Cancers, Vol. 15, Pages 131: Sulfatase 2 Affects Polarization of M2 Macrophages through the IL-8/JAK2/STAT3 Pathway in Bladder Cancer

1. IntroductionBladder cancer (BCa) is the most common malignant tumors of the urinary system and ranks four among the most common malignant tumors in men worldwide. It is estimated that nearly 40,000 patients die from BCa every year, and 549,393 new cases were reported in 2018 [1]. Bladder cancer can be divided into nonmuscle invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) according to the depth of tumor invasion. In the past 30 years, there have been few advances in the research and treatment of BCa. With the understanding of the biological functions and immune microenvironment of BCa coupled with large-scale gene sequencing, effective targeted therapies and immunotherapies are used for BCa [2,3].Currently, the role of the tumor microenvironment (TME) in the progression and metastasis of BCa has received tremendous attention [4]. The TME includes tumor cells, macrophages, fibroblasts, endothelial cells, and noncellular substances [5]. The response of macrophages to microenvironmental signals is usually polarized into two phenotypes, namely M1 and M2 macrophages. M1 macrophages have antitumor and immune promotion effects. In contrast, M2 macrophages promote tumor immune escape, angiogenesis, tumor growth, and metastasis [6,7]. Hence, it is important to explore the molecular mechanism of the communication between tumor cells and macrophages in the microenvironment of BCa.Heparin sulfate proteoglycan (HSPG) binds to various ligands, such as heparin-binding growth factor, fibroblast growth factor, and vascular endothelial growth factor, to perform a wide variety of biological functions [8,9]. Recent studies showed that the proteoglycan structure undergoes profound aberrations, and this leads to uncontrolled tumor proliferation, immune escape, metastasis, and differentiation of tumors, such as rectal cancer [10]. These structural modifications are mainly attributed to abnormal changes in the expression of heparin sulfate biosynthetic or catabolic enzymes, such as sulfatase. SULF1 and SULF2 belong to the Sulfatases family. SULF1 and SULF2 are extracellular heparan sulfate 6-O-endosulfatases with unique structural characteristics, enzyme activities, and signaling functions [11]. Studies have shown that SULF1 can bind to FGF, HGF, and VEGF and regulate the proliferation of a variety of solid tumors, including bladder cancer [12,13]. SULF2 also can modulate the expression of cell surface signal molecules on tumor cells by regulating the sulfation of the glycosyl part of HSPGs. Presently, studies have indicated that SULF2 is a potential tumor driver gene for cholangiocarcinoma and glioblastoma [14,15]. However, its role in BCa is yet unclear. Therefore, in this study, we aimed to investigate the mechanism by which overexpression of SULF2 in BCa cells promotes TME BCa progression. 2. Materials and Methods 2.1. Patient Collection

Ninety BCa tissues were collected from patients who underwent either TURBT or radical cystectomy at the Shanghai Tenth People’s Hospital (STPH) from November 2019 to September 2020. Prior informed consent was obtained from the patients (SHSY-IEC-4.1/19-120/01).

The protocols of total RNA extraction, paired-end libraries synthetization, and RNA sequence were available in our previous methods [16]. 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.The FPKM standardized mRNA expression data and clinical information of BLCA patients were downloaded from the Cancer Genome Atlas (TCGA, https://gdc-portal.nci.nih.gov/) (Accessed on 9 January 2020). 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/) (Accessed on 9 January 2020). 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 (Accessed on 9 January 2020). The clinical characteristics of BCa patients in each cohort are shown in Supplementary Table S1. 2.2. Cell Lines

Human BCa cell lines, namely T24, UMUC3, and J82, normal urothelial cell line SV-HUC-1, mouse BCa cell line MB49, and human acute monocyte THP-1 leukemia cells were purchased from Chinese Academy of Sciences, Institute of Chemistry and Cell Biology (Shanghai, China). Cell lines were cultured in either DMEM or RPMI-1640 medium with 10% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA). All cells were cultured in a 37 °C incubator with 5% CO2.

