Oral Presentation Australasian Extracellular Vesicles Conference 2020

LIVE VIDEO PRESENTATION: From nanoscale to organisms: a multi-resolution imaging system of endogenously released extracellular vesicles with bioluminescence resonance energy transfer (#35)

Charles Pin-Kuang Lai 1 2 3 4 , Anthony Yan-Tang Wu 1 2 4 5 , Yun-Chieh Sung 2 6 , Vanessa Guo 4 , John Jun-Sheng Ko 2 4 , Alan Ling Yang 2 4 , Yunching Chen 2
  1. Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan
  2. Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu , Taiwan
  3. National Taiwan University and Academia Sinica, Taipei, Taiwan
  4. Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan
  5. Department and Graduate Institute of Pharmacology, National Taiwan University, Taipei, Taiwan
  6. Department of Chemical Engineering, National Tsing Hua University, Hsinchu , Taiwan

Extracellular vesicles (EVs), including exosomes and microvesicles, are nanosized, membrane-bound vesicles released in abundance by cancer cells. These EVs are capable of delivering a select subset of proteins and RNAs designed to promote tumor growth and metastases. However, the exact time, location (i.e. spatiotemporal properties) and cargo delivery capacity of EVs remain largely unknown due to a lack of available methods to detect them.  To overcome these limitations, here we developed a next-generation reporter to enable EV imaging from organismal to super-resolutions by engineering enhanced green fluorescent protein fused to a palmitoylation signal and nanoluciferase, termed PalmGp. HEK293T and HCA-1 cells were lentivirally transduced to stably expressed PalmGp as EV donor cells. In vivo EV and nanoscopic imaging were achieved by in vivo imaging system (IVIS) and super-resolution radial fluctuations nanoscopy, respectively. PalmGp predominantly labels EV inner membrane, and exhibited a robust bioluminescent and BRET-GFP signals for EV imaging upon addition of its substrate, furimazine. IVIS imaging of liver cancer HCA1-PalmGp bearing mice showed sustained bioluminescent and fluorescent signals at the primary and metastatic sites, indicating the high sensitivity of PalmGp to enable biodistribution and clearance of endogenously released EVs. PalmGp-EVs could further be visualized at super-resolution (50-150 nm) to monitor EV subcellular trafficking. To our knowledge, this is the first report of a multi-resolution reporter strategy that enables EV imaging. Efforts are currently underway in employing the PalmGp EV reporter to elucidate the spatiotemporal property and mechanism(s) of cancer EVs during disease progression.