Abstract
Recent studies showed an unexpected complexity of extracellular vesicle (EV) biogenesis pathways. We previously found evidence that human colorectal cancer cells in vivo release large multivesicular body-like structures en bloc. Here, we tested whether this large extracellular vesicle type is unique to colorectal cancer cells. We found that all cell types we studied (including different cell lines and cells in their original tissue environment) released multivesicular large EVs (MV-lEVs). We also demonstrated that upon spontaneous rupture of the limiting membrane of the MV-lEVs, their intraluminal vesicles (ILVs) escaped to the extracellular environment by a ”torn bag mechanism”. We proved that the MV-lEVs were released by ectocytosis of amphisomes (hence, we termed them amphiectosomes). Both ILVs of amphiectosomes and small EVs separated from conditioned media were either exclusively CD63 or LC3B positive. According to our model, upon fusion of multivesicular bodies with autophagosomes, fragments of the autophagosomal inner membrane curl up to form LC3B positive ILVs of amphisomes, while CD63 positive small EVs are of multivesicular body origin. Our data suggest a novel common release mechanism for small EVs, distinct from the exocytosis of multivesicular bodies or amphisomes, as well as the small ectosome release pathway.
Competing Interest Statement
EIB is a member of the Advisory Board of Sphere Gene Therapeutics Inc. (Boston, MA, USA), ReNeuron (UK) and the Ludwig Boltzmann Institute for Nanovesicular Precision Medicine.
Footnotes
In the revised manuscript, for clarity, we rephrased several parts of the text for clarity, and we added some missing information. We mentioned in the revised text that because of 0.22 micron filtration of FBS, bovine large EVs were not present in our studies. For clarity, we modified the description of data shown in Fig2 in the revised manuscript. We also added the information that when we demonstrated the TEM of isolated EVs, we consistently used serum- free conditioned medium (Fig2 P-S, Fig2S5 J, O). Original LASX files have been provided in a database: https://orcid.org/0000-0001-5513-2471. We repeated one of our WB experiments and replaced one of the Western blot images with a new photo. To provided evidence that additional cell types release amphiectosomes; we have added new confocal microscopic images to Fig2_S3 showing amphiectosomes released also by the H9c2 (ATCC) cardiomyoblast cell line. In the revised manuscript, an additional Fig.1_S1 is provided. Here, we show by TEM the release of MV-lEVs both by an endothelial and a sub-endothelial cell (Fig.1_S1G) and we demonstrate the simultaneous presence of both MV-lEVs and sEVs in the circulation. Importantly, in the revised manuscript, we present additional super-resolution microscopy (STED) data. The intracellular formation of amphisomes, the fragmentation of LC3B-positive membranes and the formation of LC3B-positive ILVs were captured (Fig. 3B-F). In addition, we labelled the surface of live HEK293 cells with wheat germ agglutinin (WGA) and showed that the budding amphiectosome had WGA positive membrane. This provides evidence that its external membrane had a plasma membrane origin (Fig.3G). For clarity, we rephrased the figure legend to explain that Fig3_S2B shows the complete Western blot membrane, which was cut into 4 pieces, and the immune reactivity of different antibodies was tested. The actin band was included on the anti-LC3B blot. Grids separating the lanes have been eliminated on Fig.2_S4 (now Fig.2_S5 in the revised manuscript). Finally, based on the suggestion of one of the Reviewers, we added a sentence to the text regarding the disruption of the amphiectosome membrane. Here we mention that we hypothesize that this disruption only occurs after separation from the cell to avoid the release of ILVs inside the cell.