2.3. Cell Proliferation Assay

The effect of SULF2 on cell proliferation was assessed 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. For the CCK-8 assay, cells were seeded into 96-well plates at a density of 2 × 103 cells/well, 10 μL of CCK-8 reagent (Yeasen Biotechnology, Shanghai, China) was added in each well, and the cells were maintained at 37 °C for 1.5 h. The absorbance was measured at 450 nm using a microplate spectrophotometer. Each experiment was performed in triplicate.

2.4. Western Blot and Co-IPUsing RIPA lysis buffer, we extracted total protein from cells or tissues and then quantified the lysate by the bicinchoninic acid (BCA) protein assay. Next, 30 μg protein sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then proteins were transferred to nitrocellulose membrane (Sigma-Aldrich; Merck KGaA). The membrane was blocked with 5% skim milk at room temperature for 1 h, and then immunoblotting was performed with primary antibody at 4 °C overnight. Subsequently, the membrane was incubated with the HRP-labeled secondary antibody for 1 h. The Super ECL Detection (Yeasen Biotech Co. Ltd., Shanghai, China) was used to visualize and quantify the immunoreactive bands. The results were visualized and quantified by Tanon 5200 system (Tanon, Shanghai, China). GAPDH was used as an internal reference. Antibodies are listed in Supplementary Table S2. For Co-IP, lysates of 1 × 107 Bca cells were immunoprecipitated with IP buffer containing IP antibody-coupled argarose beads, and the complexes were used to western blot. IgG was used as a negative control. Original blots see supplementary File S1. 2.5. RNA Extraction and Real-Time Polymerase Chain Reaction (qPCR)TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from human tissues or cells according to the manufacturer’s instructions. A reverse transcription system kit (Vazyme Biotech Co., Ltd., Shanghai, China) was used to generate the first strand of cDNA. The ChamQ Universal SYBR qPCR Master Mix and ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) were used for qPCR. The qPCR parameters were as follows: 40 cycles of 30 s at 95 °C, then 10 s at 95 °C, and 30 s at 60 °C. GAPDH was used as an endogenous control (Supplementary Table S3). The relative fold change was analyzed by the 2-ΔΔC(t) method. Each experiment was performed in triplicate. 2.6. Reagents and Enzyme-Linked Immunosorbent Assay (ELISA) Assay

Human recombinant interleukin 8 (IL-8; PeproTech, Rocky Hill, NJ) was dissolved in trehalose at a concentration of 100 μg/mL and stored at −20 °C. At the time of use, the final concentration of IL-6 was adjusted to 100 ng/mL in the appropriate medium. AG490 (MedChemExpress, Monmouth Junction, NJ, USA), a tyrosine kinase inhibitor that can inhibit the signal transducer and activator of transcription 3 (STAT3) signaling pathway, was dissolved in dimethyl sulfoxide at a concentration of 5 mg/mL, stored at −20 °C, and protected from light. The 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, Burlington, MA, USA) for 48 h to obtain macrophage-like differentiated THP-1 cells. Then, the medium containing PMA was replaced with fresh medium to obtain resting state of macrophages (M0). Next, to differentiate into M1 phenotype, we added 20 ng/mL IFNγ and 1 mg/mL LPS (Sigma-Aldrich), and for M2 phenotype we added 20 ng/mL interleukin 4 (IL-4; PeproTech, Rocky Hill, NJ, USA).

The Human Cytokine Elisa kit (Zorin Biological, Shanghai, China) was used to detect the concentration of the supernatant cytokines in the coculture system. Cytokine and chemokine levels were determined with standard curves developed for each experiment according to the standards provided by the manufacturer. The relative expression levels were calculated from the experimental results.

2.7. Animal Models

For orthotopic bladder cancer model: C57 female mice were used for in situ injection of MB49 BCa cells, and 1 × 106 treated cells were injected into the bladder through the urethra, five in each group (NC and shSULF2). After four weeks, the mice were sacrificed, and in situ tumors were taken out and immunized by flow cytometry cell infiltration situation.

For subcutaneous xenotransplant tumors: 1 × 106 treated T24/UMUC3 cells were injected into the right armpit of four-week-old female BALB/c nude mice, five in each group. After four weeks, the mice were sacrificed, and the subcutaneous tumors were removed. The mouse body weight, tumor diameter, and tumor volume were monitored every seven days as per the formula (tumor volume = π/6 × length × width2).

