Extracellular vesicles shed by multidrug resistant cells contribute to the identification of SRC inhibitors as chemosensitizers in non-small cell lung cancer

Abstract

Aim: Non-small cell lung cancer (NSCLC) represents most lung cancer cases and remains associated with poor outcomes, mainly due to multidrug resistance (MDR). Extracellular vesicles (EVs) are crucial for intercellular communication and significantly influence chemotherapy resistance. This study aimed to characterize the EVs proteome of drug-sensitive and MDR NSCLC cell lines to identify therapeutic targets to counteract MDR.

Methods: EVs derived from NSCLC cells were isolated by ultracentrifugation and analyzed for size by nanoparticle tracking analysis, for morphology by transmission electron microscopy, and for EVs markers by Western blotting (WB). Proteomic profiling was performed using liquid chromatography-mass spectrometry (LC-MS), followed by WB validation of relevant proteins. Cell growth and viability were assessed using sulforhodamine B or CellTiter-Glo assays. P-glycoprotein [P-gp, also known as ABCB1: adenosine triphosphate (ATP)-binding cassette subfamily B member 1] activity was determined by rhodamine-123 accumulation assay. SRC-related signaling was investigated by WB.

Results: EVs from the multidrug resistant (MDR) derivative of NCI-H460, a human NSCLC cell line, displayed nine up- and eight down-regulated proteins compared with the drug-sensitive parental cells, including reduced SRC. WB results showed higher phosphorylated form of SRC (p-SRC) expression in MDR cells than in sensitive cells. In contrast, EVs from both cell lines had similar expression levels, suggesting selective intracellular retention in MDR cells. The SRC inhibitor bosutinib potentiated the activity of chemotherapeutics that are P-gp substrates in 2D and in 3D spheroids, without affecting the viability of the human lung fibroblast cell line MRC-5. Moreover, bosutinib reduced P-gp activity, likely by downregulating of phosphorylated caveolin-1.

Conclusion: These findings show reduced selective packaging of p-SRC into EVs shed by MDR cells (MDR-EVs), suggesting an important role for this protein in the MDR phenotype and its potential as a molecular target. Bosutinib, an SRC inhibitor, might be useful as a chemosensitizer of MDR cells.

Keywords

Multidrug resistance / non-small cell lung cancer / extracellular vesicles / SRC inhibitors / chemotherapy

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