Sema4D silencing increases the sensitivity of nivolumab to B16-F10 resistant melanoma via inhibiting the PI3K/AKT signaling pathway

Cell culture and B16-F10 resistance cell line establishment

293T cells, B16-F10 were purchased by Nanjing Cobioer Biotechnology Co., LTD (Nanjing, China), cultured at 37 °C in 5% CO₂ in RPMI-1640 (Life Technologies, Invitrogen, Canada) with 10% FBS (Gibco, Billings, MT, USA). The combination of high dose shock and gradually increasing dose was applied to establish the cell lines insensitive to nivolumab (anti-PD-1). The B16-F10 cell line in the logarithmic growth phase were cultured with nivolumab (AbMole Bioscience Inc., Shanghai, China) 10 ng/mL and TIL at a 1:5 ratio. After 24 h, the solution was changed, and dead cells were discarded. The remaining cells were then cultured with RPMI-1640 without nivolumab, when the remaining cells have stabilized, then cultured with nivolumab 25 ng/ml and repeat these steps until the cells could grow continuously and stably in the medium with nivolumab 50 ng/mL, and then cultured drug-free for 1 month, B16-F10 resistance cell line (B16-F10R) were obtained. B16-F10R also needs with nivolumab 5 ng/mL to maintain the resistance (Maeda, Lafreniere & Jerry, 1991).

Tumor infiltrating lymphocyte (TIL)

The tumors were removed from the Balb/c mice, about 2–3 cubic millimeters in size, and cultured with two mL complete medium in 24 well plates. The complete medium included 6,000 IU/mL IL-2 which purchased from Novartis (Basel, Switzerland), RPMI-1640 and 2.05 mM L-glutamine were the major components and purchased from Thermo Fisher Scientific (Waltham, MA, USA), there is also a need to add 10% heat-inactivated human AB serum (Omega Scientific, Westlake Village, CA, USA), 55 µM 2-mercaptoethanol and 10 mM of HEPES Buffer (Mediatech, Hsinchu, Taiwan), gentamicin (50 µg/mL), penicillin (100 I.U./mL), streptomycin (100 µg/mL) were purchased from Invitogen and prevent infection (Arneth, 2019).

Western blotting

We investigated the expression of PD-L1, Sema4D, Plexin-B1, PI3K, p-PI3K, AKT and p-AKT. Antibodies against these proteins were purchased from Abcam Shanghai Trading Co., LTD and cell signaling technology, Inc. (Shanghai, China). Total protein was extracted and BCA protein detection kit (Beyotime, Jiangsu, China) was used to evaluate the protein concentration. after electrophoresis, transfer membrane, antibody incubation process, the dilute concentration of primary antibody is PD-L1(1:500), Sema4D(1:1000), Plexin-B1(1:1000), PI3K(1:100), p-PI3K(1:1000), AKT(1:1000), p-AKT(1:500), an ECL chromogenic substrate was applied for detecting the signals (Beyotime, Jiangsu, China).

RT-qPCR

Total RNA was obtained by processing the samples with TRIzol reagent and Rnase-free DNase Set (Beyotime, Jiangsu, China). The synthesis of complementary DNA was performed using a cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). Finally, RT-qPCR analysis was performed using an ABI PRISM 7900-HT sequence detection system (Applied Biosystems, Waltham, MA, USA). For RT-qPCR, the following primers were used.

