antibody companies and product number used

The mouse orthotopic bladder cancer model and in vivo growth assays

C57 female mice were used for in situ injection of BCa cells --MB49, and 1×10⁶treated cells were injected into the bladder through the urethra, 5 in each group. Four weeks later, the mice were sacrificed and the tumors in situ were taken out and immunized by flow cytometry Cell infiltration situation. For subcutaneous transplanted tumors, 1×10⁶treated T24 cells were injected subcutaneously into the right armpit of 4-week-old female BALB/c nude mice, 5 in each group. The mice were sacrificed four weeks later, and the subcutaneous tumors were removed. Monitor the mouse body weight, tumor diameter, and tumor volume every three days according to the formula (tumor volume = π/6 × length × width ²). In order to establish a metastasis model, 1×10⁶ overexpressing or knocking down SULF2 cell lines were injected into the tail vein of 4-week-old nude mice, with 3 mice in each group. The in vivo imaging system (IVIS) was used every week to observe the tumor metastasis after 3 weeks.

Flow cytometry

In order to analyze the immune cells infiltrating the mouse orthotopic bladder cancer model, the mice were sacrificed at the 4th week after MB49 was injected into the C57 mice bladder, and the tumor tissue was cut and combined with collagenase (100 μg/ml) Incubate at 37°C for 30 minutes. The separated tumor cells were filtered with a 70um mesh, red blood cells were removed using red blood cell lysis buffer, the cells were resuspended in PBS, and then incubate with flow cytometry antibodies. After 30 minutes, the cells were washed 3 times with PBS and resuspended in 500ul of PBS buffer for flow cytometry analysis. For the cell cycle, cells were collected, trypsinized to form a cell suspension, and then fixed with 75% ethanol at 4°C overnight. On the second day, the cells were washed three times with PBS, and then RNase containing iodide (PI, 40%, Sigma- Aldrich) incubated for 30 minutes, washed the cells with PBS three times, and then used flow cytometry (BD Biosciences, USA). The experiment was repeated three times. The results are analyzed using Flowjo.

Co-cultivation system

In order to co-culture BCa cells and macrophages, we used a 0.4um Transwell chamber (Corning, Lowell, MA), the lower chamber is a THP1 cell line, and the upper chamber is a BCa cell lines that overexpresses or knocks down SULF2, as shown in Fig. 6A. The co-cultivation time is 48h. Collect the required cells for the next experiment.

Colony formation

For colony formation analysis, the SULF2 overexpression or knockdown cell line was seeded into a six-well plate at 1 × 10 3 /well, and cultured in an incubator for 14 days. After that, it was washed 3 times with cold PBS, fixed with 75% ethanol, and stained with 0.1% crystal violet. The image of the stained tumor cell colony was recorded with a digital camera and statistically analyzed.

Cell migration and invasion

Use Transwell chamber (Corning, Lowell, MA) to detect cell migration ability. Mix 5×10 4 cells in 200ul of FBS-free RPMI-1640 medium and add them to the upper chamber of the transwell chamber, and add 600 μl of cell culture medium containing 10% FBS to the lower chamber. Incubate in a 37°C incubator for 16 hours. Wash 3 times with cold PBS after 16 hours. Carefully wipe the cells that have not passed through the upper chamber with a wet cotton swab, fix with 75% ethanol for 30 minutes, and then stain with 0.1% crystal violet for 10 minutes. Observe cell migration under a microscope (Olympus Corporation). For tumor cell invasion experiments, Transwell filters are pre-coated with Matrigel (BD Biosciences, Franklin Lakes, NJ), and cells are allowed to invade for 24 hours. The rest of the experiment process is the same as the tumor cell migration experiment. Each experiment was performed in triplicate.

Immunohistochemistry (IHC)

Fix the tissue (human BCa tissue or mouse bladder tissue) in cold 4% paraformaldehyde. The fixed tissue samples were embedded in paraffin and sectioned. After hydration, antigen retrieval, and blocking procedures, incubate with the desired antibody overnight at 4°C. Next, the sections were incubated with biotinylated goat anti-rabbit antibody IgG for 20 minutes at room temperature, and then incubated with streptavidin-horseradish peroxidase for 30 minutes. Subsequently, diaminobenzidine-H2O2 was used as a substrate for peroxidase. When the cytoplasm of the cancer cells was stained yellow, two pathologists read the pathological sections and confirmed a positive result.

