The RNAseq data for lung adenocarcinoma (LUAD) and Lung squamous carcinoma (LUSC) were obtained from the Cancer Genome Atlas database (TCGA, https://portal.gdc.cancer.gov/). We extracted the data of SEMA7A expression and then drew a picture to describe the expression pattern of SEMA7A in LUAD and LUSC using R package. We used the tissue samples from the Human Protein Atlas (HPA) database to assess the local expression pattern of SEMA7A in NSCLC. CIBERSORT (a computational tool that uses gene expression data to estimate the abundance of cell types in a mixed cell population) was used to analyze immune cell infiltration in NSCLC from the TCGA databases.

The mouse NSCLC cell line and normal lung epithelial cell line MLE-12, as well as human NSCLC cell line H1299 and normal lung epithelial cell line BEAS-2B were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Murine macrophage cell line RAW264.7 was purchased from Fenghui (CL0266; Fenghui Biotechnology, Hunan, China). Cells were cultured routinely in Dulbecco’s modified Eagle’s medium (DMEM, Pricella, Wuhan, China), supplemented with 10% fetal bovine serum (FBS, Pricella, Wuhan, China) and 1% penicillin/streptomycin (Gibco) at 37°C with 5% CO₂. For coculture experiments, 5×10⁵ of shSEMA7A-transfected cells were seeded in a 6-well plate, followed 5×10⁵ of RAW264.7 cells were seeded onto the membranes of the top chambers of a 0.4-mm pore Transwell chamber (Corning). After incubation for 48 hours, the macrophages were collected for further studies.

To silence the expression of SEMA7A in LLC and H1299 cells, specific small interfering RNA (siRNA) targeting mouse and human SEMA7A were designed and synthesized by Sangon Biotech Technologies, Shanghai, China ( Supplementary Table 1 ). A total of 3×10⁵ cells were seeded into each well of 6-well plates. After culturing for 24 h, siRNA or negative control (NC) siRNA were transfected into the cells with Lipofectamine 3000 transfection reagent (L3000001; Thermo Scientific, USA) according to the manufacturer’s protocol. Purified lentiviral vectors with puromycin resistance (PLV-puro) for silencing SEMA7A were provided by Sangon Biotech Technologies (PLVE3866, Shanghai, China). After transfection, the stable cells with a silenced target gene were obtained using puromycin selection. The sequences of the designed oligonucleotides against SEMA7A and NC were listed in the Supplementary Table 1 .

The Cell-Counting Kit-8 (CCK-8) method was used to analyze the effect of knockdown of SEMA7A on tumor cell proliferation. The migration ability of the NSCLC cells was evaluated via a scratch wound-healing assay after disturbing the expression of SEMA7A in the cells. The role of SEMA7A in cell invasion was detected via Transwell 24-well plates (8 μm wells; Corning).

Specific-pathogen-free (SPF) male C57BL/6J mice (6–8 weeks of age) were purchased from Huafukang Biotechnology (Beijing, China), and maintained with fresh water and autoclaved food at the Laboratory Animal Center, China Medical University. To establish a mouse model of NSCLC, Approximately 1×10⁶ LLC cells or shSEMA7A-LLC cells in 100μl of PBS were implanted subcutaneously into the right axillary region of the mice. Tumor volumes were measured every 2 day, and calculated using the formula: tumor volume=W²×L/2, where L represents the longest diameter and W represents the shortest diameter of the tumor. All animal experiments were approved and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of China Medical University (Approval No. 20240684).

Total RNA was extracted using the TRizol reagents (Vazyme, China) and was converted to cDNA using the Prime Script RT Reagent Kit (RR047A; TaKaRa, Japan) according to the manufacturer’s instructions. Then, PCR was run in a LightCycler^® 480 (Roche Molecular Biochemicals) under identical amplification conditions. The results were normalized to β-actin expression and presented as fold change (fold change = 2^−ΔΔCT). The primer sequences were listed in the Supplementary Table 2 .

Tumor mass were collected on day 21 after LLC implantation, and digested with 2 mg/mL of collagenase D (Sigma) and 100 ng/mL of DNase I (Roche) at 37°C for 1 h. After passing the digested tumor tissue through a 70mm cell strainer, the red blood cells in the single-cell suspension were lysed with 0.15 M ammonium chloride and 1 mM potassium bicarbonate. Then, the cells were washed twice with PBS and were resuspended in RPMI 1640 medium.

