Page 188 - Haematologica Vol. 107 - September 2022

ARTICLE - NOTCH2 in myeloma-derived extracellular vesicles D. Giannandrea et al.
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Figure 3. NOTCH receptors and ligands in extracellular vesicles. (A) Western blot analysis for NOTCH2-FL (full length), NOTCH2- TM (transmembrane form), and NOTCH2-IC (active intracellular NOTCH2) expressed in 7 different human multiple myeloma cell lines (HMCL) and the respective produced extracellular vesicles (EV). b-actin and TSG101 were used as loading controls for cells and vesicle protein extracts, respectively. In order to perform all the hybridizations, two western blots were performed with cell and EV extracts loaded with an identical amount of protein. (B) Western blot analysis shows the expression of NOTCH2-TM and NOTCH2-IC in EV populations of different sizes. Large and small EV were isolated from RPMI8226 and OPM2 cells by sequential ultracentrifugation at 20,000 g (20K) and 110,000 g (110K), and the expression of the two NOTCH2 forms was separately assessed by western blot analyses using specific antibodies for NOTCH2 and NOTCH2-IC; TSG101 was used as control for vesicle protein extracts. (C) EV-mediated cell-to-cell transfer of NOTCH2: the donor HEK293 cell line was forced to express HA-tagged NOTCH2 carried by pCDNA3.1 or the corresponding empty vector (negative control); EV secreted by donor cells were collected by ultra- centrifugation and used to treat receiving HEK293 cells for 24 hours. Cell and EV protein extracts were analyzed by western blot using a specific primary antibody anti-HA. α-tubulin and TSG101 were used to normalize cellular and vesicular protein extract loading, respectively. NOTCH2-IC identified by rehybridization of the same membrane with anti-NOTCH-IC (see the Online Sup- plementary Figure S3) is indicated by an asterisk.
the presence of the HA-signal in donor cells carrying NOTCH2-HA,isolatedEV,andreceivingcells.Thisdemon- strates that EV can transfer NOTCH2 between distant cells. In this model system, the EV cargo include NOTCH2-TM and NOTCH2-FL, while both donor and receiving cells show also the presence of NOTCH2-IC. The absence of NOTCH2-IC in HEK293-derived EV might be due to a lower level of NOTCH activation in HEK293 cells in comparison to HMCL.
Multiple myeloma-derived extracellular vesicles activate NOTCH signaling in receiving cells
In order to address if the variation of NOTCH2 levels in MM cells may affect MM-EV-mediated communication, we studied the effect of NOTCH2 silencing in RPMI8226 and OPM2 cells. These cells were transduced with the pTRIPZ lentiviral vector conditionally expressing shRNA for NOTCH2
(HMCLN2KD) or the scrambled sequence (HMCLSCR) and single cell clones were isolated. Figure 4A confirms that RPMI8226 and OPM2 cells are knocked down (KD) for NOTCH2 and clearly shows that also the produced MM-EV displayed a reduced level of NOTCH2. Also NOTCH2-IC de- creased in OPM2 cells with a corresponding decrease in the shed EV, while NOTCH2-IC decrease in EV release from RPMI8226 was less evident (Online Supplementary Figure S4). Through alignment search tool BlastN (USA National Center for Biotechnology Information) we excluded re- gions of local similarities between the used shRNA and the sequences of other NOTCH receptors and ligands (E values range between 1 and 15). However, the high se- quence homology between the four NOTCH receptors prompted us to analyze by western blot the expression of the other NOTCH receptor isoforms. The Online Supple-
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