Objective To investigate the mechanism of C-C motif chemokine ligand 5(CCL5)modulation by extracellular matrix stiffness in non-small cell lung cancer(NSCLC)and to determine the effect of CCL5 on the immunotherapy response of NSCLC.Methods The correlation between extracellular matrix stiffness and CCL5 expression in NSCLC was analyzed with the TCGA database.Polyacrylamide hydrogels with different stiffness were designed according to the extracellular matrix stiffness of NSCLC,and H1299 cells responding to the mechanical loading of hydrogel matrix stiffness were subjected to transcriptome sequencing.High matrix stiffness was verified to promote the expression of CCL5 by using sequence.Results High extracellular matrix stiffness upregulated CCL5 expression,and interferon-γ mediated signaling pathway might be involved in the process.NSCLC patients with high CCL5 expression had a greater abundance of cytotoxic T-cells in tumor tissue and reacted favorably to anti-programmed cell death protein 1 treatment.Conclusion Increased extracellular matrix stiffness promotes CCL5 synthesis,and CCL5 enhances immunotherapy responsiveness in NSCLC by increasing cytotoxic T cell infiltration in tumor tissue.