The single-cell suspension from fresh tumor tissues was incubated firstly with anti-mouse CD16/32 mAbs (BD Bioscience) for blocking the Fc receptor, and stained with anti-mouse fluorescein-conjugated antibodies against surface antigens for 30 minutes on ice. The antibodies used are shown in the Supplementary Table 3 .

The single-cell suspension from tumor tissues obtained by 30-70% percoll gradient centrifugation. Single-cell suspension was centrifuged at 300g for 10 min at RT. Cell pellets were resuspended in a 30% percoll gradient in a 15 ml polypropylene tube. Take a new 15ml centrifuge tube and add 5ml of 70% percoll separation solution. Use a pipette to slowly add the 30% percoll separation solution containing cells from the previous step into the 70% percoll solution. Gradients were centrifuged without brake for at 500g 20 min at 4°C. After centrifugation, cells were collected from the middle layer (5 ml interphase) in the 30–70% percoll gradient. Then, magnetically macrophages were isolated by using F4/80 labelled beads according to the manufacturer’s protocols (130-110-443, Miltenyi Biotec, Nordrhein-Westfalen, Germany). The sorted macrophages were seed into 6-well plates and stimulated with 100 ng/mL of recombinant SEMA7A protein (HEK293; MCE, USA) in the presence or absence of etomoxir (200 μmol/L) (HY-50202; MCE, USA) for 24 h. After incubation, the macrophages were collected for further analysis.

The sorted tumor-associated macrophages and RAW264.7 cells were denatured in RIPA buffer (Beyotime, Shanghai, China). Proteins were separated via SDS-PAGE and were transferred into polyvinylidene fluoride membranes (Millipore, Shanghai, China). After blocked with 5% nonfat milk at room temperature for 2 h, the membranes were incubated with primary antibodies such as Rabbit anti-SEMA7A (1:500; bs-2702R, Bioss), Rabbit anti-ERK1/2 (1:1,000; YT1625, Immunoway) and Rabbit anti-p-ERK1/2 (1:1,000; YP0101, Immunoway) at 4°C overnight. Then, GAPDH were used as loading controls for different protein analysis. The immunocomplexes were visualized with the BeyoECL Plus Chemiluminescent Substrate. Protein band intensity was quantified with Image J software.

The levels of IL-1β, TNF-α, IL-10 and TGF-β in the tumor tissues were measured via the ELISA kits (Elabscience) according to the manufacturer’s instructions. The absorbance at 450 nm was measured by a microplate reader.

The sorted macrophages were seeded in 6-well plates (1×10⁵ cells/well) and treated with or without recombinant SEMA7A protein (100 ng/mL) and etomoxir (200 μmol/L) for 48 h. After incubation, the cells were changed to unbuffered assay media, and were incubated in a non-CO2 incubator at 37 °C for 1 h. Oxygen consumption rates (OCR) were measured via an O2k-FluoRespirometer after the sequential addition of oligomycin (1 mmol/L, Sigma), trifluoromethoxy carbonylcyanide phenylhydrazone (1.5 mmol/L, Sigma), and antimycin/rotenone (2 mmol/L, Sigma).

Macrophages were sorted from the tumor tissues of the tested mice, and lysed in the extracting solution. The cell lysate was centrifuged at 8000 rpm for 10 minutes before the supernatant was collected. A FFA detection kit (boxbio, China) was used to detect the levels of free fatty acids. The absorbance at 550 nm was measured by a microplate reader.

All experiments have been repeated at least three times. Values were presented as mean ± standard deviations (SD). One-way analysis of variance (ANOVA) was used to compare differences between three or more groups. Student’s t-test was applied to analyze the differences between the two groups. p-values < 0.05 were considered statistically significant. The data were analyzed using GraphPad Prism8 software.