Statistical analysis

SPSS (version 22; SPSS, Inc., Chicago, IL) and R statistical software (version 3.6.1 R, https://www.r-project.org/) were used to analyze the data. The Kaplan-Meier (log-rank tests) was used to compare the OS (Overall survival) and DFS of the BCa patients between low and high expression groups. The differences between two groups were evaluated using Student's t-test, the chi-square test, or the Mann–Whitney U test, as appropriate. One-way ANOVA followed by Bonferroni’ test was used to compare multiple group comparisons. P-value <0.05 were considered statistically significant difference

SULF2 was highly expressed in BCa and associated with poor prognosis of patients

We extracted RNA from the tumors and adjacent tissues of 20 patients with BCa.And we found that the expression of SULF2 in cancer tissue was significantly increased by using qPCR (Fig. 1A). Then,RNA sequencing results of 90 patients with cancer tissues showed that MIBC patients had higher SULF2 expression than NMIBC patients (P=0.003) (Fig. 1B), and BCa patients in high SULF2 expression group had poorer DFS than those in low SULF2 expression group (P=0.038) (Fig. 1C and Supplementary Table2). In addition, Kaplan-Meier analysis and univariate Cox analyses showed that SULF2 expression was an adverse prognostic factor in TCGA, IMvigor210 trial and GSE32894 (Fig. 1D, E and F).

Meta-analysis further revealed that SULF2 expression was significantly associated with poor OS among 6 studies with 1308 BCa patients (random effects model, HR=1.64, 95% CI=1.14-2.36). No publication bias was measured by Egger’s funnel plot (P>0.05)(Fig. 1G and H).

In order to further explore the expression of SULF2 in BCa, we verified the expression of SULF2 in patient tissues and adjacent tissues through IHC and Western Blot. Consistently, we found that SULF2 is highly expressed in BCa tissues (Fig. 2A and B). Furthermore, we verified the expression of SULF2 in BCa cell lines by qPCR and Western Blot, and confirmed that the expression of SULF2 in a variety of cell lines is higher than that of immortalized normal urothelial cells (SV-HUC-1). So, we selected T24 and UMUC3 for the next experiment (Fig. 2C and D).

SULF affects the proliferation, migration and invasion of BCa

Next, we evaluated the effect of SULF2 expression on the proliferation of BCa cells. We knocked down the expression of SULF2 in the T24 cell line, and selected shRNA-#2 with the highest knockdown efficiency among the three shRNAs to construct stable transgenic strains through lentivirus.Meanwhile, we constructed a stable transgenic strain overexpressing SULF2 in the UMUC3 cell line through lentivirus (Fig. 2E,F and G).

In vivo experiments, we used T24 or UMUC3 stable transgenic cells to subcutaneously inject into nude mice to establish a xenograft mouse model. The results showed that the tumors in the T24 group of sh-SULF2 were significantly smaller, while the tumors in the UMUC3 group of oe-SULF2 were significantly larger (Fig. 3A,B and C). Immunohistochemistry of subcutaneous tumor tissue showed that the expression of SULF2 was significantly reduced in sh-SULF2, as was the expression of the proliferation marker Ki67. However, the results were reversed in the oe-SULF2 group (Fig. 3E-F and Fig S1A) CCK8, EDU and colone formation assays were found that knocking down SULF2 significantly inhibited cell proliferation, while overexpression of SULF2 significantly promoted cell proliferation(Fig 3D,G and H).

