Multiple myeloma associated angiogenesis: the notch pathway in the interplay between myeloma and endothelium

Multiple myeloma(MM) is a still incurable cancer that strongly depends on interactions with bone marrow(BM) microenvironment. Patients display high level of angiogenesis due to endothelial cells(ECs) malignant behavior as compared to healthy counterpart(1). Moreover, MM cells show dysregulation in Notch signaling pathway due to Jag ligands overexpression. This condition brings to the formation of a sustaining loop between MM cells with surrounding cells including bone marrow stromal cells(BMSCs) and osteoclasts. Although the role of Notch in MM progression, it is not clear its involvement in MM-EC crosstalk. Thus, this project aims to investigate this aspect of MM. Notch ligands, Jag1 and 2, were silenced in RPMI8226 cell line using short hairpin RNAs (RPMI8226shJAG1/2). For ECs, Human pulmonary aortic endothelial cells (HPAEC) was used and for BMSCs, the GFP+HS5 cell line. Matrigel and Wound Healing assays were set up to study Notch involvement in modulating angiogenesis potential of MM cells co-cultured with HPAEC and ECs motility in response to MM-produced soluble factors. To mimic MM niche, we generated an organoid exploiting a decellularized extracellular matrix as scaffold. We evaluated apoptosis of MM cells alone or in co-culture with BMSCs and with ECs by flow cytometry. Matrigel assay of HPAEC cultured with RPMI8226scrb cells showed that direct contact increased angiogenic potential of ECs; this effect was reduced in absence of Jag ligands, indicating an active role of Notch in ECs stimulation. Wound Healing assay demonstrates that the silencing of Notch signaling also influences HPAEC motility. Concerning the organoid, our results indicate that the dECM is suitable for cells seeding and 3D structure generation. Moreover, apoptosis assays show that MM cells display and increased survival in presence of BMSCs, confirming their protective role; we do not obtain significant difference in MM apoptosis in presence, or not, of ECs. Surprising, we observe that MM cells protect ECs suggesting that myeloma can improve angiogenesis also by preventing ECs apoptosis. These results indicate a novel and active role of Notch in MM-EC crosstalk; 3D organoid can mimic microenvironment and it can be used as novel tool to study BM microenvironment beyond animal models.