To evaluate SEMA7A expression in the NSCLC, we examined the expression of SEMA7A in human tumor tissues and non-carcinoma adjacent tissues, using the TCGA databases. The results showed that the expression levels of SEMA7A were significantly elevated in LUAD and LUSC tumor tissues compared with normal tissues ( Figure 1A ). Log-rank survival analysis revealed that NSCLC patients with higher SEMA7A expression had poorer overall survival (OS) and progression free survival (PFS) rates than those with lower SEMA7A expression ( Figures 1B, C ). In order to confirm the higher expression of SEMA7A in NSCLC at the tissue protein level compared to normal breast tissue, we used immunohistochemical results from the HPA database. The results showed that the protein expression level in NSCLC tissue was significantly higher than in normal tissue. Typical microphotographs of IHC are shown in Figure 1D . In addition, a progressively increased expressions of SEMA7A mRNA were detected in the tumor tissues of Lewis lung carcinoma (LLC) tumor-bearing mice ( Figure 1E ). Meanwhile, an elevated SEMA7A protein levels were detected in the tumor tissues on day 21 after tumor implantation ( Figure 1F ). These results suggest that SEMA7A might be related to the progression and outcomes of NSCLC.

To confirm whether SEMA7A plays a key role in NSCLC tumor progression, siRNAs were used to silence the SEMA7A gene in the mouse NSCLC cell line LLC and the human NSCLC cell line H1299 ( Supplementary Figures 1A, B ), which have relatively high SEMA7A expression at both the mRNA and protein levels ( Figure 2A ). The results showed that SEMA7A deficiency may attenuate the proliferation but enhance the apoptotic capabilities of LLC and H1299 cells ( Figures 2B, C ). Meanwhile, the invasion and migration activities of these tumor cells were markedly reduced after silencing the SEMA7A gene ( Figures 2D, E ). Moreover, knockdown of SEMA7A in LLC cells resulted in a diminished tumor growth characterized by reduced tumor weight and tumor volume, as well as prolonged survival of the tumor-bearing mice ( Figures 2F–H ). IHC staining also revealed a significant reduction in the proliferative activities of tumor cells in the tumor tissues obtained from the mice implanted with SEMA7A-knockdown-LLC ( Figure 2I ). These experimental results suggest that SEMA7A may contribute to the progression of NSCLC.

By using bioinformatics analysis, we found the levels of SEMA7A might influence the cell composition in the TME of NSCLC ( Figure 3A ). In fact, a significant increase in the percentages and absolute numbers of TAMs, identified as CD11b⁺F4/80⁺ cells, as well as M1-like macrophages, identified as CD11b⁺F4/80⁺CD86⁺ cells, were observed in the TME of tumor-bearing mice inoculated with SEMA7A-knockdown LLC cells ( Figures 3B, C ), but the percentages and absolute numbers of M2-like macrophages, identified as CD11b⁺F4/80⁺CD206⁺ cells were significantly decreased ( Figure 3C ), resulting in an increase in the M1/M2 ratio ( Figure 3D ). In addition, elevated expressions of antitumor factors such as IFN-γ and TNF-a, as well as decreased expression of tumor promoting factors such as IL-10 and TGF-β were detected in the tumor tissues of SEMA7A-knockdown LLC tumor-bearing mice ( Figure 3E ). However, it should be noted that knockdown of SEMA7A did not influence the percentages and absolute numbers of CD4⁺T, CD8⁺T and Treg cells in the TME of tumor-bearing mice ( Supplementary Figures 2A, B ), suggesting that macrophages might be the main target cells of SEMA7A.

To further confirm the effects of SEMA7A on macrophage polarization, coculture of RAW264.7 cells and LLC cells in vitro were performed. As shown in Figure 3F , the proportion of CD206⁺ macrophages were decreased, whereas the proportion of CD86⁺ macrophages were increased in RAW264.7 cells that were cocultured with SEMA7A-knockdown-LLC cells, compared with that cocultured with the normal LLC cells ( Figure 3F ). Moreover, the mRNA levels of M1 factors, such as iNOS and TNF-α, were remarkably upregulated, whereas the expression levels of M2 factors, such as Arg-1 and IL-10, were downregulated in RAW264.7 cells cocultured with SEMA7A-knockdown-LLC cells ( Figure 3G ). These results suggest that SEMA7A may promote the polarization of TAMs toward M2 phenotype.