The effect of SULF2 on bladder cancer migration and invasion was assessed by transwell assay. The results showed that the migration and invasion of the sh-SULF2 were significantly reduced compared with the negative control (Fig. 4 A), and the migration and invasion of the oe-SULF2 group were significant increase compared with the negative control (Fig. 4 B and C). At the same time, EMT-related markers were detected by western blot, and the results show that the expression of E-cadherin (epithelial cell marker) in the sh-SULF2 was highly expressed, while the expression of N-cadherin and vimentin (mesenchymal cell marker) were decreased. (Fig. 4 D). Flow cytometry analysis showed that knockdown of SULF2 caused S phase arrest (Fig. 4 E and F) . In addition, we constructed a tail vein metastasis model. In vivo imaging system(IVIS) showed that the expression of SULF2 affects tumor metastasis (Fig. 4 G and H).

SULF2 promotes the polarization of macrophages to M2 in the microenvironment of BCa

For further explore the role of SULF2 in BCa, studies have found that SULF2 has been proven to affect changes in the microenvironment [12, 13]. Therefore, we further investigated whether SULF2 affects the microenvironment of BCa. Bioinformatics analysis shows that macrophage expression in bladder cancer microenvironment is significantly abnormal, and M2 macrophages are significantly higher in bladder cancer tumors than in normal tissues based on TCGA database(Fig. 5A and B).we analyzed the relationship between the expression of SULF2 in BCa and immune cells in the immune microenvironment of BCa. We found that the level of SULF2 expression is closely related to macrophages (Fig. 5 C). In addition, GSEA analysis on 90 RNA-sequence samples showed that several macrophage-related signaling pathways were significantly enriched in SULF2 high expression group(Fig. 5 D). As show in Fig. 5 E,We constructed a knockdown SULF2 stable transgenic cell line of BCa mouse MB49, and constructed a bladder orthotopic cancer model in C57 mice., Flow cytometry demonstrated that the proportion of M2 macrophages in macrophages was significantly down-regulated after knockdown SULF2 (Fig. 5F). Meanwhile, we took the orthotopic cancer tissue for immunohistochemistry, which also indicated the decrease of M2 macrophage markers (CD163 and CD206) (Fig. 5F). Therefore, we constructed a co-culture system (Fig. 6A),We co-cultured knockdown or overexpression cell lines with THP1, and found changes in CD163 and CD206(Fig. 6B). All these suggest that SULF2 may regulate the changes in the microenvironment of BCa

SULF2 affects the secretion of IL-8 through β-catenin

Next, in order to find the mechanism of SUL2 affecting the polarization of macrophages, we analyzed the the expression of IL-6, IL-8, IL10, CXCL1 and other cytokines after knockdown or overexpression of SULF2 by qPCR. The results found that IL-8 was significantly down-regulated in the sh-SULF2 cells. Similarly, compared to the oe-SULF2 cells, IL-8 was significantly increased (Fig. 6C-D). The TCGA database also confirmed that SULF2 and IL-8 were significantly correlated (Fig. 6G and Fig. S1B). We added IL-8 to the sh-SULF2 and oe-SULF2 and co-cultured with THP1, and found that no matter in the knockdown group or overexpression group, the exogenous addition of IL-8 promoted the expression of CD163 and CD206(Fig6 E and F)

Through the GSEA analysis of RNA-sequence, we found that SULF2 and wnt/β-catenin pathway is closely related (Fig. 6 G and Fig. S1C). According to previous studies, SULF2 can bind to wnt3a to prevent β-catenin from being degraded and recruit other transcriptio n co-activators to promote the transcription and translation of downstream genes [14]. Therefore, we hypothesized that β-catenin regulated by SULF2 could promote the secretion of IL-8. It was found that β-catenin decreased significantly after knocking down SULF2. Similarly, β-catenin expression was up-regulated in UMUC3 overexpressing SULF2 cell lines (Fig. S2A and B). The combination of SULF2 and wnt3a was verified by CO-IP in BCa (Fig. S2C). Then we knocked down the expression of β-catenin by siRNA in T24 cells, after co-cultivation with M0 cells induced by THP1 cells, we found that the β-catenin knockdown group was down-regulated in the expression of CD163 and CD206 (Fig. 6H).

