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Prostate cancer

Cathepsin K regulates the tumor growth and metastasis by IL-17/CTSK/EMT axis and mediates M2 macrophage polarization in castration-resistant prostate cancer

Data collection

We screened datasets from Gene Expression Omnibus (GEO), and The Cancer Genome Atlas to explore [16]. In this article, we selected datasets of localized samples and CRPC samples from the GEO database. Finally, GSE32269, GSE32982, and GSE70770 met our inclusion criteria. Details of these three datasets are listed in the Supplementary Table. We used the SVA package in R language to combine the data into a database containing 276 samples, of which 45 were CRPC samples and 231 were localized samples.

Screening and identification of the differentially expressed genes (DEGs)

We compared the protein expression data of the localized samples with those of metastatic samples using limma package of R (3.48.3) to identify the significant DEGs (|Log2FC | > 1.3, adjusted p value < 0.05) [17]. Volcano plots and heatmaps were used to characterize the DEGs, and the KEGG pathway database was used to choose the signal pathways in DEGs enrichment [18, 19].

WGCNA

At first, we selected DEGs between localized and metastatic samples from the combined set of GSE32269, GSE32982, and GSE70770. Thereafter, we executed the weighted gene co-expression network analysis (WGCNA) (1.70-3) on the basis of DEGs in R. The DEGs were hierarchically clustered into eight gene modules when the β value was defined as 6.

Survival analysis

We downloaded the data from GSE32269 dataset and analyzed the ROC curve in SPSS to identify the specificity and sensitivity. Survival analysis was performed on the GSE16560 dataset.

Statistical analysis

Continuous data were described using mean ± standard deviation or median (interquartile range). The t-test was used to compare normal data while the Mann–Whitney U test was used to compare non-normal data. Spearman correlation analysis was used to analyze the correlations in the cross-sectional study. The receiver operating characteristic (ROC) curve and area under the curve were used to calculate the best predictive cut-off value; p values < 0.05 were considered statistically significant. Statistical analyses of the data were performed using GraphPad Prism version 8.0 and SPSS version 25.0.

Serum sCD206 levels of patients with PC

All serum samples were routinely collected from patients before administering treatments, during hospitalization, and stored at −80 °C. The concentrations of sCD206 were measured using commercial enzyme-linked immunosorbent assay kits (Human MMR ELISA Kits, RayBiotech, Norcross, GA). First, 100 μl of standard solutions or samples was added to each and incubated for 2.5 h. After four washes, 100 μl of prepared biotin antibodies was added to each well. After 1-h incubation, 100 μl of prepared streptavidin solution was added and incubated for 45 min. The mixture was washed four times, and 100 μl of TMB one-step substrate reagent was added to each well and incubated for 30 min, followed by another four rounds of washing. Finally, 50 μl of stop was added to each well.

Patients

Prostate tissue specimens, used in this study, were surgical specimens from patients with PC haying complete clinicopathological data. ADPC specimens were acquired by radical prostatectomy, and BPH/CRPC specimens were acquired by transurethral resection of the prostate. These samples were paraffin-embedded and subjected to IHC with standard DAB staining protocols. All tissue samples were obtained from patients with PC, who had undergone surgical operation in the Second Affiliated Hospital of Tianjin Medical University (Tianjin, China), and were inspected by three qualified pathologists to acquire accurate grades. The main site of metastasis was bone tissues in patients with mPC.

Cell lines

Benign prostatic hyperplasia cells (BPH) and various PC cell lines (22Rv1, C4-2, PC3, LNCaP, and DU145) were acquired from ATCC. The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified environment containing 5% CO2 at 37 °C.

Western blot (WB)

Proteins from BPH, 22Rv1, C4-2, PC3, LNCaP, DU145, and different carcinomas were extracted using PMSF and RIPA. BCA kit was used to detect the concentration of different proteins. Protein samples were separated by SDS-polyacrylamide gel electrophoresis in 10% acrylamide gel and then transferred to polyvinylidene fluoride membrane. Next, the was blocked with skimmed milk powder (5%) and incubated with primary antibodies [GAPDH (1:1000) (ab9485), AR-V7 (1:1000) (ab198394), vimentin (1:1000) (ab92547), β-catenin (1:1000) (ab32572), β-tubulin (1:1000) (ab78078), E-cadherin (1:1000) (ab40772), claudin-1 (1:1000) (ab211737), slug (1:1000) (ab180714), twist (1:1000) (ab175430), snail (1:1000) (ab180714)] at 4 °C overnight. The membrane was washed twice with PBS, and incubated with anti-mouse IgG/anti-rabbit IgG at normal temperature for 1 h. The membrane was washed twice with PBS once again. Finally, wb automatic chemiluminescence imaging system was used to detect the bands.

