Collagen type I (Col I) is one of the major extracellular matrix proteins in the cancer tissue. Previously, we have reported that Col I induces epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance by mTOR activation through Akt and ERK1/2 independent pathway. In this study, we aimed to elucidate the molecular mechanism of Col I induced EGFR-TKI resistance. First, we demonstrated the uptake of fluorescently labeled Col I by EGFR-mutated lung cancer cell line PC-9 cells using confocal microscopy and flow cytometry. Metabolome analysis revealed that the metabolic profiles of PC-9 cells was influenced by Col I treatment. Uptake of Col I into PC-9 cells was not inhibited by MMP inhibitor, GM6001, and endocytosis inhibitors, Pitstop2 and Dyngo4a; however, macropinocytosis inhibitor EIPA prevented its uptake. Moreover, the combination of EIPA and EGFR-TKI abrogated Col I-induced EGFR-TKI resistance in PC-9 cells. Inhibition of Racl, which is essential for micropinocytosis, also decreased the uptake of Col I in PC-9 cells and restored their sensitivity to EGFR-TKI. Thus, EGFR mutated lung cancer cells could develop EGFR-TKI resistance by Col I uptake by macropinocytosis route. (C) 2020 Elsevier Inc. All rights reserved.