Pan-cancer analyses were performed to compare the expression of SEMA4B in the tumor samples of genotype-tissue expression (GTEx) combined with TCGA. UCSC TOIL was used to correct for batch effects, and to allow for sample merging. In total, 33 different TCGA projects, each representing a specific cancer type, were analyzed. The difference between tumor and normal samples was tested by the Wilcoxon rank sum test.
SEMA4B differential expression in TCGA LUAD data
Gene expression data from RNA-Seq used in this study were collected from TCGA and GTEx projects (including 515 LUAD tissues and 347 normal tissues). RNAseq data in level 3 HTSeq-FPKM format was converted into transcripts per million (TPM) reads format and was log2 transformed for all downstream analyses. Boxplots and scatter plots were generated to compare differential expression of SEMA4B between tumor or normal tissues. The diagnostic performance of SEMA4B was estimated using receiver operating characteristic (ROC) curves. DESeq2 (3.8) package was used to identify DEGs between SEMA4B-high and SEMA4B-low patients from TCGA datasets . The adjusted. p value<0.05 and |log2(fold change)| > 2 were defined as cutoff values for DEGs. All the DEGs were presented in a heat map and a volcano plot.
Functional enrichment and analysis of immune cell infiltration
GO analysis and KEGG analysis were conducted using R cluster Profiler package (3.14.3) to predict the SEMA4B-related phenotypes and signal pathways [23, 38,39,40]. An false discovery rate (FDR) q-value < 0.2, fold change≥2, and p < 0.05 were considered significant statistically. In GSEA analysis, a permutation test with 1000 times was used to identify the significantly changed pathways. An FDR < 0.25 and adjusted p < 0.05 were identified as significant related genes. Statistical analysis and graphical plotting were conducted and visualized using R package ggplot2 (3.3.3) . The tumor infiltration of 24 immune cell types were quantified by single-sample GSEA (ssGSEA) using the R GSVA package based on TCGA . The TIMER2.0 database was then used to analyze the correlation between the expression of SEMA4B and infiltration of CD8+ T cells, B cells, MDSCs, T-regs, cancer associated fibroblast (CAFs) and macrophages. The degree of significance (p value) between SEMA4B and immune cell infiltration was < 0.01. A |Spearman’s rank correlation coefficient|>0.2 was considered as positive correlation.
Evaluation the relationship between SEMA4B expression and LUAD prognosis
The relationship between clinical pathologic features and SEMA4B was analyzed with the Wilcoxon signed-rank sum test and logistic regression. Univariate and multivariate analysis with Cox’s regression model were used to statistically identify the best combination of risk factors to predict prognosis. The OS difference of between patients with high and low SEMA4B expression was calculated by the Kaplan–Meier method with the two-sided log-rank test using two R packages (survival 3.2–10 and survminer 0.4.9). A p value<0.05 was considered as significance in all tests.
Clinical relevance of SEMA4B expression in LUAD patients
A total of 10 human paraffin-embedded tissue samples obtained by surgical resection from LUAD patients were collected from the Department of Thoracic Surgery of Tangdu Hospital. Tissue samples were then investigated for SEMA4B expression by immunohistochemistry (IHC). The rabbit anti-SEMA4B antibody (Sigma, #HPA013372, 1:200) was purchased from Sigma, St. Louis, MO, USA. FOXP3+ T-reg cells and CD11b+ MDSCs were also stained with Rat anti-CD11b (Abcam, ab8878,1:200) and anti-FOXP3 (Abcam, ab215206,1:100) antibody.
Cell culture and preparation
LLC and HEK293T cells were purchased from Shanghai Institutes for Biological Sciences (Chinese Academy of Sciences, Shanghai, China). The LLC cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS, Biological industries, Beit Ha’emek, Israel), penicillin 100 U/mL and streptomycin 100 U/ml. HEK 293 T cells were cultured in DMEM medium with 10% FBS, penicillin 100 U/mL and streptomycin 100 U/ml. Luciferase plasmid was constructed based on the pLenti6.3 lentiviral vector. Next, HEK293T cells were transfected with luciferase plasmid (4μg) along with the helper plasmid psPAX2 (3μg), and pMD2.G (1μg) using lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA). The viral supernatants were collected in 48 h and 72 h after transfection and used for infection of LLC cells to produce luciferase-labeled LLC cells, which were maintained in 1640 medium supplemented with 10% FBS and 250 μg/ml Hygromycin B (Roche, Switzerland). Lentiviral plasmids encoding shSEMA4B/shCtrl were constructed based on the pLKO.1 vectors. 4 ng of shSEMA4B/shCtrl plasmids was co-transfected into HEK293T cells with helper plasmids using lipofectamine 2000. The viral supernatants were collected in 48 h and 72 h after transfection and used for infection of luciferase-labeled LLC cells with 8 μg/mL polybrene (Sigma, St. Louis, MO, USA). The transduced cells were selected using 2μg/ml Puromycin (Gibco, UK).