Flow cytometry

For flow cytometry analysis, the tumor mass was dissociated into single cells. Prior to antibody staining, red blood cells were removed with ammonium chloride-potassium lysis buffer for 3 min at room temperature, followed by incubation or staining with cell surface antibody [APC anti-mouse CD206 (BioLegend)] for 30 min on ice. The cells were then washed twice and re-suspended in FACS buffer. Flow cytometry was performed using a CytoFLEX flow cytometer (Beckman Coulter), and the resulting data were analyzed using CytExpert software.

IHC

After clinical and animal laboratory surgery, we obtained human tissue specimens and mouse tumor specimens, which were then fixed with formalin. We prepared pathological sections of the specimens by freezing, paraffin fixing, and sectioning. Next, we put the pathological sections into the oven at 60 °C for 60 min, dewaxed the slices in xylene, and used graded alcohol for rehydration. Thereafter, we used PBS to wash the pathological sections twice, and used citric acid buffer to recover the pathological sections (7 min on high fire and 10 min on medium fire). The pathological sections were washed twice with PBS, and endogenous peroxidase was added to sections for 20 min. Finally, primary antibody was added to the pathological section and left in the refrigerator at 4 °C overnight. The next day, we uesd secondary antibody to detect after washing the pathological sections with PBS twice. DAB chromogen was used for detection, and hematoxylin was used for redyeing it after washing with tap water. After dehydration, the plates were sealed with neutral gum and photographed under a microscope.

Wound healing

We seeded PC cells into 6-well plates. Next, we drew a straight line in the middle of the plates with the tip of the 10-μl microsphere after transfecting negative control siRNA, negative control siRNA + IL-17A, CTSK siRNA, and CTSK siRNA + IL-17A into PC cells (after 24 h). Thereafter, we washed the plates twice with PBS and cultured the cells in a cell incubator for 24–72 h. Photographs were taken under a microscope at 0, 24, and 72 h, respectively.

Clone formation assay

We seeded digestive cells (2.0 × 103 DU145 cells, 2.0 × 103 LNCaP cells) into a 6-well plate. Then, we transfected negative control siRNA, negative control siRNA + IL-17A, CTSK siRNA, or CTSK siRNA + IL-17A into the cells after 24 h. After 1–2 weeks of culturing the PC cells, the plates were washed twice with PBS. Next, we fixed the cells with paraformaldehyde and and used PBS to wash the plates again twice. Finally, the cells were stained with crystal violet for 0.5 h, and the plates were washed twice with PBS and dried thereafter.

Transwell migration

We transfected CTSK-siRNA or negative control siRNA into DU145 or LNCaP cells, respectively. We added 2 × 104 cells and 1640 (10% FBS) to the top of transwell insert, and 1640 (10% FBS) to the bottom chamber. Then, we cultivated the cells for 48 h at 37 °C, and used PBS to wash the chambers twice. Next, we fixed the cells with paraformaldehyde and used PBS to wash the chambers twice. Finally, the cells were stained with crystal violet for 1 h.

Tumor xenografts mouse model

Male mice were injected with 2 × 106 PC3 cells, suspended in 150 μl of Matrigel and 1640 medium, under the skin of the abdomen in control, control + IL-17A, shCTSK, and shCTSK + IL-17A groups. Tumor volume data were collected for at least 2 weeks, being measured at the same time every day. Finally, the mice were sacrificed and weight of the tumor were measured with precision. Parts of the fresh specimens were examined by flow cytometry to verify the immune-related indicators. Rest of the mouse tumors were fixated with paraformaldehyde, and immunohistochemical staining was performed for markers of CTSK, β-catenin, vimentin and E-cadherin. All procedures involving mice were approved by the University Committee on Use and Care of Animals at the Tianjin Medical University and met all regulatory standards.