Since SEMA7A exerts its immune function via the receptor integrin β1 (ITGB1) (12) and macrophages express relatively high levels of ITGB1 (26), it is possible that SEMA7A may promotes TAM polarization through the ITGB1 receptor. We found that the coculture of ITGB1-silenced RAW264.7 cells with LLC cells may result in a decrease in the proportion of CD206⁺ macrophages but an increase in the proportion of CD86⁺ macrophages ( Figure 4A ). Furthermore, reduced expressions of mRNAs for CD206, Arg-1 and IL-10 were detected in the ITGB1-silenced RAW264.7 cells ( Figure 4B ), suggesting that SEMA7A may promote M2 polarization in an ITGB1-dependent manner.

Increasing evidence highlights the crucial role of metabolic reprogramming in macrophage activation (29). Therefore, it is necessary to clarify whether SEMA7A-associated M2 polarization may be related to the metabolic reprogramming in the macrophages. Then, we analyzed the expression of genes involved in lipid and glucose metabolism in TAMs sorted from the tumor tissues of the mice on day 21 after SEMA7A-silenced LLC or control LLC implantation. The results showed that most of the genes involved in fatty acid oxidation (FAO) were downregulated in the TAMs sorted from the SEMA7A-silenced LLC-bearing mice compared with those sorted from the control LLC tumor-bearing mice ( Figures 5A, B ). Notably, no significant differences in the genes related to glucose metabolism were detected in these TAMs ( Supplementary Figures 3A–C ), suggesting that fatty acid oxidation (FAO) might be an important pathway for SEMA7A-mediated M2 polarization. In fact, a decreased lipid accumulation ( Figure 5C ) and a lower concentration of free fatty acid (FFA) ( Figure 5D ) were observed in the TAMs isolated from the tumor tissues of SEMA7A-silenced LLC-bearing mice. In addition, the quantity of mitochondria in these TAMs was limited ( Figure 5E ), followed by a decrease in the mitochondrial oxygen consumption rate (OCR) ( Figure 5F ). Blockade of FAO by treatment of the sorted TAMs with etomoxir, an inhibitor of a rate-limiting enzyme of mitochondrial fatty acid oxidation-CPT1A, may restore the reduced OCR caused by knockdown of SEMA7A in the TAMs ( Figure 5G ). Moreover, blocking FAO may significantly decrease the expression levels of mRNAs for M2 factors such as Arg-1 and IL-10 but increase the levels of mRNAs for M1 factor iNOS ( Figure 5H ). In addition, the percentage of M2 macrophages was decreased but the proportion of M1 macrophages was increased, resulting in an elevated M1/M2 ratio after treatment of SEMA7A protein-stimulated TAMs with etomoxir ( Figure 5I ). Together, these results indicate that FAO may be essential for SEMA7A induced M2 polarization in the TME of tumor-bearing mice.

By performing GSEA, we found that the ERK1 and ERK2 signaling pathway might be related to the expression levels of SEMA7A ( Figure 6A ). In fact, the protein levels of phosphorylated ERK1/2 were significantly lower in the TAMs sorted from SEMA7A-silenced LLC-bearing mice than in those sorted from control LLC-bearing mice ( Figure 6B ). In vitro coculture experiments further confirmed that knockdown of SEMA7A may reduce the LLC-induced increase in phosphorylated ERK1/2 proteins in the cocultured RAW264.7 cells ( Figure 6B ). Blockade of the ERK1/2 pathway with the inhibitor PD98059 may decrease the expression levels of mRNAs for FAO-related genes in the RAW264.7 cells cocultured with LLC cells ( Figure 6C ). Besides, the accumulation of lipid droplets and the mitochondrial OCR in the cocultured RAW264.7 cells were also reduced after blocking the ERK1/2 pathway in the cells ( Figures 6D, E ). Notably, treatment of the cocultured RAW264.7 cells with the inhibitor PD98059 may decrease the percentages of CD206⁺ macrophages and the expression levels of mRNAs for M2 markers, such as Arg-1 and IL-10, but increase the percentages of CD86⁺ macrophages and the expression levels of mRNAs for M1 markers, such as INOS, in the cocultured RAW264.7 cells ( Figures 6F, G ). Taken together, these results suggest that SEMA7A may regulate fatty acid oxidation in the macrophages via the ERK1/2 pathway.