P-206 3D in vitro modelling of relapsed/refractory multiple myeloma to unveil mechanisms of acquired resistance to therapy

Introduction: Relapsed/refractory (R/R) multiple myeloma (MM) patients have limited therapeutic options and poor outcome. A major limitation for the development of targeted therapies in this setting is the poor predictive value of preclinical models. In fact, standard two-dimensional (2D) cultures do not recapitulate the complexity of the tumour microenvironment (TME) that has a crucial role in cancer progression and therapy response. This limitation most likely explains the massive failure of the approval rate of novel treatments from international medical agencies. Here we developed in vitro three-dimensional (3D) scaffolds with biomimetic tissue like properties and leveraged this platform for drug testing and for the identification of TME-mediated mechanisms of drug resistance in R/R MM. Methods: Bone marrow-like 3D scaffolds (BM3Ds) were obtained through a crosslinking and a freeze-drying process by combining collagen type I and hydroxyapatite (30:70 wt% ratio). Human Stromal (HS5), Human Umbilical Vein Endothelial Cells (HUVEC) and MM cell lines H929 or JJN3 were co-cultured in BM3Ds, imaged by confocal microscopy after immunofluorescence staining or embedded in paraffin for immunohistochemistry. Cells were treated with increasing concentrations of Bortezomib (BTZ) and Lenalidomide (LEN) to calculate the half maximal inhibitory concentration (IC50) by cell viability assays. Results: BM3Ds displayed a highly reproducible micro-architecture resembling clinical BM specimens and a tunable structure enabling applications in 96/wells format. HUVEC and HS5 widely colonized the inner space of the scaffold with a multilayer cell organization. Conversely, MM cell lines grouped between the collagen fibers supported by the layers of stroma and endothelium when in co-cultures. By comparing 2D and 3D conditions we observed no differences in the growth rate of MMs, while HUVEC and HS5 showed a decreased proliferation at 72 hours from seeding when cultured in BM3Ds (P < 0.001). In BM3Ds, H929 cells showed decreased IC50 to both LEN and BTZ (0.60 μM and 0.0009 μM) compared to 2D culture (1.58 μM and 0.0019 μM). JJN3 did not respond to LEN and showed a doubled IC50 when treated with BTZ in BM3Ds (0.002 μM) with respect to 2D culture (0.001 μM). Then we optimized a 5 days protocol for a standardized process of drug discovery/testing. Specifically, HUVEC and HS5 are seeded in BM3Ds (3 x 105/each) at day 0 followed by JJN3 or H929 (5 x 105) at day 3. Treatments are carried out at day 4 prior to the drug sensitivity evaluation at day 5. Conclusions: BM3Ds is an alternative biomimetic 3D approach to model R/R MM for preclinical drug screening and drug discovery application. Ongoing single cell RNASeq experiments of stromal, endothelium and MM cells co-cultured in 2D or in BM3Ds after BTZ and LEN treatment will inform on molecular pathways driving mechanisms of drug resistance. This platform is expected to increase the consistency of preclinical data and to foster the clinical translation of new discoveries.