Fig 2: The impact of ELF4/IL1RN axis on macrophages and mouse intestinal epithelial cells. (A) The mRNA levels of ELF4 and IL1RN in each group of BMDM cells were detected by RT-qPCR; (B) The protein levels of ELF4 and IL1RN in each group of BMDM cells were detected by Western blot; (C) The polarization of M1 and M2 in each group of BMDM cells was detected by flow cytometry; (D) The protein levels of M1 marker iNOS and M2 markers Arg1 and CD163 in each group of BMDM cells were detected by Western blot; (E) The apoptosis of each group of MODE-K cells was detected by flow cytometry (The sum of the blue quadrant areas represents the apoptotic rate); (F) The protein levels of apoptosis factors Bcl-2, Bax, cleaved caspase-1, pro caspase-1,caspase 3 and Cytochrome c in each group of MODE-K cells were detected by Western blot; (G) The levels of inflammatory factors Treml, IL-6, TNF-α, and IL-1β in each group of MODE-K cells were detected by ELISA. * indicates P < 0.05 compared with oe-NC + sh-NC, and # indicates P < 0.05 compared with oe-ELF4 + sh-NC. All cell experiments were repeated three times.

Fig 3: TKI treatment promotes T-cell infiltration by inhibiting LAMC2 protein expression. (A) Density (cells/mm2) of CD8+, CD8+GB+, CD68+, and CD68+CD163+ cells in tumors and stroma, quantified by multiplex immunofluorescence in paired pretreatment tumor samples and resected tumors. (B) PC-9 cells and H3122 cells were injected into Hu-HSC mice (n=3 mice per group) on day 0, and osimertinib or crizotinib was administered as indicated. (C) Tumor growth curves of cell-derived xenograft mouse models treated as indicated. Tumor volume is shown as the mean±SEM (n=3). The tumor weight (g) of each mouse is shown. (D) Immunohistochemistry analysis of CD8 expression in tumor sections from the different groups. Representative images are shown. (E) LAMC2 expression levels in the indicated cells, analyzed by western blotting. (F) ELISA analysis of LAMC2 levels in PC-9 cells and H3122 cells following treatment for 24 hours. (G) Schematic diagram of a T-cell chemotaxis assay directly regulated by the addition of medium from PC-9 and H3122 cells. (H) Representative images of infiltrating T cells stained with CFSE dye. (I) Immunofluorescence staining with antibodies against CD8 and LAMC2 in tissues harboring EGFR mutations and ALK fusions. (J) LAMC2 expression levels in cancer cells treated with different concentrations of TKIs for 12 hours, analyzed by western blotting. (K) Representative images of infiltrating T cells stained with CFSE dye, with TKI inhibition directly regulated by the recombinant protein Ln-γ2. (L) Immunofluorescence staining with antibodies against CD8 and LAMC2 in non-small cell lung cancer tissues harboring EGFR mutations and ALK fusions following treatment with TKIs. ALK, anaplastic lymphoma kinase; CFSE, carboxyfluorescein diacetate succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LAMC2, laminin subunit γ−2; PBS, phosphate-buffered saline; TKI, tyrosine kinase inhibitor.

Fig 4: TKI therapy alters the tumor immune microenvironment. (A) Representative mIF images of pretreatment tumor and resected samples analyzed for immune-related biomarkers. (B) Densities (cells/mm2) of CD8+, CD8+GB+, CD8+PD-1+, CD163+, CD68+, and CD163+CD68+ by mIF quantification in paired pretreatment tumor samples and resected tumors. (C) Cell viability CCK-8 assay for cells treated with TKIs (osimertinib: 10 nM, lorlatinib: 10 nM), activated T cells (1:1 ratio to cancer cells), or the combination. (D) T cell-mediated cancer cell-killing assay. PC-9 and H3122 cells co-cultured with activated T cells for 48 hours with or without TKIs (osimertinib: 10 nM, lorlatinib: 10 nM) were subjected to crystal violet staining. Ratio of cancer cells to T cells: 1:1. (E) Ki67 incorporation assay on PC-9 and H3122 cells treated as indicated. Activated T cells (1:1 ratio to cancer cells) or TKIs (osimertinib: 10 nM, lorlatinib: 10 nM) were added to the culture medium for 48 hours. Cells were then counterstained with DAPI. (F) PC-9 cells were injected into mice (n=3 mice per group) on day 0, hu-PBMCs were injected into mice via the tail vein on day 7, and osimertinib was administered as indicated. (G) Macroscopic appearance of tumors after drug application for 4 weeks. (H) The tumor weight (g) for each mouse is shown. *p<0.05, **p<0.01. ns, no significance. (I) Immunofluorescence staining with an antibody against CD8 to detect T cells and antibodies against CD68 and CD206 to detect macrophages in TKI-resistant non-small cell lung cancer tissues (11 cases of EGFR-TKI resistance, 5 cases of ALK-TKI resistance). Scale bar: 50 µm. ALK, anaplastic lymphoma kinase; DAPI, 4′,6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; ALKi,anaplastic lymphoma kinase inhibitor; EGFRi,epidermal growth factor receptor inhibitor; hu-PBMC,human-Peripheral blood mononuclear cell; mIF, multiplex immunofluorescence; PD-1, programmed cell death protein 1; TKI, tyrosine kinase inhibitor.

Fig 5: G-CSF polarizes macrophages to an anti-inflammatory phenotype to promote tissue regeneration(A) Dot plot demonstrating average expression and percentage of immune cells expressing Csf3r.(B) Analysis of key cell-to-cell interactions between immune cells in the ear skin of WT (salmon color) and CXCR2 KO (blue color) mice. Mac, macrophage; T, T cell.(C) Dot plot demonstrating average gene expression of key genes between WT and CXCR2 KO macrophages.(D) GSEA of macrophage populations in WT and CXCR2 KO wounded skin.(E) Representative images and quantification of immunofluorescence of WT and CXCR2 KO wounded skin for CD80 (n = 4), COX2 (n = 4), CD163 (n = 12 for WT and n = 11 for KO), MRC1 (n = 10 for WT and n = 6 for KO), and ARG1 (n = 10 for WT and n = 6 for KO). Cell percentages are calculated with total DAPI+ cells as the denominator. Scale bars, 100 μM. Unpaired two-tailed Student’s t test.(F) Representative H&E immunostaining images and quantification of PBS- and G-CSF-injected stented back wounds of WT mice for CD163 (n = 7 for PBS and n = 5 for G-CSF), MRC1 (n = 5 for PBS and n = 7 for G-CSF), CD31 (n = 8 for PBS and n = 7 for G-CSF) and Ki67 (n = 5 for PBS and n = 4 for G-CSF). Cell percentages are calculated with total DAPI+ cells as the denominator. Scale bars, 100 μM. Unpaired two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.01. Mean ± SEM are plotted.