For tail vein lung metastasis model: 1 × 106 cells with either overexpression or knockdown of SULF2 were injected into the tail vein of four-week-old nude mice, with six mice in each group. The in vivo imaging system (IVIS) was used weekly after three weeks to observe the tumor metastasis.

2.8. Flow Cytometry

To analyze the immune cells infiltrating the mouse orthotopic BCa model, the mice were sacrificed after four weeks of MB49 injection into the C57 mice bladder. The tumor tissues were taken out, cut, and incubated with collagenase (100 μg/mL) at 37 °C for 30 min. The separated tumor cells were filtered through a 70 µm mesh, and red blood cells were removed using red blood cell lysis buffer. The tumor cells were resuspended in PBS and then incubated with flow cytometry antibodies. After 30 min, the cells were washed thrice with PBS followed by resuspension in 500 µL of PBS buffer for flow cytometry analysis. For gating strategy, live–dead dye (APC-Cy7) can separate living cells. Then, CD45 (FITC) can separate immune cells from living cells, CD11b (percp5.5) and F4/80 (BV421) can separate macrophages, CD86 (APC) can separate M1 macrophages, and CD206 (PE) can separate M2 macrophages.

For cell cycle analysis, the tumor cells were collected, trypsinized to form a cell suspension, and then fixed with 75% ethanol at 4 °C overnight. Next day, the cells were washed thrice with PBS followed by incubation with RNase containing iodide (PI, 40%, Sigma-Aldrich) for 30 min and then washed thrice with PBS and used for flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). The experiment was repeated thrice. The results were analyzed using FlowJo.

2.9. Establishment of Stable Cell LinesThe specific shRNAs targeting SULF2 were cloned into the lentivirus vector pLKO (Zorin, Shanghai, China) (Supplementary Table S3). The full-length cDNA amplified by PCR was inserted into the lentivirus vector pCDH (Zorin, Shanghai, China) to produce a specific plasmid overexpressing SULF2. Then, 293T cells were transfected with specific sequence pCDH or pLKO and packaging plasmid. Virus particles were generated and collected 48 h after transfection. The virus was used to infect bladder cancer cell lines for 24 h and then used puromycin to screen stable cell lines. 2.10. Cocultivation System

The BCa cells and macrophages were cocultured using a 0.4 µm Transwell chamber (Corning, Lowell, MA, USA). The THP1 cell line was placed in the lower chamber while BCa cell line with either overexpression or knockdown of SULF2 was placed in the upper chamber. The cocultivation time was 48 h. The required cells were collected for the next experiment.

2.11. Colony Formation

For colony formation analysis, cell lines with either SULF2 overexpression or knockdown were seeded into a six-well plate at a density of 1 × 103 cells/well and cultured in an incubator for 14 days. After this, the cells were washed thrice with cold PBS, fixed with 75% ethanol, and stained with 0.1% crystal violet. The images of the stained tumor cell colony were recorded with a digital camera and statistically analyzed.

2.12. Cell Migration and Invasion

The cell migration ability was detected using a Transwell chamber (Corning, Lowell, MA). Then, 5 × 104 cells were added to 200 µL of FBS-free RPMI-1640 medium and transferred to the upper chamber of the transwell while 600 μL of cell culture medium containing 10% FBS was added to the lower chamber. The transwell chamber was incubated at 37 °C for 16 h and then washed thrice with cold PBS. Cells that did not pass through the upper chamber were carefully wiped with a wet cotton swab, fixed with 75% ethanol for 30 min, and then the chamber stained with 0.1% crystal violet for 10 min. The cell migration was observed under a microscope (Olympus Corporation). For tumor cell invasion experiments, the Transwell chambers were precoated with Matrigel, and the remaining experimental procedure was same as the tumor cell migration experiment. Each experiment was performed in triplicate.