PD-L1:5′-AGAACCTGGCTGCACCTAAC-3′(F),

5′-GAGAAGGTCAAACCGCCTCA-3′(R);

Plexin-B1:5′-AAGCCCAGCCTACTAACAACC-3′(F),

5′-CAGCCCCACTGTACTGACTG-3′(R);

Sema4D:5′-TCAAAGCAGACGGAATGCCTA-3′(F),

5′-CCCCAACCATGACTGATGTGTA-3′(R);

GAPDH:5′-TTGTCATGGGAGTGAACGAGA-3′(F),

5′-CAGGCAGTTGGTGGTACAGG-3′(R).

shRNA and expression plasmid transfection

Short hairpin RNA (shRNA) targeting Sema4D and the lentivirus containing this shRNA were designed and synthesized by Genepharm Co. (Shanghai, China), sh-sema4D (5′-GGATGGGACTG TCTATGATGT-3′) and the control NC-sema4D (5′-TTCTCCGAACGTGTCACGT-3′). The shuttle plasmid and three other packaging plasmids (pGag/Pol, pRev, pVSV- G) were co-transfected into 293T cells to generate a lentivirus expressing Sema4D shRNA or a control lentivirus expressing shRNA. Cells were inoculated into 6-well plates at 1 × 10⁵ and infected with lentivirus while 5 µg/mL of Polybrene was added. The culture media was changed after 24 h of infection, and the infected cells were screened with 1 µg/mL of puromycin for 7 days after the culture media change to obtain stable lentiviral shRNA expressing cell lines for subsequent experimental analysis (Moore et al., 2010).

Sema4D overexpression

B16-F10 cells were treated with RPMI 1640 containing 10% fetal bovine serum culture medium was placed in 5% CO₂ incubator at 37 °C, and logarithmic growth was taken long-term cells were used in the experiment. Construct Sema4D lentivirus overexpression vector LV-Sema4D and negative control LV-NC, these vectors were commissioned by Gemma Pharmaceutical Technology Co., LTD. (Shanghai, China) to synthesize. B16-F10 cell was inoculated in 24-well plates and cultured for 24 h multiplicity of infection, complete culture medium was replaced for further culture 48 h, the expression of Sema4D was detected by RT-qPCR.

MTT

Cell viability was measured by Methyl Thiazolyl Tetrazolium (MTT) assay. MTT was dissolved in DMSO and 0.5 mg/mL MTT diluted in 1 × PBS for 4 h, Turn on Benchmark Plus microplate reader (Bio-Rad, Hercules, CA, USA) and detect absorbance at 570 nm (Algazi et al., 2020).

Cell apoptosis analysis

Cell apoptosis were detected by AnnexinV-AlexaFluor647/PI Kit Apoptosis Assay Kit (Biotech, Minneapolis, MN, USA). 5 × 10⁵ B16-F10R cells were seeded in 10-cm culture dishes (Corning, Corning, NY, USA) and incubated overnight. 5 × 10⁵ treated cells were collected and resuspended with 500 µL binding buffer. 5 µL Annexin V/AlexaFluor647 were incubate for 5 min at room temperature, then add 10 µL propidium iodide (PI), fluorescent dyes should be protected from light. All operations are performed as per manufacturer instructions.

Wound healing and invasion analysis

B16-F10R cells invasion and migration was analyzed by Matrigel-coated invasion chambers and scratch assays respectively. The initial number of cells cultured was 5 × 10⁵, a sterile 10 µL pipette tip was used to mark a line in the monolayer of each well, the wounds were observed at 0, 24 and 48 h under a microscope (Nikon, Japan). ImageJ software was used to measure the wound areas. Cell free migration area =(cell free area 0 h - cell free area 24h)/cell free area 0 h (Ji et al., 2018). 5 × 10⁵ cells were placed into the Matrigel-coated invasion chambers (Corning, USA) to incubation, the upper and lower cultures were separated by polycarbonate membranes, and the cells under study were planted in the chamber, after 48 h, the top of Matrigel and invading cells were fixed and stained with 0.1% crystal violet. The microscope (Nikon, Japan) was used to photograph invading cells, and 3 fields were randomly selected and counted each time.

