In Vitro and In Vivo Analysis of Extracellular Vesicle‐Mediated Metastasis Using a Bright, Red‐Shifted Bioluminescent Reporter Protein

Abstract

Cancer cells produce heterogeneous extracellular vesicles (EVs) as mediators of intercellular communication. This study focuses on a novel method to image EV subtypes and their biodistribution in vivo. A red-shifted bioluminescence resonance energy transfer (BRET) EV reporter is developed, called PalmReNL, which allows for highly sensitive EV tracking in vitro and in vivo. PalmReNL enables the authors to study the common surface molecules across EV subtypes that determine EV organotropism and their functional differences in cancer progression. Regardless of injection routes, whether retro-orbital or intraperitoneal, PalmReNL positive EVs, isolated from murine mammary carcinoma cells, localized to the lungs. The early appearance of metastatic foci in the lungs of mammary tumor-bearing mice following multiple intraperitoneal injections of the medium and large EV (m/lEV)-enriched fraction derived from mammary carcinoma cells is demonstrated. In addition, the results presented here show that tumor cell-derived m/lEVs act on distant tissues through upregulating LC3 expression within the lung.

A Dual-Reporter Platform for Screening Tumor-Targeted Extracellular Vesicles

“…We have previously utilized PalmReNL [ 30 ] as a dual-reporter for bioluminescence and fluorescence tracking of EVs in vitro and in vivo. In the current study, we generated HEK293FT cells stably expressing PalmReNL and selected the EV donor cells that maintained a high level of PalmReNL expression ( Figure 1 A,B).…”

“…Moreover, PalmReNL can be used as a fluorescent EV reporter for standard microscopy and flow cytometry as well. We previously demonstrated that the labeling efficiency of PalmReNL was 1.2% for small EVs as determined by flow cytometry [ 30 ]. However, individual EVs carry probe molecules not detectable by flow cytometry due to the background autofluorescence.…”

“…However, individual EVs carry probe molecules not detectable by flow cytometry due to the background autofluorescence. Hence, their labeling efficiency may be higher as assessed by bioluminescence measurement [ 30 ].…”

“…For the EV reporter, PalmReNL, a palmitoylation sequence (MLCCMRRTKQ) [ 26 , 33 ] was genetically fused to Red enhanced Nano-lantern (ReNL [ 31 ]; Addgene plasmid (Watertown, MA, USA) #89536, gift from Takeharu Nagai) by PCR and subcloned into the Sleeping Beauty transposon vector [ 34 ] as we previously reported [ 30 ]. HEK293FT cells were cotransfected with a plasmid encoding PalmReNL and pCMV(CAT)T7-SB100 (Addgene #34879, gift from Zsuzsanna Izsvak).…”

“…Since recent studies revealed that endocytosed EVs were rapidly translocated into endosomal compartments [ 28 , 29 ], acidic pH-sensitive eGFP (p K a 6.0) is not an ideal reporter for tracking cellular EV uptake. Therefore, in this study, we used a novel BRET EV reporter, PalmReNL [ 30 ], consisting of a tdTomato-NanoLuc fusion protein [ 31 ] (p K a for tdTomato: 4.7) and a palmitoylation signal sequence [ 26 ], to characterize cellular EV uptake as a dual-reporter platform. We developed tumor-targeted EVs using several tumor-homing peptides (THPs) [ 32 ] coupled to lipid nanoprobes for noncovalent postproduction EV engineering, and assessed their increased capability of cancer cell targeting by bioluminescence measurement and fluorescence microscopy in vitro.…”