Page 187 - Haematologica Vol. 107 - September 2022

ARTICLE - NOTCH2 in myeloma-derived extracellular vesicles D. Giannandrea et al. AB
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carrying NOTCH receptors and, in particular the over- expressed receptor NOTCH2,26 as part of their cargo. By western blot analysis, we compared NOTCH2 expression in protein extracts from seven HMCL, namely AMO1, JJN3, H929, RPMI8226, LP1, KMS12, OPM2, and EV isolated from HMCL conditioned media (CM). Figure 3A shows that MM- EV are able to carry NOTCH2, whose relative amount re- flects that expressed in the different protein extracts of the HMCL. The analysis of the different forms of NOTCH2 indicated that MM-EV could carry not only the transmem- brane NOTCH2 (NOTCH2-TM) but also the full-length un- cleaved NOTCH2 (NOTCH2-FL). Since the cleavage operated by γ-secretase on the intracellular portion of NOTCH takes place inside the endocytic bodies27 and exo- somes arise from late endosomes,28 we investigated whether the active cleaved intracellular NOTCH2-IC may be included in MM-EV cargo by using a specific antibody. Results in Figure 3A indicate that MM-EV cargo also carries NOTCH2-IC. Online Supplementary Figure S2 shows that also NOTCH1 is widely represented in MM-EV, while the presence of other two isoforms in MM-EV is less no- ticeable.
In order to assess which EV fraction expresses NOTCH2,
Figure 2. Multiple myeloma cell-released extracellular vesicles can be taken up by osteoclasts and endothelial cells. Osteo- clast (OCL) progenitor and endothelial cell (EC) uptake of multiple myeloma cell-released extracellular vesicles (MM-EV) from RPMI8226 cells. Raw264.7 cells and primary human pul- monary arterial cells (HPAEC) were treated with CM-DIL stained MM-EV or the negative control for 4 hours (h) at 37°C or 4°C. (A and B) Representative flow cytometry dot plots show the CM- DIL-labeled RPMI8226-EV uptake by Raw264.7 cells (A) and HPAEC (B) by measuring CM-DIL-positive cells in the PE chan- nel. OPM2-EV provided similar results (not shown). Data are presented as the mean values of 3 independent experiments. (C) Maximum intensity projection of CM-DIL-positive RPMI8226-EV internalization in Raw264.7 cells and HPAEC after 4 h of incubation at 37°C and 4°C. Red fluorescence: RPMI8226- EV labeled with CM-DIL dye; green fluorescence: CFSE+ labeled cells; blue fluorescence: nuclei with DAPI (63x magnification).
we performed a western blot analysis on large and small vesicles collected from the HMCL CM by sequential ultra- centrifugation at 20,000 g (20 K) and 110,000 g (110 K). We found that NOTCH2-TM and NOTCH2-IC were present both in large and small vesicles (Figure 3B). Interestingly, NOTCH2-IC level was increased in 110K MM-EV fraction. EV allow distant cells to communicate between each other, thus modifying their behavior. In order to demon- strate that NOTCH2 may be involved in these processes and can be transferred to distant cells via EV, we devel- oped a model system of HEK293 donor and receiving cells (Figure 3C). The first were forced to constitutively express NOTCH2 tagged with HA at the C-terminus (NOTCH2-HA)29 to distinguish it from the endogenous NOTCH2. In addi- tion, the position of the HA-tag at the C-terminus of NOTCH2 enabled us to detect NOTCH2-FL, the NOTCH2- TM portion of the heterodimeric NOTCH2 form, matured in the trans-Golgi network upon the cleavage by a furin- like convertase,30 and the mature NOTCH2-IC, due to homotypic activation mediated by ADAM10 and the γ-sec- retase.29 EV-donor cells were added to the culture medium of receiving HEK293 cells. Figure 3C shows a western blot analysis performed with an anti-HA antibody, confirming
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