Page 191 - Haematologica Vol. 107 - September 2022

ARTICLE - NOTCH2 in myeloma-derived extracellular vesicles D. Giannandrea et al.
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Figure 5. NOTCH2 contributes to the protumorigenic communication of multiple myeloma cell-released extracellular vesicles toward osteoclasts and endothelial cells. (A) The effect of MM-EVSCR and MM-EVN2KD collected from the osteoclastogenic cell line RPMI8226. The Raw264.7 cell line was treated with or without the osteoclastogenic RANKL (30 ng/mL), multiple myeloma cell-released extracellular vesicles (MM-EV) or control fresh medium (w/o EV). After 7 days TRAP-positive multinucleated cells (≥3 nuclei) were enumerated (TRAP-positive multinucleated cells are indicated by an arrow). Representative images are shown for each condition on the left (4x magnification); a graph on the right represents the mean value of the absolute number of TRAP+ multinucleated cells +/- standard error of the mean. Statistical analysis by a one-way ANOVA with Tukey post-test; *P<0.05. (B) Tumor angiogenesis induced by MM-EVSCR and MM-EVN2KD. Tube formation assay performed for 13 hours with primary human pulmonary arterial cells (HPAEC) laid on a matrigel-coated support stimulated with MM-EVSCR and MM-EVN2KD collected from RPMI8226 and OPM2 cells or control fresh medium (w/o EV). The graphs show the mean values of areas and nodes (branch points) enumerated in four quadrant of the well +/- standard error of the mean. Statistical analysis was performed by ANOVA and Tukey post-test; *P<0.05, **P<0.01. Representative images are shown below for each condition (4x magnification).
ure 6D shows that MM-BM-EV boost the angiogenic po- tential of HPAEC while MGUS-BM-EV showed a non-stat- istically significant increasing trend. Importantly, the inhibitory effect of DAPT is statistically significant when added to HPAEC treated with MM-BM-EV. These results confirm the increasing angiogenic potential of EV released in the BM during MM progression, the role played by NOTCH delivered via MM-BM-EV and strengthen the po-
tential of a NOTCH-directed therapeutic approach to block the support of MM microenvironment to the disease progression.
Discussion
The pathological interplay between malignant cells and
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