Figure 6.

ATR inhibition by VX-970 synergizes with melphalan in inducing apoptosis in multiple myeloma patient cells independently of the level of DNA damage. (A) Schematic representation of the experimental procedure in the 3D bioreactor. (B-C) CD138⁺ primary cells from multiple myeloma (MM) patients were seeded in gelatin scaffolds pre-seeded with CD73⁺ bone marrow stromal cells (BMSC) and cultured in the 3D bioreactor system either in untreated conditions (NT) or in the presence of VX-970 (0.3 µM), melphalan (1.2 µM), or a combination of both drugs. (B) Seventy-two hours after the beginning of the treatment, cells retrieved from scaffolds were stained with Annexin V and anti-CD38 antibody before flow cytometric (FACS) analysis. The table underneath the graph summarizes the Bliss and Loewe synergy scores calculated for the combined treatments in each patient. Due to the limited number of cells that could be recovered from MM patient bone marrow (BM) samples, it was possible to assay just one concentration for each drug, in each patient. (C) Representative immunohistochemical (IHC) analyses performed on scaffolds populated with primary MM cells and retrieved at the end of the culture period, showing the distribution of CD138⁺ MM and CD73⁺ BMSC cells and the effect of the co-treatment specifically on MM cells. (D) Representative IHC images form the BM biopsies of MM patients analyzed for γH2AX and pCHK1 expression (original magnification 40X; samples presented are the same used for experiment in (B)). The inset in each panel indicates the percentage of tumor cells positive for the indicated marker. The percentage of neoplastic plasma cells (PC) is shown above each panel.