Animal and tumorigenesis

Balb/c mice with 12-week-old were purchased from Chendu dossy experimental animals CO., LTD (SCXK (Chuan) 2020-030, Chengdu, China). The mice were fed unrestricted diet and water intake, and were kept under specific pathogen-free conditions in a laboratory where a 12-hour light/dark cycle was strictly enforced (SYXK(Chuan) 2018-065, Luzhou, China). For each group, at least 5 mice were used (Sanmamed et al., 2015). Tumor volumes were measured on 1, 3, 6, 9, 12, 15 and 18 days with caliper, at days 18, mice were sacrificed, tumor weight was measured, tumor volumes were calculated with the formula: tumor volume = 0.52 × length × width² (mm³) .

Data collection

Melanoma gene expression profiling data were extracted from the Cancer Genome Consortium (TCGA) database using the TCGAbiolinks software package. Expression profiles were converted to transcripts per million mapped reads per kilobase exon model (TPM) and normalized by log2.

Weighted co-expression network analysis

To explore the signaling pathways involved in Sema4D, gene co-expression network analysis (WGCNA) was first conducted to determine genes with significant change between high- and low-expression of Sema4D through the WGCNA package on R project. Topological overlapping measurement was utilized to identify modules most associated with Sema4D expressions. Correlation between modules and traits is calculated by Spearman correlation analysis.

Protein-protein interaction

To explore the potential functions played by Sema4D in various biological processes, we constructed Sema4D-related protein-protein interaction network by STRING (https://cn.string-db.org/).

Gene set variation analysis

Gene set variation analysis was conducted to reveal the molecular mechanisms under the different Sema4D expression populations in melanoma. The h.all.v7.5.1.symbols.gmt gene set was used to enrichment analysis and was downloaded from MSigDB (https://www.gsea-msigdb.org/gsea/index.jsp).

Functional enrichment analysis of Sema4D

To explore the mechanism of action of Sema4D involved in PD-1 inhibitor resistance, enrichment analysis on hub genes was performed. “ClusterProfiler” package in R project was utilized to identify the signaling pathways involved in Sema4D within TCGA cohort (which contains 471 melanoma samples).

Statistical analysis

GraphPad Prism V.7.00 software was utilized for statistical analysis. Different statistical methods were selected according to different experimental purposes, compare the differences between two groups, we chose Student’s t test, one-way ANOVA was used to compare the differences between the overall differences. P < 0.05 is considered to be statistically significant.

Sema4D, Plexin-B1 and PD-L1 upregulation in nivolumab resistance B16-F10 cells

To investigate the mRNA and protein expression of Sema4D, Plexin-B1and PD-L1 in sensitive cell line and insensitive cell line of melanoma, mRNA and protein expression of Sema4D, Plexin-B1 and PD-L1 of B16-10 and B16-F10R were detected. Compared with B16-10 group, mRNA and protein expression of Sema4D, Plexin-B1and PD-L1 were significantly upregulation in B16-F10R group (Figs. 1A–1G).

Sema4D downregulation inhibit PD-L1 expression upon anti-PD-1 therapy

We analyzed the correlation of PD-L1 and Sema4D with bioinformatics, it showed that PD-L1 and Sema4D are positively correlated (Fig. 2A) in TCGA-cohort. Compared with Sema4D-NC group, protein expression of Sema4D in Sema4D-shRNA group was significantly downregulated, it indicated that Sema4D silencing is success (Figs. 2B and 2C). Compared with Sema4D-NC group, mRNA and protein expression of PD-L1 in Sema4D-shRNA group was significantly downregulated after nivolumab treatment, which was consistent with the results of bioinformatics (Figs. 2B, 2D and 2E). Therefore, Sema4D silencing significantly repressed the mRNA and protein expression of PD-L1.

