# Profiling of Extracellular Vesicles of Non‐Small Cell Lung Cancer Reveals Proteins Associated With Osimertinib Resistance

## TL;DR

Researchers identified proteins in extracellular vesicles linked to resistance to the lung cancer drug osimertinib, suggesting potential biomarkers for treatment response.

## Contribution

The study identifies a novel set of EV-associated proteins that may predict osimertinib resistance in non-small cell lung cancer.

## Key findings

- Proteins like CSPG4, HSPG2, and TNC were found in EVs associated with osimertinib resistance.
- PEA profiling revealed immune-related proteins like PD-L1 and CD73 linked to treatment response and survival.
- CSPG4 suppression reduced NSCLC cell viability, suggesting its role in drug resistance.

## Abstract

Precision cancer medicine with small tyrosine kinase inhibitors (TKIs) directed toward oncogenic drivers, are important treatment regimens for solid tumours. The epidermal growth factor receptor (EGFR)‐TKI osimertinib is a preferred therapy for patients with non‐small cell lung cancer (NSCLC) driven by activating mutations in EGFR, unfortunately responses are heterogeneous. This calls for non‐invasive methods to predict or monitor treatment response, for example, via biomarker analyses in blood. To reveal such putative biomarkers, we analysed the proteome of extracellular vesicles (EVs) from osimertinib resistant or responsive NSCLC cells in vitro and from EVs isolated from serum samples of NSCLC patients treated with osimertinib in second line within the phase II clinical trial TREM. The protein cargo of the EVs was analysed by mass spectrometry (MS) and proximity extension assay (PEA). Western blotting, ELISA and single vesicle analysis was performed to validate and further confirm the expression of certain proteins. MS profiling of the NSCLC cells and their released EVs revealed a protein signature associated with osimertinib refractoriness. Among them were CSPG4, HSPG2, MCAM, L1CAM, TAGLN, THBS1 and TNC. GO‐pathway analysis related several of these proteins to the focal adhesion and proteoglycan in cancer pathways. Some of these proteins, including CSPG4, which when suppressed by transient siRNA transfection in NSCLC cells resulted in reduced cell viability, were expressed also in EVs from serum of the NSCLC patients. Moreover, PEA profiling of the serum‐isolated EVs revealed signatures associated with immune cells, best response and/or progression‐free survival, including PD‐L1, CD73/NT5E, FR‐alpha/FOLR1, LAMP3, FASLG1 and ANXA1. In summary, we demonstrate that protein profiling of EVs in relation to osimertinib refractoriness has the potential to identify possible biomarkers that can indicate osimertinib treatment resistance, for example, CSPG4, HSPG2, TAGLN, TNC, THBS1, ANXA1 and CD73/NT5E. Studies in expanded cohorts should be conducted to further validate these putative osimertinib biomarkers.

Non‐invasive biomarkers are needed to further personalize EGFR tyrosine kinase inhibitor (EGFR TKI) osimertinib treatment in non‐small cell lung cancer (NSCLC) patients. By analysing the proteome of extracellular vesicles (EVs) isolated from an osimertinib resistant NSCLC cell line and from serum of patients given osimertinib in second line within the TREM trial (NCT02504346), we here revealed proteins putatively associated with response and outcome to osimertinib treatment.