2.13. Immunohistochemistry (IHC)

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

2.14. Statistical AnalysisSPSS version 22 (SPSS, Inc., Chicago, IL, USA) and R statistical software (https://www.r-project.org/) (Accessed on 9 January 2020) were used for data analysis. The Kaplan-Meier (log-rank test) was used to compare the overall survival (OS) and DFS of the patients with BCa between low and high expression groups of SULF2. Patients were divided into low and high SULF2 expression groups based on the optimal cutoff values in each dataset calculated by the ‘survminer’ R package v0.4.8. The differences between the groups were evaluated using Student’s t-test, chi-square test, and the Mann–Whitney U test, as appropriate. One-way ANOVA followed by Bonferroni test was used to compare multiple groups. A p-value of 4. DiscussionMacrophages are the main components of the TME. They are transformed into different states due to differences in the microenvironments, such as the increase or decrease of local cytokines, hypoxia, necrosis, or changes in metabolites [22]. Currently, the polarization of macrophages can be divided into the M1-type (classically activated macrophages) and the M2-type (alternatively activated macrophages), which have differential effects on tumor progression. M2 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 [23,24,25].In this study, we found that there were significant differences in M2 macrophages in the immune cell infiltration of the BCa microenvironment. This was also confirmed by tumor formation in situ in mice. Additionally, we found that the knockdown of SULF2 in the MB49 cells decreased the proportion of M2 macrophages in the BCa microenvironment. Xu et al.’s research on the analysis of immune cells in BCa also corroborated the phenomenon of increased macrophages in BCa [26]. Our previous clinical study on SULF2 showed that IHC analysis and clinical prognostic follow-up of SULF2 expression in 203 patients with bladder cancer confirmed that SULF2 is highly expressed in bladder cancer and may be associated with lymphatic metastasis, which has clinical significance for evaluating prognosis. In addition, several studies have shown that adiponectin, an inhibitor of SUFL2, may be a potential tumor therapeutic drug [27]. The role and mechanism of adiponectin in bladder cancer still need further verification.SULF2 not only changes the function of HSPG by regulating 6-O-sulfation but also plays an important role in the occurrence and development of tumors, such as participating in the tumor cell cycle, proliferation, apoptosis, and other biological functions [28]. Therefore, we performed RNA sequencing of BCa tissues obtained from 90 patients with BCa in our hospital. Upon analysis, we found that SULF2 expression was significantly increased, and it is closely related to the OS and PFS of patients with BCa. However, the role and function of SULF2 in the TME of BCa have not been studied yet. Biroccio et al. found that the TSS upstream region of SULF2 contains a binding site for TRF2. The combination of TRF2 and its target motif enhances the promoter activity, and ultimately increases the expression of SULF2, which further promotes VEGF-A secretion and thus affects angiogenesis in the TME [29]. Another study reported that M2 macrophages increase the transcription levels of SULF1 and HSPG2 in bone marrow fibroblasts and affect the microenvironment of prostate cancer bone metastasis [30].As per previous studies, SULF2 affects the Wnt pathway [31,32]. We also confirmed this in our experiments. Meanwhile, using ELISA, we detected changes in cytokines, including those considered HSPG-binding factors, such as IL6, IL8, and IL10. We found that IL-8 secretion was reduced after SULF2 knockdown. Therefore, we speculated that SULF2 may affect the secretion of IL-8 through β-catenin. IL-8, also known as a leukocyte chemical attractant, recruits macrophages into the TME in a paracrine manner [33,34]. Interestingly, we found that tumor-derived IL-8 promotes phenotypic conversion during the recruitment process to promote M2 macrophage polarization. Upon adding IL-8 to THP1 cells, the 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 [35,36]. Therefore, SULF2 is highly expressed in BCa and increases IL-8 secretion, which promotes the polarization of macrophages to the M2-type and enhances the malignant progression of the tumor through the communication between BCa cells and macrophages.The JAK/STAT signal transduction pathway is stimulated by cytokines. It is involved in 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 [37]. In mastitis, researchers identified a crosstalk signaling pathway between breast epithelial cells and macrophages. The exosomal miR-221 promotes the polarization of macrophages through SOCS1/STATs [38]. In this study, we tested the activities of STAT1, STAT3, STAT5a, and STAT6 after cocultivation in THP1 cells. We found that STAT3 was activated in THP1, and the secretion of IL-8 regulated by SULF2 promotes the phosphorylation of JAK2 (Tyr1008) and STAT3 (Tyr705). These results indicate that the effect of IL-8 on promoting the polarization of M2 macrophages depends on the regulation of the JAK2/STAT3 signaling pathway.

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