Quantitative real-time PCR
Total RNA was extracted by TRIzol (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA with RT reagent Kit (Takara Bio Inc. Japan). The difference in expression level of SEMA4B was compared by qPCR (ROCHE lightcycle 96, Indianapolis, IN, USA). Correlation data was calculated based on the ΔΔCT of the target gene and the internal reference actin. SEMA4B-F: 5′-TAGCTTCCAGGGAAACGACC-3′; SEMA4B-R:5′-CCCGTCTCGCTGAAGAAGAA-3′. hACTIN-F: 5′-CGGCACCACCATGTACCCTG-3′;hACTIN-R: 5′- ACACGGAGTACTTGCGCTCA′-3′.
Cell proliferation assay
Cells proliferation capacity was evaluated with CCK-8 and Edu assay. Firstly, 5 × 103 cells/well LLC cells expressing shSEMA4B or shCtrl were seeded at 96-well plates, and cultured at 37 °C. The viability of the cells was detected according to the manufacturer’s instructions. 10 μL Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) was added at the indicated time points and cells were incubated for 2 h at 37 °C. Finally, the absorbance of each well at 450 nm was measured with a microplate reader (Tecan, Männedorf, Switzerland).
EdU staining was conducted according to the manufacturer’s instruction (US everbright inc, Suzhou, China). Briefly, 1 × 104 LLC cells expressing shSEMA4B or shCtrl were seeded at 96-well plate, and (3 sub-wells were set up for each group). After incubated at 37 °C for 24 hours, each well was added with 100 μl of solution A with a final concentration at 5%. 4 hours later, cells were fixed with 4% paraformaldehyde for 15 min, and then neutralized with 100 μl of glycine (2 mg/ml) for 5 min, and an addition of 100 μl 0.5% TritonX-100 for 20 min. 80 μl of C solution (1×), 4 μl of D solution, 0.2 μl of B solution, 10 μl E (1×) was added to each well. After being stained in darkness for 30 min at room temperature, cells were incubated with 100 μl of 1% F solution for 30 min, and counted under a fluorescence microscope.
Plate clone formation assay
Briefly, cells expressing shSEMA4B or shCtrl were seeded in 6-well plates at a density of 103 cells/well for 7–14 days and then fixed with 4% paraformaldehyde for 20 min. After being stained with 0.1% crystal violet for 30 min, cells were rinsed and scanned to grayscale image by Odyssey infrared imaging system (LICOR, USA).
5-week-old of female C57BL/6 mice were purchased from the Animal Experimental Center of the Fourth Military Medical University. 2 × 106 LLC tumor cells expressing shSEMA4B or shCtrl were implanted subcutaneously in the thigh of C57BL/6 mice. 2 to 3 weeks later, the tumor masses maintained in mice were observed after being intraperitoneally injected with 150 mg/kg Dluciferin. Living images were captured by an IVIS imaging system (PerkinElmer, life sciences, USA). All the experimental procedures involving animals were conducted under a protocol reviewed and approved by the Ethics Committee of Tangdu Hospital, Fourth Military Medical University.
Flow cytometry and immunohistochemistry assay
After being harvested and weighed, tumor tissues were cut into small pieces and mixed with IV collagenase for 2 h. Tissues were homogenized and filtered through a 70 μm filter to remove remaining undigested tissue. The single cell suspension was centrifuged at 500 g for 5 min, incubated with 2 μl extracellular antibody for 30 min, and then fixed with 4% paraformaldehyde for 20 min. Finally, the suspension was incubated with intracellular antibody for another 30 min before being tested by flow cytometry (NovoCyte, ACEA Biosciences, San Diego, USA). APC-Anti-CD4 (#553051), PE-Cy7-anti-CD25(#552880), PE-anti-FOXP3(#563101), PE-Cy7-anti-CD11b (#552850) and FITC-anti-Gr-1(#553126) were purchased from BD pharmingen™.
All experiments were performed in triplicate on three independent occasions. Data are expressed as mean ± S.D. One-way ANOVA was employed for statistical analysis by SPSS 22.0 (SPSS, Chicago, USA). The differences between means were tested by an independent sample t-test. The association between SEMA4B expression and clinico-pathological parameters was examined by χ2 test. A p value < 0.05 was considered significant. * p < 0.05, ** p < 0.01, *** p < 0.001.