Culture of cell lines and isolation of primary stromal cells or pericytes
Human retinal pericytes cell line HRMVP and mouse RCC cell line Renca were purchased from aoyinbio Co., Ltd (Shanghai, China). Mouse prostate cancer (PCa) cell line RM1 and mouse melanoma cell line B16F10 were purchased from zqxzbio Co., Ltd (Shanghai, China). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS) (#A3161002C, Gibco) and 1% penicillin–streptomycin (#15070063, Gibco). Primary stromal cells or pericytes were isolated from the tumor tissues of ccRCC patients or patients with lung cancer, from May 2021 to June 2021 in Xijing Hospital, Fourth Military Medical University (Xi'an, China) according to previously described protocols[8, 22].
Flow cytometry analysis of clinical ccRCC sample
This study was approved by the ethics committee of Fourth Military Medical University. The ccRCC samples were obtained from 80 patients in Xijing Hospital, Fourth Military Medical University (Xi'an, China) from February 2021 to June 2021. Clinical information was collected from the electronic medical records. The type I and type IV collagenase were used as digestive enzyme to turn the fresh surgical ccRCC specimens into single-cell suspension. Cells were divided into 4 parts and incubated with 4 groups of antibodies, respectively. Group 1 includes anti-CD4, anti-CD25, anti-CD127 and anti-Foxp3 antibodies (#A42924, Invitrogen). Group 2 includes anti-CD3, anti-CD4, anti-CD8, anti-CD279, anti-CD366 and anti-CD152 antibodies (#11003742 #11004842 #12008842, Invitrogen, #329916 #345014 #369612, Biolegend). Group 3 includes anti-CD326, anti-CD31, anti-CD45 and anti-EN antibodies (#17579182, Invitrogen, #303105 #304029, #949902, Biolegend). Anti-EN antibody IgG78 was conjugated by FITC kit (ARL0021K, Frdbio) before being used for analysis. Cells were incubated for 30 min at 4℃ in darkness before flow cytometry analysis.
Western blot
Protein samples was separated by 8% SDS-PAGE and transferred into a PVDF membrane (ThermoFisher Scientific), before the membrane was incubated with primary antibodies overnight at 4℃, followed by incubation with corresponding HRP-conjugated secondary antibodies and visualization by chemiluminescence. The primary antibodies used were: anti-EN (#ab48185, Abcam), anti-GAPDH (#10494-1-AP, Proteintech), anti-p-MAPK (#4370T, CST) and anti-MAPK (#4695T, CST).
Animal experiments
Animal experiments in this study were approved by the Laboratory Animal Welfare and Ethics Committee of Fourth Military Medical University. C57BL/6 endosialin knockout (ENKO) mice were purchased from Shanghai model organism company (#NM-KO-200094). BALB/c mice (6–8 weeks old, female, body weight 18–23g) were purchased from the animal center of Fourth Military Medical University (Xi'an, China) and maintained in a 12h light/12h dark cycle with free access to food and water. Each mouse was subcutaneously inoculated with 2×106 tumor cells in the back. For the antibody treatment, mice were treated with anti-EN antibody (mIgG78, 5mg/kg) and anti-mouse PD-1 antibody (50mg/kg, #BE0033-2, Bio X Cell) or anti-mouse PD-1 antibody alone, every four days, for a total of four times. Mice were sacrificed when tumor diameter reached 2 cm or severe weakness was observed, and tumors were isolated for subsequent experiments. The isolated tumor tissues were digested into single cell suspension, before flow cytometry was applied to examine T cells using fluorescent labeled anti-CD3, anti-CD4, anti-CD8 and anti-CD69 antibodies (#100306 #100412 #100708, Biolegend).
RNA-seq and single cell RNA-seq analysis
RNA was isolated from tumor derived pericytes which were harvested from ccRCC tissues in EN-high or EN-low groups and subjected to RNA sequencing. The two-terminal sequencing model of the Illumina Hi-Seq sequencing platform was used for high-throughput sequencing of multiple samples (Genergy bio). ScRNA-seq data of ccRCC were obtained from 48 samples of 38 patients[23-26].
