Multiple myeloma-endothelium: 2D and 3D systems to study notch signaling

INTRODUCTION: Multiple myeloma (MM) is characterized by the accumulation of monoclonal plasma cells within bone marrow (BM), where they interact with surrounding BM cells. MM-associated angiogenesis is key mechanism, which correlates with disease progression. Notch pathway deregulation in MM is due to Notch receptors and Jagged ligands overexpression, has pro-tumor effects and is involved in MM crosstalk with different cell types. Here, we investigate the role of Notch pathway in the crosstalk between MM cells and endothelial cells (ECs). MATERIALS AND METHODS: RPMI8226 cells were transduced with lentiviral vectors carrying shRNAs for Jagged1 and 2 (RPMI8226-JAG1/2) or scrambled shRNAs (RPMI8226scrb). Angiogenic potential was assessed by Matrigel assay using Human Pulmonary Artery Endothelial Cells (HPAECs) and RPMI8226-JAG1/2 or RPMI8226scrb cells (ratio 1:2). 3D-co-culture was set up by loading BMSCs (HS5), MM cells (RPMI8226) and ECs (HPAEC) (ratio 1:2:1) in decellularized extracellular matrix produced by NIH3T3 cells. Apoptosis was assessed by AnnexinV staining and flow-cytometry. RESULTS: RPMI8226scrb cells display angiogenic activity lost by RPMI8226-JAG1/2. We set up 3D-organoids that that recapitulate the interaction of MM cells with key BM cellular components (ECs and mesenchymal cells) and result in angiogenesis and prevention of tumor cell apoptosis. DISCUSSION and CONLUSION: Overexpressed Jagged ligands in MM cells are key in activating Notch signaling in nearby ECs and the consequent MM-associated angiogenesis. 3D-organoids are a useful tool to study MM-associated angiogenesis and reveal that MM cells can sustain vessels formation. We are going to investigate in details the role of Notch by 2D and 3d assays.