Fig 1: Pericyte‐IL32 expression is associated with cancer progression in NSCLC patients harboring EGFR mutations. A) Visualizing a heatmap comparing secretome analysis between EGFR mutant cancer cell line HCC827 and PCs derived from 3 different NSCLC patients harboring EGFR mutations. Highlighting profound alterations in cytokine secretion in PCs in comparison to cancer cells. B,C) KEGG and GO biological process analyses of the secretomics data in (A). The pathways related to cell‐cell communications and EGFR TKI resistance are highlighted in red. D) Representative images of proteome profiler cytokine XL array from the conditioned medium (CM) of PC and HCC827 cells are given. The expression of IL‐22, IL32, CCL5, IL17A, and CXC12 was up‐regulated in the CM of PCs as compared to that of HCC827 cells. E) Heatmap showing the RT‐PCR analysis of the relative expression of indicated cytokines in PCs as compared to HCC827 or PC9 cells. F) Western blot analysis was performed to evaluate IL32 expression in cancer cells and PCs derived from tumors of three EGFR‐mutated NSCLC patients. The quantification of the Western blot results is provided below the blots. The data are normalized to the control group. G) ELISA measurements were conducted to assess IL32 expression in the CM obtained from normal fibroblast (NF), human umbilical vein endothelial cell (EC), cancer cell lines (HCC827/PC9/NCI‐H1975), and pericyte (PC). H–J) Single‐cell RNA sequencing analysis of the published dataset (GSE171145) derived from NSCLC patients harboring EGFR mutations. UMAP visualization map of all cell type clusters in tumors derived from EGFR mutant NSCLC patients. Each color represents the annotation phenotype of a single cluster (H). The circle plot shows the communication strength between interacting cells. The thicker the line represented, the higher the number of interactions (I). KEGG pathway enrichment analysis in IL32+Pericytes (J). K) The bar chart illustrates the percentage of pericyte‐IL32 positive blood vessels for each patient. L) Representative triple immunostaining images of IL32, α‐SMA, and CD34 on tumor sections from NSCLC patients with EGFR mutations, showcasing low or high pericyte‐IL32 expression, are provided. M) A high percentage of pericyte‐IL32 positive blood vessels correlated with poor progression‐free survival in EGFR‐mutated NSCLC patients (n = 20 patients, our cohort). N) ELISA IL32 assays of serum samples from EGFR‐mutated cancer patients responsive or non‐responsive to third‐generation TKI treatment (n = 20 patients, our cohort). Violin plots represent mean ± S.E.M are given. NS: non‐significant difference. *p < 0.05, ***p < 0.001. (G) One‐way ANOVA. (M) Log‐rank (Mantel‐Cox) test. (N) Student's t‐test. Scale bars in (L) represent 100 µm.
Fig 2: Disrupting IL32‐β5‐integrin paracrine signaling prevents pericyte‐mediated TKI tolerance in EGFR mutated cancer cells. A) Western blot and RT‐PCR analysis of the expression of IL32 in PCs after transfected with IL32 targeting siRNA‐1/‐2 or non‐silencing siRNA (siNSC). The quantification of the Western blot results is provided below the blots. The data are normalized to the control group. B) Almonertinib/Osimertinib IC50 experiments of HCC827 cells treated with conditioned medium (CM) from PCs transfected with either siNSC or IL32 targeting siRNA‐1/‐2. C) Representative fluorescent images of GFP fluorescently labeled HCC827 cells co‐cultured with RFP fluorescently labeled siNSC or siIL32‐1/‐2 transfected PCs in the presence of Almonertinib/Osimertinib. Bar charts show the relative number of GFP or RFP‐positive cells in each group. D,E) Colony formation assays of HCC827 cells after being treated with CM from PCs transfected with either siNSC or IL32 targeting siRNA‐1/‐2 in the presence/absence of Almonertinib/Osimertinib. Bar charts show the relative colony number in each group. F,G) Transwell invasion assays of HCC827 cells after being treated with CM from PCs transfected with either non‐silencing control (siNSC) (PC‐siNSC‐CM) or IL32 targeting siRNA‐1/‐2 (PC‐siIL32‐1/‐2‐CM) in the presence/absence of Almonertinib/Osimertinib. Bar charts show the relative number of invaded cells in each group. H) Almonertinib/Osimertinib IC50 experiments of HCC827 cells after being treated with CM from PCs together with or without Cilengitide. I) Western blot and RT‐PCR analysis of the expression of β5‐integrin in each group. J) Almonertinib/Osimertinib IC50 experiments of HCC827 cells stably transfected with scramble (HCC827‐scr) or β5‐integrin targeting shRNA (HCC827‐shβ5‐integrin) treated with CM from PCs. K) Colony formation assays of HCC827 cells after being treated with CM from HCC827 or PCs in the presence of Almonertinib/Osimertinib together with or without Cilengitide. Representative images of crystal violet‐stained colonies in each group are given. Bar charts show the relative colony number in each group. L) Transwell invasion assays of HCC827 cells after being treated with CM from HCC827 or PCs in the presence of Almonertinib/Osimertinib together with or without Cilengitide. Bar charts represent the relative number of invaded cells in each group. NS: non‐significant difference. ** p < 0.01, ***p < 0.001. A,C–G,K,L) One‐way ANOVA. I) Student's t‐test. Scale bars in (C) represent 200 µm. F,G,L) 100 µm.
Fig 3: Pericyte‐secreted IL32 regulates β5‐integrin‐Src‐Akt pathway in EGFR mutated cancer cells, influencing TKI sensitivity. A) The volcano plot depicts the proteomics analysis of HCC827 cells following treatment with CM from HCC827 cells or PCs in the presence of Osimertinib. B,C) KEGG and GO pathway analyses of the proteomics data in (A). Pathways related to cell signaling, cell‐cell communications, and drug response are highlighted in red. D) Western blot analysis of the indicated proteins in HCC827 cells after being treated with CM from HCC827 or PCs in the presence or absence of Almonertinib/Osimertinib. The quantification of the Western blot results is provided below the blots. E) Western blot analysis of the indicated proteins in HCC827 cells after exposure with CM from PCs transfected with siNSC or siIL32 in the presence or absence of Almonertinib/Osimertinib. F,G) Western blot analysis was conducted to assess the indicated proteins in HCC827 cells following treatment with or without Almonertinib/Osimertinib, in combination with or without the β5‐integrin inhibitor Cilengitide. H) Western blot analysis was performed on the indicated proteins in HCC827 cells transfected with β5‐integrin‐targeting shRNA (shβ5‐integrin) or scramble shRNA (scr) after being treated with CM from PCs in the presence of Almonertinib/Osimertinib. I) Representative immunohistochemical staining images of p‐Src and p‐Akt in tumor sections from each group are shown. Violin plot shows the staining intensity of p‐Src/p‐Akt in each group (n = 20 patients, our cohort). J) Correlation study between tumor‐p‐Src/p‐Akt and pericyte‐IL32 levels in our EGFR‐mutated NSCLC patient cohort (n = 20 patients, our cohort). I) Student's t‐test. J) Pearson correlation coefficient. Scale bars in (I) represent 100 µm.
Fig 4: qRT‐PCR expression profile of the most intercorrelated genes found in microarray analysis. (A) IL32, (B) UBD, (C) CXCL9, and (D) CXCL10 were significantly up‐regulated in patients with NAFLD, whereas (E) IGFBP2 and (F) HPRT1 were significantly down‐regulated compared to controls. Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 5: IL32 protein expression in liver biopsies from Cont and Ob_NASH patients and effect of recombinant IL32 on insulin signaling in PHHs. (A) Immunoblots of protein extracts from patients with NASH and control liver biopsies showing an increase in IL32 expression in NASH. (B) Recombinant IL32 treatment induces decreased insulin signaling in protein lysates from PHHs as demonstrated by the robust decrease in AKTT308 phosphorylation after insulin stimulation. Abbreviations: Cont, controls; NT, nontreated; *, non specific.
Supplier Page from Abcam for Anti-IL-32 antibody