Analyses of scRNA-seq datasets were performed primarily using the Seurat package (v4.0.5) in R (v4.1.0)[27]. First, individual gene expression matrices of scRNA-seq were used to create distinct Seurat objects. Second, strict quality control procedures were performed. Data were filtered to exclude cells that expressed fewer than 200 genes, more than 6000 or 8000 genes, greater than 20% mitochondrial genes, and more than 0.1% or 1% hemoglobin genes. Meanwhile, genes expressed in fewer than three cells were removed. Third, all Seurat objects were merged to generate a combined dataset, and the merged dataset was normalized and scaled separately using the NormalizeData and ScaleData functions. The FindVariableFeatures function was applied to identify the variable genes. Then principal component analysis (PCA) was conducted with the RunPCA function based on the variable genes. Importantly, the datasets and corrected the batch effects from different samples were integrated using the Harmony package (v0.1.0)[28]. Finally, clustering was conducted using the FindNeighbors and the FindClusters functions with resolution = 0.8. Visualization was implemented via t-distributed stochastic neighbor embedding (tSNE).
Marker genes of cell clusters were identified using the FindAllMarkers function via Wilcoxon rank-sum tests. Then each cell cluster was renamed to the specific cell type according to classical marker genes: B cells (marked with CD79A and MS4A1), plasma cells (marked with CD79A, IGKC, and IGLC2), CD4+ T cells (marked with CD3D, CD4, and IL7R), CD8+ T cells (marked with CD3D, CD8A, and GZMB), dendritic cells (DCs, marked with CD1C and CD1E), endothelial cells (marked with PECAM1 and vWF), epithelial cells (marked with EPCAM and KRT18), macrophages (marked with CD68 and CD163), mast cells (marked with CPA3 and KIT), monocytes (marked with CD14 and S100A8), natural killer cells (NK cells, marked with NKG7 and GNLY), and stromal cells (marked with ACTA2 and RGS5).
Immunohistochemistry (IHC) staining
The primary antibodies used for IHC staining were as following: ani-human endosialin (#ab204914, Abcam) and anti-CD8 (#70306S, CST). The expression of endosialin and CD8 were assessed according to the percentage and intensity of IHC staining[29, 30].
T cell infiltration assay
Si-EN or control HRMVP cells (7.5×105) were plated in pericyte medium on the top of the 24-mm chamber with 8.0-mm pore Matrigel-coated Transwell filters (Corning) and cultured for 24 h. Then, CD3+ T cells (5×105) isolated from human peripheral blood mononuclear cell (PBMC) were added to the top chamber in DMEM supplemented with 2% FCS, DMEM supplemented with 20% FCS was added in the bottom chamber. After another 24 h, cells in the bottom chamber were collected for flow cytometry analysis using anti-CD8 antibody (#344717, Biolegend). Human PBMC were isolated from healthy donors, and CD3+ T cells were isolated using Dynabeads™ FlowComp™ human CD3 kit (#11365D, Invitrogen).
Cell proliferation and migration assay
To measure cell proliferation, cell counting kit 8 (CCK8) assay was applied. Si-EN or control HRNVP cells were seeded into 96-well plates at the density of 3×103 cells each well and incubated for indicated time. At indicated time point, 10 μL CCK8 reagent (mishubio, Xi’an, China) was added into each well and incubated for at 37 °C for 1 h. Plates were then gently shaken on a shaker for 5 minutes in the dark before the absorbance at 450 nm was measured by a microplate reader.
To measure cell migration, wound healing assay was applied. At 48 h after siRNA transfection, or after incubation with anti-EN antibody or control IgG for 45 min, HRNVP cells were reseeded in a six-well plate at the density of 5×105 cells per well. Twelve hours later, a scratch was made using a 200 μl pipette tip, then culture medium was changed into serum-free medium. At this time, 0 h images were captured, then the plates were returned to the incubator, and images were taken again after 12 h, 24 h or 48 h.
Tube formation assay
Matrigel (BD Biosciences) was added into 48-well plate (150 μL/well), and incubated at 37℃ for 30 min. Primary stromal cells were suspended by brief exposure to 0.25% trypsin (Invitrogen) and incubated with human anti-EN antibody or control IgG for 45 min in 50% PBS/45% basal media/5% FBS before they were seeded on the Matrigel covered 48-well plate. Si-EN or control HRNVP cells or primary pericytes (21,000 cells per well) were plated onto the Matrigel covered 48-well plate in 300 μL of PBS/media/2% FBS. Cells were incubated at 37 °C in the humidified incubator for 16 h. The tubes/networks were imaged using Metamorph image analysis software (Molecular Devices, Sunnyvale, CA).
Statistics
All statistical analysis was performed using IBM SPSS statistical software (version 23) or GraphPad Prism 8 software. Results were depicted as mean ± SD Comparisons were analyzed by two-tailed Student t test or one-way ANOVA followed by Dunnett’s post hoc test. Differences were considered significant when P < 0.05.