SULF2 promotes macrophage polarization through the IL-8/JAK2/STAT3 pathway

Previous studies reported that the STAT family is critical to the polarization of macrophages. GSEA also suggests that SULF2 may affect the JAK-STAT pathway (Fig. 7A). Therefore, in order to study the mechanism of SULF2 affecting the polarization of macrophages, we detected the knockdown or overexpression of SULF2 after co-culture with THP1, and the phosphorylation levels of STAT1, STAT5, STAT3, STAT6 are evaluated (Fig. S2 D). We found that after knockdown of SULF2, the phosphorylation level of STAT3 decreased significantly. Therefore, we suggested that SULF2 affects the expression of STAT3 in macrophages by regulating the secretion of IL-8. At the same time, we verified that the phosphorylation level of JAK2 was significantly reduced in the co-culture system that knocked down SULF2(Fig. 7B).

For further verification, we added IL-8 exogenously to THP1 cells. The results showed that the phosphorylation of JAK2 and STAT3 changed significantly (Fig. 7C and D). When STAT3 inhibitors (AG490) were used, we found that C163 and CD206 were significantly inhibited (Fig. 7 E and F). These data indicate that SULF2 reveals a new way for tumor immune escape during the progression of BCa (Fig. 7G).

Macrophages can be a main component of the tumor microenvironment. Due to the different microenvironments, such as increase or decrease of local cytokines, hypoxia, necrosis, or changes in metabolites, macrophages will transform into different states to further promote or inhibit tumor progression [15]. At present, the polarization of macrophages can be divided into M1 type (classical activated macrophages), M2 type (alternatively activated macrophages) and so on. M2 type macrophages are closely related to the malignant progression of tumors, and are associated with reduced survival rates in pancreatic cancer, kidney cancer, and breast cancer [16–18].

In this study, we got some interesting results. Through the analysis of TCGA and GEO, we found that in the immune cell infiltration of the BCa microenvironment, macrophages increased, and there were significant differences in M2 macrophages. Secondly, tumor formation in situ in mice also confirmed this point. Xu et al.'s research on the analysis of immune cells in BCa also corroborated the phenomenon of increased macrophages[19]. At the same time, we also found that MB49 knocking down SULF 2 in the mouse bladder planted changed the proportion of M2 macrophages in the microenvironment.

SULF2 not only exerts its own biological functions, such as degrading endosulfatase activity proteins, but also plays an important role in the occurrence and development of tumors, such as participating in tumor cell cycle, proliferation and apoptosis and other biological functions. Therefore, we performed RNA-sqe sequencing on 90 BCa patients in our hospital and analyzed that SULF2 expression was significantly increased in BCa, and it is closely related to the OS and PFS of BCa patients. However, the role and function of SULF2 in the tumor microenvironment have not been studied. A study by Biroccio et al. found that the TSS upstream region of SULF2 contains the binding site of TRF2. The combination of TRF2 and its target motif can enhance promoter activity, and ultimately lead to increased expression of SULF2, which further promotes the secretion of VEGF-A affects angiogenesis in the tumor microenvironment[20]. Studies have also reported that M2 type macrophages can increase the transcription levels of SULF1 and HSPG2 in bone marrow fibroblasts, affect the microenvironment of prostate cancer bone metastasis, promote the expression of Wnt3a and mediate the proliferation of prostate cancer[21].

As reported in the literature, SULF2 affects the changes in the wnt pathway. We have also confirmed this in our experiments. At the same time, in the next experiment, we detected the changes of some cytokines through qPCR, including what are considered HSPG-binding factors, (such as IL6, IL8, IL10, etc.) and found that the abnormal secretion of IL-8 was knocking down at the same time. Therefore, we speculate that SULF2 may affect the secretion of IL-8 through β-catenin. IL-8, also known as leukocyte chemical attractant, recruits macrophages into the tumor microenvironment in a paracrine manner [22, 23]. Subsequently, we discovered an interesting phenomenon that tumor-derived IL-8 promotes phenotypic conversion during the recruitment process to promote macrophage M2 polarization. When IL-8 is added to THP1 cells, thr expression of CD206 and CD163 increased. These results are consistent with previous studies on IL-8 in liver cancer, oral squamous cell carcinoma and papillary thyroid cancer [24–26]. Therefore, IL-8 secreted by the highly expressed SULF2 of BCa promotes the polarization of macrophages to M2 type, and enhances the malignant progression of the tumor through the communication between BCa cells and macrophages.

