Fig 2: VBMECs induced NSCLC cell adhesion through the CX3CL1/ICAM-1/LFA-1 pathway in the presence or absence of platelets. (A) Schematic illustration of the parallel-plate flow chamber system for adhesion assays. (B) Genetic inhibition of LFA-1 in A549 cells (shLFA-1). (C, D) Quantitative analysis of NSCLC cell adhesion to indicated VBMECs in the presence or absence of platelets. Data represent the mean ± SEM (n = 3). **P < 0.01, and ***P < 0.001. (E-G) Overexpression of ICAM-1 in VBMECs remarkably rescued the efficacy of CX3CL1 inhibition to decrease NSCLC cell adhesion to VBMECs. Data represent the mean ± SEM (n = 3). **P < 0.01, ***P < 0.001, and NSP > 0.05. (H-J) Overexpression of ICAM-1 in VBMECs significantly reversed the decrease in NSCLC cell adhesion to VBMECs induced by LFA-1-KD treatment. Data represent the mean ± SEM (n = 3). **P < 0.01, ***P < 0.001, and NSP > 0.05.

Fig 3: ICAM-1/LFA-1 expression is associated with CX3CL1-mediated NSCLC spinal metastases. (A, B) Quantitative IHC analysis of ICAM-1 protein expression in cancellous bone of vertebrae (vertebrae, n = 5), cancellous bone of lower femur (limb, n = 5), and normal lung tissue (lung, n = 5). Representative micrographs of these samples are shown. Data represent the mean ± SEM. *P < 0.05 and **P < 0.01. (C) ICAM-1 mRNA levels in vertebrae, limb, and lung tissues. Data represent the mean ± SEM. **P < 0.01. (D) Western blotting analysis of ICAM-1 protein levels in vertebrae, limb, and lung tissues. (E) RT-qPCR of the ICAM-1 mRNA expression levels in VBMECs and LBMECs. Data represent the mean ± SEM. *P < 0.05. (F) CX3CL1 protein levels in VBMECs and LBMECs detected by western blotting. (G) Genetic ablation of CX3CL1 (siCX3CL1) significantly decreased ICAM-1 expression in VBMECs indicated by western blotting. (H, I) Quantitative IHC analysis of LFA-1 protein expression in normal lung tissues (normal, n = 11), primary tumors (primary, n = 11), and spinal metastatic samples (SM, n = 11). Data represent the mean ± SEM. *P < 0.05 and **P < 0.01. (J) RT-qPCR of the LFA-1 mRNA expression levels in NSCLC spinal metastases, primary tumors and normal lung tissues. Data represent the mean ± SEM. **P < 0.01 and ***P < 0.001. (K) Western blot analysis of LFA-1 levels in NSCLC spinal metastases, primary tumors and normal lung tissues.

Fig 4: NECTIN1-deficient cells activate an integrin-dependent cell-to-matrix adhesion program.a, Volcano plot representing the significance and relative abundance of cell-surface proteins in A375 human melanoma cells in the presence or absence of NECTIN1. A P value (two-tailed t-test) threshold of 0.05 was chosen (red: P < 0.05, black: P > 0.05). Some of the most differentially expressed proteins are indicated. b–e, Adhesion of A375 human melanoma cells stably expressing an shRNA directed against NECTIN1 (shNECTIN1) relative to cells expressing a control shRNA (shCTRL) to collagen-I-, fibronectin-, laminin- or vitronectin-coated surfaces at various timepoints after seeding. Data represent mean ± s.d. of four independent experiments (paired two-tailed t-test). f, Western blot analysis of ITGB levels in the cells described in panel b transfected with a control siRNA (C) or siRNAs targeting ITGB1 (1), ITGB2 (2), ITGB3 (3), ITGB4 (4) or ITGB5 (5). Data are representative of four independent experiments. g, Migration of the cells described in panel f relative to cells stably expressing a control shRNA (shCTRL) and transfected with a control siRNA in a transwell assay after 12 h of serum starvation. Cells were allowed to migrate for 6 h. Data represent mean ± s.d. of four independent experiments (paired two-tailed t-test; P values are shown for the comparisons siITGB versus siCTRL (shNECTIN1)). h, Migration of A375 human melanoma cells stably expressing an shRNA directed against NECTIN1 (shNECTIN1) relative to cells expressing a control shRNA (shCTRL) in a transwell assay upon treatment with an integrin α6β4 blocking antibody (ITG Ab, GoH3, 40 μg ml−1). Cells were allowed to migrate for 6 h. Data represent mean ± s.d. of four independent experiments (paired two-tailed t-test). IgG, immunoglobulin G. i, Migration of A375 human melanoma cells stably expressing an shRNA directed against NECTIN1 (shNECTIN1) relative to cells expressing a control shRNA (shCTRL) in a transwell assay upon treatment with two integrin αvβ3 and αvβ5 inhibitors (ITGi#1: SB273005; ITGi#2: echistatin). Cells were allowed to migrate for 6 h. Data represent mean ± s.d. of four independent experiments (paired two-tailed t-test).Source data