Petra Hååg

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.

Extracellular vesicles (EVs) are lipid membrane-bound nanoscale (20 nm - 1000 nm) objects shed by all cells. As EVs play an important role in intercellular signaling, these have emerged as promising biomarkers for many diseases including cancer and neurodegenerative disorders. A major bottleneck in research into EVs and their smooth translation to clinic as disease biomarkers is a lack of access to affordable and user-friendly technologies to quickly isolate EVs from complex biological and clinical samples with acceptable purity and yield. Here, we report two different designs of inertial microfluidic devices (e.g. LoAR-Exo and HiAR-Exo) that can isolate EVs from different kinds of biological samples in a single label-free step using a spiral microchannel design with different aspect ratios. LoAR-Exo has a height-to-width aspect ratio ~1, while HiAR-Exo has a height-to-width aspect ratio of 2.5, optimized by COMSOL simulations. We tested both designs by separating polystyrene particles of size < 1 micrometer from a heterogeneous mixture. We then benchmarked the performance of the microfluidic chips against ultracentrifugation and a precipitation kit by isolating EVs from the cell culture-conditioned media (CCM) of MDA-MB-231 cells. We also compared the microchip with SEC by using the CCM from H1975/OR cells. The size distribution of EVs isolated by the microfluidic chip was comparable with ultracentrifugation, precipitation and SEC. In summary, both devices isolated EVs from as little as 1 mL of sample volume using a label-free technique in a continuous manner and without any user intervention. Both microfluidic platforms offer a simple, efficient, and scalable alternative to conventional methods for EV isolation.

Supplementary Table 2: NMR data of (3S)-14-methyldocos-4E-en-1-yn-3-ol (2).