The JAK/STAT signal pathway is a signal transduction pathway stimulated by cytokines. It is involved in many important biological processes, such as proliferation, differentiation, apoptosis and immune regulation. Ying et al. found that broussonin E can promote the phenotypic transition of macrophages by inhibiting ERK and p38 MAPK and activating the JAK2/STAT3 pathway [27]. In mastitis, researchers discovered a crosstalk signaling pathway between breast epithelial cells and macrophages. The exosomal miR-221 promotes the polarization of macrophages through SOCS1/STATs [28]. In this study, we tested the activities of THP1 that mediate STAT1, STAT3, STAT5a and STAT6 after co-cultivation. We found that STAT3 is activated, and the secretion of IL8 regulated by SULF2 promotes the phosphorylation of JAK2 (Tyr1008) and STAT3 (Tyr705). Studies have shown that STAT3 is one of the main transcription factors for the polarization of macrophages to the M2 phenotype. Once JAK2/STAT3 is activated, it will transfer to the nucleus, resulting in the production of CD163, CD206 and Arg-1. In addition, JAK2/STAT3 is used Pathway inhibitor AG490 treatment can eliminate the upregulation of CD206 and CD163 caused by IL-8. These results indicate that the effect of IL-8 in promoting the polarization of M2 macrophages depends on the regulation of the JAK2/STAT3 signaling pathway.

In summary, we found a new mechanism of cross talk between BCa and macrophages in the tumor microenvironment through in vivo and in vitro experiments and bioinformatics verification. The highly expressed SULF2 in BCa can promote IL-8(HSPG-binding factors) secretion through β-catenin; IL-8 secreted into the microenvironment can activate JAK2/STAT3, leading to the upregulation of CD163 and CD206 transcription, which is the conversion of macrophages to M2 phenotype, and promotes the malignancy of BCa progress.

BCa:Bladder cancer; NMIBC:non-muscular invasive bladder cancer; MIBC:muscular invasive bladder cancer; SULF2: Sulfatase2, TCGA: The Cancer Genome Atlas; GSEA: Gene Set Enrichment Analysis; OS: overall survival; IHC: immunohistochemistry; shRNA: small hairpin RNA; ROC: receiver operating characteristic; AUC: Area Under Curve; DSS: diseasespecific survival; PFI: progression-free interval.

Acknowledgments:

The authors would like to thank Prof.Yan Lv, Prof.Meiyu Bao for excellent technical support.

Fundings:

This work was supported in part by a grant from the National Natural Science Foundation of China (#81472389); the Shanghai Science Committee Foundation (#19411967700); National Natural Science Foundation of China, Youth Project (#81602469; #81802554, #82101838); Shanghai Pujiang Talent Program (#20PJ1412400) and General Project of Shanghai Natural Science Foundation of China (#20ZR1443000).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate

For Human:This study was reviewed and approved by the Ethics Committee of Shanghai Tenth People’s Hospital(SHYS-IEC-4.1/19-120/01,2019). This study was also performed in accordance with the ethics guidelines of the World Medical Association (Declaration of Helsinki).

For animals: This study was reviewed and approved by the Ethics Committee of Shanghai Tenth People’s Hospital(SHDSYY-2021-3028,2021)，and this study also line in the Basel Declaration.

Consent for publication

Not applicable

Competing interests

The authors declare no confict of interest

Author contributions:

ZWT ,YFH wrote the manuscript, ZZT, LC and MSY generated the figures. WY and WRL contributed to the clinical and experimental data, ZJF, ZY and WH analyzed the data, LW,HJH and YXD contributed to editing the manuscript. All authors read and approved the final manuscript.

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Sulfatase2 affects the polarization of M2 macrophages through the IL-8/JAK2/STAT3 pathway in bladder cancer

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