Sema4D repression renders B16-F10R cells sensitive to PD-1 treatment

To explore the relationship between Sema4D downregulation and nivolumab treatment efficacy in B16-F10 and B16-F10R cells, we investigated the cell viability and cell apoptosis. First, we observe the effects of nivolumab on the cell viability of B16-F10 and B16-F10 resistant cell lines. Compared with the B16-F10 cell lines, nivolumab had inhibitory effect on B16-F10 cell lines (p = 0.0018), it is suggested that nivolumab has inhibitory effect on sensitive cell lines. Compared with the B16-F10+nivolumab group, the inhibitory effect of nivolumab on drug-resistant cell lines was decreased significantly (p = 0.0025), it is suggested that nivolumab has weak inhibitory effect on resistance cell lines (Fig. 3A). Next, we explored the effects of nivolumab on the cell viability of Sema4D overexpressed in B16-F10 cells. First, the mRNA expression of Sema4D was detected, and the results showed that Sema4D expression of LV-Sema4D group was increased significantly compared with the LV-NC group(p = 0.0083), it indicates that Sema4D transfection is successful (Fig. 3B). Through MTT, compared with LV-NC group, nivolumab have little inhibitory effect on B16-F10 cells with Sema4D overexpression (p < 0.0001), this suggest that Sema4D overexpression could weaken nivolumab effect, so Sema4D was related to nivolumab sensitivity to B16–F10 cells. (Fig. 3C). We further explored the effect of nivolumab on the cell viability of Sema4D knockdown B16-F10R cell lines, the results showed Sema4D downregulation potentiates anti-PD-1 treatment efficacy in B16-F10R cells. Compared with Sema4D-NC group, the cell viability of Sema4D-shRNA group in combination with nivolumab was decreased significantly (p = 0.0186) (Fig. 3D) and rate of apoptosis cell in Sema4D-shRNA group after treatment with anti-PD-1 was notably increased (p = 0.0027) (Fig. 3E). Finally, we tested the effect of nivolumab on Sema4D-knockdown melanoma in mice. In vivo experiment, we transplanted B16-F10R cells in the mice and observed the size and weight of tumors treated with nivolumab, tumor volume and weight were significantly delayed in Sema4D-shRNA group mice as compared to Sema4D-NC group mice (Figs. 3F, 3G and 3H).

Sema4D repression potentiates the effect of anti-PD-1 on cell invasion and migration

The effect of Sema4D repression with anti-PD-1 treatment on B16-F10R cell invasion and migration was analyzed by Matrigel-coated invasion chambers and scratch assay. The results showed that when knockdown of Sema4D, cell migration of Sema4D-shRNA group was decreased (p < 0.0001), the cell-free area between migrating cells from Sema4D-shRNA group was wider than the gap between cells of the Sema4D-NC group (Figs. 4A and 4B). In addition, the number of invaded cells was significantly less in Sema4D-shRNA group in contrast to Sema4D-NC group (Figs. 4C and 4D), these data suggesting that lowering Sema4D could enhance the inhibitory effect of anti-PD-1 on cell migration and invasion.

Downregulation of Sema4D inhibit PI3K/AKT expression

The brown module was determined to be most significantly associated with Sema4D expression (r =  +  − 0.67, p-value = 1e−62), which contained a total of 778 genes (Figs. 5A–5B). Gene set variation analysis (GSVA) revealed several significantly upregulated malignancy-associated events (IL-6, JAK, STAT3, MTORC1, and KRAS signaling pathways) in the Sema4D high expression group as well as downregulation of p53 signaling (Fig. 5C). Sema4D was significantly associated with oncogenes RHOA, RRAS and immunomodulatory-related genes PTPRC and PLXNC1 (Fig. 5D). Pathway enrichment analysis revealed that Sema4D is involved in multiple immune-related pathways as well as the PI3K/AKT pathway (Fig. 5E). Numerous studies have demonstrated that excessive activation of PI3K/AKT signaling pathway is associated with excessive proliferation of tumor cells, inhibition of apoptosis and attenuation of the efficacy of immune checkpoint inhibitors (Zhang, Richmond & Yan, 2022). The expression of PI3K/AKT protein was explored by Western blot. p-PI3K/PI3K, p-AKT/AKT of Sema4D-shRNA group were decreased than those of Sema4D-NC group, which suggesting aberrant activation of the PI3K/AKT signaling pathway in the Sema4D-NC group (Fig. 5F).