A Highly Reproducible 3D Culture Approach for Functional Drug Testing on Primary Multiple Myeloma Cells.

Introduction
One of the main challenges in multiple myeloma (MM) management is the emergence of multi-drug resistance, which eventually leads to disease relapse. Clonal evolution of malignant plasma cells over the course of treatment leads to outgrowth of clones whose drug sensitivity is difficult to predict, as the factors conditioning this evolution are poorly understood and may greatly differ between patients. Until recently, studies on primary MM cells have been limited due to the short lifespan of malignant plasma cells outside their niche. Therefore, development of novel culture systems able to preserve primary MM cell viability is needed as a tool to better understand the survival requirements for malignant plasma cells and to test drug sensitivity in a patient-specific manner.

Methods and Results
We have tested cell viability and drug sensitivity of primary MM cells in different 2D and 3D culture setups and identified a 3D culture system in which viability of primary MM cells is preserved significantly better than in conventional 2D cultures. This 3D setting, based on a self-assembling peptide hydrogel in the presence of pro-survival cytokines IL-6 and APRIL, was able to keep primary MM cells alive up to multiple weeks even in the absence of supportive cell subsets, such as mesenchymal stromal cells (MSC). In contrast to matrigel, the nature of this specific peptide hydrogel allows easy isolation of the primary cells after culture for further analysis. In this setup primary MM cells, as well as human myeloma cell lines, were significantly more resistant to apoptosis induced by conventional or novel MM drugs, including bortezomib, dexamethasone, melphalan, venetoclax, or MCL-1-specific inhibitors, in 3D- than in 2D-culture. In addition, we found that both T cells and/or NK cells can be co-cultured in this 3D setup.

Conclusions
We have established a fully controllable 3D culture approach able to preserve primary MM cell survival in the absence of additional bone marrow-derived cell subsets. This method allows testing of (novel) MM drugs and, when co-cultured with immune cells, can be used to test immunomodulatory drugs (IMiDs) and immunotherapeutic approaches. Combined, this experimental setup will lead to a better understanding of MM cell-intrinsic and -extrinsic mechanisms to escape apoptosis, and may be a useful tool to predict, in a patient-specific manner, the most effective drug combinations to prevent disease relapse.