TRPC3 signalling contributes to the biogenesis of extracellular vesicles

Abstract Extracellular vesicles (EVs) contribute to a wide range of pathological processes including cancer progression, yet the molecular mechanisms underlying their biogenesis remain incompletely characterized. The development of tetraspanin‐based pHluorin reporters has enabled the real‐time analysis of EV release at the plasma membrane. Here, we employed CD81‐pHluorin to investigate mechanisms of EV release in ovarian cancer (OC) cells and report a novel role for the Ca2+‐permeable transient receptor potential (TRP) channel TRPC3 in EV‐mediated communication. We found that specific activation of TRPC3 increased Ca2+ signalling in SKOV3 cells and stimulated an immediate increase in EV release. Ca2+‐stimulants histamine and ionomycin likewise induced EV release, and imaging analysis revealed distinct stimulation‐dependent temporal and spatial release dynamics. Interestingly, inhibition of TRPC3 attenuated histamine‐stimulated Ca2+‐entry and EV release, indicating that TRPC3 is likely to act downstream of histamine signalling in EV biogenesis. Furthermore, we found that direct activation of TRPC3 as well as the application of EVs derived from TRPC3‐activated cells increased SKOV3 proliferation. Our data provides insights into the molecular mechanisms and dynamics underlying EV release in OC cells, proposing a key role for TRPC3 in EV biogenesis.


Immunogold labelling
For immunolabelling of CD81 on SKOV3-EVs, 8µl of EV concentrate was deposited on glow discharged, carbon-coated nickel grids (Agar Scientific) for 2 min before grids were blocked in 1% BSA in PBS for 1hr at RT. Grids were then incubated with mouse anti-CD81 (1:100, Abcam, ab79559) in 1% BSA in PBS for 1h at RT. Grids were next washed three times for five minutes per wash in 1% BSA in PBS, before being incubated for 1h at RT with 10nm gold-conjugated anti-mouse (1:50, Abcam, ab27241) in 1% BSA in PBS.After washing steps were repeated, samples were fixed in 2.5% glutaraldehyde (EM grade) in 0.1M phosphate buffer.Finally, grids were washed twice in ddH2O and negatively stained with 2% uranyl acetate for 10 sec.Grids were left to airdry before imaging.Immunogold-labelled EVs were imaged on a Jeol JEM-1400 Flash Transmission Electron Microscope, using a Gatan OneView 16 Megapixel camera at 100kV.
For H1HR immunostaining, SKOV3, OVCAR3 and OVCAR5 cells were seeded, fixed, permeabilised and blocked as above, without Membrite staining.Cells were incubated overnight at 4°C with mouse anti-HRH1 primary antibody (1:50, Santa Cruz Biotechnology, sc-374621).Anti-mouse Alexa Fluor® 594 (Thermo Fisher Scientific, A-11005) secondary antibody was added to cells the following day and incubated for 30 min at RT, before coverslips were mounted onto glass slides using Prolong Gold Antifade DAPI mounting media (Thermo Fisher Scientific).Cells were imaged using a Zeiss Axio Imager 72 upright microscope fitted with an ORCA-Flash 4.0 Digital CMOS camera (Hamamatsu).

qPCR
Total RNA was extracted from SKOV3, OVCAR3 and OVCAR5 cell lines using Direct-zol RNA MiniPrep Kit (ZymoResearch) following the manufacturer's instructions.A total of 300ng RNA was DNAse I treated prior to reverse transcription, which was performed using the HighCapacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific).PCR reactions were performed with iTaq Universal SYBR Green Supermix (Biorad) using the CFX96 TouchTM thermal cycler (Bio-Rad) with the following thermal profile: 30s hot start at 95°C, 35 cycles of 5s at 95°C and 30s at 60°C.Primers for H1HR and β-Actin (ACTB) are shown in Table S1.
The fold difference in expression between H1HR and ACTB was calculated using the ΔΔCq method.

Primer
Sequence 5' to 3' Figure S1.CD81 is enriched on the surface of SKOV3-EVs.Representative immunogold (anti-CD81) negative stained electron micrographs of SKOV3 EVs.EV diameter measured and shown in yellow.Arrows indicate 10nm gold particles.Scale bars 100nm and 1000nm (far right image only).