Page 193 - Haematologica Vol. 107 - September 2022

ARTICLE - NOTCH2 in myeloma-derived extracellular vesicles D. Giannandrea et al.
the surrounding non-tumoral BM cells promotes neo- plastic cell growth and survival as well as key events of tumor progression including bone disease and angiogen- esis. These lines of evidence suggest that an effective therapeutic approach should not be focused merely on the MM cells, but it should target their interaction with the surrounding BM niche.
Recently, EV have been reported as critical players in the communication between MM cells and the nearby BM cells and leading to MM progression. Indeed, MM-EV pro- mote different events associated with MM progression, in- cluding angiogenesis11,13,34,35 and osteoclastogenesis.13,31-33 Here, we contribute to elucidate the molecular mechan- isms involved in MM-EV pathological communication with the BM microenvironment, strengthening the role of the EV pathological communication as a promising thera- peutic target in MM.
The evidence that NOTCH signaling activation, mediated by MM cell heterotypic interaction with the surrounding BM cells, plays a key role in tumor angiogenesis16,21 and osteoclastogenesis22-24 prompted us to investigate whether NOTCH signaling contributes to determine the impact of MM-EV on these processes.
Our analysis on a panel of HMCL and the respective shed EV indicate that NOTCH receptors were present in MM-EV cargo with high levels of NOTCH2 and slightly lower levels of NOTCH1, consistently with evidence from EV released by other cell types.36 We focused on NOTCH2 receptor, widely expressed in MM cell lines and in primary MM cells, particularly from high-risk patients.18,26 In details, we found that MM-EV carried the mature heterodimeric form of NOTCH2 (since we detected the NOTCH2-TM portion), the immature NOTCH2-FL and the activated NOTCH2-IC, that upon delivering to target cells might directly activate the transcription of the NOTCH target genes without requiring the interaction with ligands and the activation by ADAM protease and γ-secretase.
The expression of NOTCH receptors has been reported in exosomes37 but also in microvesicles.38 Thereby, we won- dered which of the two subpopulations of EV hosted NOTCH2. We separated large and small vesicles and found the presence of NOTCH2-TM both in large and small par- ticles, while NOTCH2-IC level was higher in 110K small EV fraction. Although it is impossible to distinguish exosome and microvesicles only on the basis of their dimension, we presume that small vesicles are enriched with exo- somes respect to large vesicles. Therefore our results suggest the presence of NOTCH2-IC within exosomes consistently with its presence in the endosomal compart- ment from which exosomes take origin.
Before validating the hypothesis that vesicular NOTCH2 contributes to molecular and biological effects on rel- evant BM cell populations as OCL and EC, we monitored if these cells uptake MM-EV, using respectively Raw264.7
cells and HPAEC. Flow cytometry detection showed a quick (4 h) uptake of MM-EV by the majority of cells in both models, confirmed by a Z-stack analysis in confocal microscopy.
Additionally, we unequivocally assessed that NOTCH sig- naling members could be transferred via EV from one cell to another using an experimental system model based on HEK293 cells forced to express NOTCH2 tagged with HA. This model system allowed us to assess that NOTCH2-HA could be released within EV and be transferred to distant cells. In this system, we have confirmed that EV carried high levels of NOTCH2-TM form and NOTCH2-FL, even if at much lower level. On the contrary, although the NOTCH2-IC form was present in MM-EV shed by HMCL, we could not detect it in HEK293-derived EV, possibly due to a lower level of NOTCH2 activation in HEK293 cells in comparison to HMCL. Receiving cells clearly took up the transmembrane form of NOTCH2-HA, but also showed a very faint band corresponding to NOTCH2-IC, consistently with a slight NOTCH2 activation after EV uptake. This re- sult provided a first indication that EV carrying NOTCH2 may activate NOTCH signaling in receiving cells.
Using a selective RNA interference of NOTCH2 in RPMI8226 and OPM2 cells, we confirmed that NOTCH2 KD in HMCL impacted the levels of vescicular NOTCH2-TM, NOTCH2-FL and NOTCH2-IC, although the decrease of the activated form was evident only in OPM2 cells. On the contrary, NOTCH2 KD did not significantly affect MM-EV size and concentration.
In order to confirm that MM-EV might activate the onco- genic NOTCH signaling in receiving cells, we tested MM- EVSCR or MM-EVN2KD in in vitro and in vivo NOTCH reporter systems. The first in vitro cellular model transfected with a Nanoluc-based NOTCH reporter vector, indicated that MM-EVSCR might activate a NOTCH-dependent gene re- porter, while MM-EVN2KD induced a significantly lower ac- tivation. This result was confirmed by a second reporter in vivo system recapitulating the complexity of a whole organism. The injection of MM-EVSCR or MM-EVN2KD in transgenic zebrafish embryo reporter for NOTCH not only confirmed the observed MM-EV-mediated NOTCH signal- ing activation but also provided evidence of MM-EV ability to induce NOTCH signaling activation at distant sites through the circulation. Indeed, MM-EV injected in the duct of Cuvier may be transported through the circulation and activate NOTCH signaling in the caudal hematopoietic tissue which represents the main hematopoietic organ in zebrafish embryo, analogous to the human BM.39 In contrast, NOTCH signaling activation mediated by MM- EVN2KD was significantly lower. MM-EV effectiveness in in- ducing NOTCH signaling activation at distant sites in zebrafish embryos carried by the blood stream suggests that MM-EV could also play an important role in the metastatic process, as reported for pancreatic cancer.40
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