Page 185 - Haematologica Vol. 107 - September 2022

ARTICLE - NOTCH2 in myeloma-derived extracellular vesicles D. Giannandrea et al.
features of MM-EV may contribute to MM dissemination at distant sites, thereby favoring skeletal metastasis formation, progression and bone disease.13
This work elucidates how MM cells exploit the aberrantly expressed NOTCH2 oncogene to shape the BM niche via MM-EV, specifically focusing on tumor angiogenesis and osteoclastogenesis.
NOTCH is a family of transmembrane receptors (NOTCH1- 4) activated by the interaction with five different mem- brane-bound ligands (JAGGED1-2 and DLL1-3-4) present on the adjacent cells. The consequence of this interaction is the activation of cleavage by γ-secretase, which re- leases the active form of NOTCH (NOTCH-IC) from the cell membrane and allows its translocation to the nucleus and the activation of the CSL transcription factor.14
NOTCH deregulation in MM cells is due to the aberrant ex- pression of NOTCH receptors and/or ligands.15 High levels of NOTCH pathway activity are associated with increased myeloma cell infiltration in BM biopsies of MM patients.16 Other studies suggest that MM cell skeletal infiltration may be due to events promoted by NOTCH, including MM cell recruitment at the BM,17 mitogenic or anti-apoptotic effect17-19 or MM stem cell self-renewal.20 Additionally, MM infiltration of BM niche is also associated with the activa- tion of NOTCH signaling in the tumor niche, which pro- motes angiogenesis,16,21 osteoclastogenesis,22-24 and bone marrow stromal cell (BMSC)-mediated release of cyto- kines involved in these events (IL-6, VEGF, IGF-1, SDF-1, RANKL, etc.).16,18,19,25
Up to now, the increased activation of NOTCH signaling in the tumor microenvironment has been attributed to the presence of high levels of MM cell-derived JAGGED li- gands. Here, we demonstrate that MM cells may trigger tumor angiogenesis and osteoclastogenesis by transfer- ring NOTCH2 receptor via EV. Moreover, we provide evi- dence that targeting the NOTCH pathway may represent a suitable strategy to hamper the MM-EV-mediated patho- logical communication with the BM niche.
Methods
Extracellular vesicles isolation from human multiple myeloma cell line and multiple myeloma patients’ bone marrow aspirates
EV were obtained from supernatants of RPMI8226 and OPM2 cells cultured for 48 hours (h) in RPMI1640 medium depleted of fetal bovine serum-derived bovine EV or from the plasma obtained by BM aspirates of monoclonal gam- mopathy of undetermined significance (MGUS) (MGUS- BM-EV) and MM patients (MM-BM-EV). The Institutional Review Board of Insubria Italy approved the design of this study (approval n. 1 on 27th February 2018). Written in- formed consent was obtained in accordance with the
Declaration of Helsinki. Clinical information of patients is reported in the Online Supplementary Table S1.
EV pellets were resuspended in the appropriate buffer/medium for subsequent studies. Further details are reported in Online Supplementary Appendix.
Production of viral supernatants and NOTCH2 knockdown
Viral supernatants were generated by calcium phosphate- DNA transfection of HEK293T cells with the Dharmacon Trans-lentiviral packaging kit containing pTRIPZ vector carrying a doxycycline-inducible system (Tet-on) express- ing short hairpin RNA (shRNA) against NOTCH2, or the cor- responding scrambled shRNA (Horizon Discovery, United Kingdom). A pilot experiment on HEK293T cells was car- ried out by transient transfection of four shRNA for NOTCH2 to select the more effective NOTCH2 shRNA (Cat.ID RHS5087-EG4853 - mature antisense sequence: ATGTCACAAGAGACATTGG). Lentiviral supernatants were used to infect and generate stable clones of RPMI8226 and OPM2 cells. shRNA expression was induced by treat- ment with 1 μg/mL doxycycline (Sigma Aldrich, Italy).
In vivo experiments
In vivo experiments were carried out on transgenic zebra- fish (Danio rerio) embryos obtained by crossing Tg(T2KTp1bglob:hmgb1-mCherry) with Tg(fli1a:EGFP) ob- tained from the Wilson lab, University College London, UK. Zebrafish embryos were raised and maintained under standard conditions and national guidelines (Italian de- cree 4th March 2014, n. 26). All experiments have been conducted within 5 days post fertilization (dpf). EV were injected into the duct of Cuvier of embryos at 48 hours post fertilization (hpf) with a manual microinjector (Ep- pendorf, Germany) using glass microinjection needles. Further details on the above procedures and information concerning cell cultures, transmission electron micro- scopy, in vitro uptake, western blot, EV-derived NOTCH2 tracking system, viral particle production, luciferase re- porter assay, in vivo experiments, osteoclastogenesis and angiogenesis assays, ex vivo experiments and statistical analyses are reported in the Online Supplementary Appen- dix.
Results
Multiple myeloma-derived extracellular vesicles are uptaken by bone marrow cell populations
EV produced by two different human MM cell lines (HMCL), RPMI8226 and OPM2, were isolated by ultracen- trifugation and fully characterized. Particle size distribu- tion assessed by nanoparticle tracking analysis (NTA) revealed that the EV populations produced by the two
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