Homosalate boosts the release of tumour‐derived extracellular vesicles with protection against anchorage‐loss property

Abstract

Eukaryotic cells, including cancer cells, secrete highly heterogeneous populations of extracellular vesicles (EVs). EVs could have different subcellular origin, composition and functional properties, but tools to distinguish between EV subtypes are scarce. Here, we tagged CD63‐ or CD9‐positive EVs secreted by triple negative breast cancer cells with Nanoluciferase enzyme, to set‐up a miniaturized method to quantify secretion of these two EV subtypes directly in the supernatant of cells. We performed a cell‐based high‐content screening to identify clinically‐approved drugs able to affect EV secretion. One of the identified hits is Homosalate, an anti‐inflammatory drug found in sunscreens which robustly increased EVs’ release. Comparing EVs induced by Homosalate with those induced by Bafilomycin A1, we demonstrate that: (1) the two drugs act on EVs generated in distinct subcellular compartments, and (2) EVs released by Homosalate‐, but not by Bafilomycin A1‐treated cells enhance resistance to anchorage loss in another recipient epithelial tumour cell line. In conclusion, we identified a new drug modifying EV release and demonstrated that under influence of different drugs, triple negative breast cancer cells release EV subpopulations from different subcellular origins harbouring distinct functional properties.

Luminescence-based screening for extracellular vesicle release modulators reveals a role for PI4KIIIβ in exosome biogenesis upon lysosome inhibition

“…This is critical since CD63 itself drives EV secretion (van Niel et al, 2011) and overexpression may cause unwanted changes in cellular physiology. Others (Grisard et al, 2022;Gupta et al, 2020) and we (data not shown) observed that in some cell lines, such as HeLa, overexpression of NanoLuc-tagged CD63 results in cleavage and secretion of the NanoLuc-tag as free soluble protein. In this case, simply measuring NanoLuc activity in the supernatant will lead to an overestimation of EV secretion.…”

“…During the course of this study the use of NanoLuc-tagged CD63, or other EV-cargo, has been proposed in various strategies to quantify EV secretion (Cashikar and Hanson, 2019; Grisard et al, 2022; Gupta et al, 2020; Hikita et al, 2018). The approach described in this study brings together two important advances.…”

“…Despite these advantages, our reporter only quantifies EV-associated CD63 secretion, and thus does not provide a direct quantification of EV number. Furthermore, compounds that interfere with CD63 trafficking or sorting into EVs, or compounds that directly inhibit NanoLuc enzyme activity (Grisard et al, 2022), may be picked up as false hits. Therefore, validation of compounds outside of the HA-NL- CD63 screen, using gold-standard EV isolation and quantification techniques according to MISEV criteria (Thery et al, 2018) is important.…”

“…Despite these notable advances, insight into the mechanisms and druggability of EV biogenesis and secretion by these screening methods has been hampered by technical and practical limitations. Recently, the ultrabright NanoLuciferase enzyme (Hall et al, 2012) has been used to create EV reporters that have the potential for robust, fast and scalable quantification of EV secretion compatible with both genetic and pharmacological screens (Bonsergent et al, 2021; Cashikar and Hanson, 2019; Grisard et al, 2022; Gupta et al, 2020; Hikita et al, 2018).…”

Excess filaggrin in keratinocytes is removed by extracellular vesicles to prevent premature death and this mechanism can be hijacked by Staphylococcus aureus in a TLR2-dependent fashion

“…Intriguingly, we did not observe any substantial differences in the filaggrin-related cargo as far as the calcium level was concerned which is unexpected given the large increase in filaggrin expression upon NHEK differentiation. To improve the sensitivity of detection we next pooled fractions 1-5 (Figure 2d) adjusting the samples against cell counts (as in [26][27][28] ), which revealed the band of ~75 kDa in size (possibly filaggrin dimer, Figure 2d) to be the most prominent; the 37 kDa filaggrin monomer band could be also sometimes detected at lower levels in the vesicles harvested from differentiated NHEKs. Unexpectedly, the differences between sEV/exosomes obtained from the two calcium conditions were relatively small.…”