Current research has demonstrated that cancer cells are not only influenced by their microenvironment, but are also able to drastically alter their surroundings to further accelerate cancer progression. Hematological malignancies create a forward feedback system with local mesenchymal stromal cells within the bone marrow, but the exact mechanisms and cellular changes within the stroma are largely unknown. Our work has aimed at understanding the bidirectional interaction between multiple myeloma (MM) cells and human bone marrow-derived mesenchymal stromal cells (hMSCs) by characterizing hMSCs derived from either MM patients or healthy individuals, using medium-throughput assays and 2D and 3D in vitro bone marrow models.
First, primary human myeloma patient MSCs (MM-MSCs) and normal donor MSCs (ND-MSCs) were characterized in terms of proliferation when cultured with and without MM.1S cancer cells in direct and indirect co-culture. Next, primary ND- and MM-MSCs were profiled for their microRNA (miRNA) (n=3 for normal, n=7 for MM) and mRNA (n=5 for normal, n=5 for MM) expression using Nanostring technologies. We analyzed 800 human miRNAs from miRBase v.18 and 230 human cancer-related genes using the nCounter® Human Cancer Reference Kit, which allowed for much greater specificity and reliability than microarray technologies. Lastly, a more representative culture system was designed to better mimic myeloma growth within the bone marrow. We developed a 3D in vitro model using hMSCS and fluorescent, luciferase-labeled MM cell lines seeded into porous, autofluorescent silk scaffolds. Scaffolds were made using silk fibroin protein isolated from silkworm cocoons and were formed into biocompatible cylinders with pores of 500–600 microns in diameter. Both ND- and MM-MSCs were labeled with fluorescent cell-tracker dyes and cultured on scaffolds with or without MM1S-GFP+/Luc+ cells. These were non-destructively assessed using confocal microscopy and bioluminescent imaging (BLI) for their ability to promote cancer cell growth and protect cancer cells from chemotherapeutics. Comparison with a conventional 2D in vitro model was performed. Scaffolds were also assessed for their ability to support in vitro culture of primary MM cells using confocal microscopy.
MM-MSCs differed from ND-MSCs at miRNA, mRNA, and functional levels. MM-MSCs proliferated slower than ND-MSCs and direct, but not indirect, co-culture of ND-MSCs with MM.1S was able to recapitulate this slowed proliferation in vitro. Indeed, we found that 22 microRNAs were significantly dis-regulated (14 up-regulated and 8 down-regulated in MM patients versus healthy individuals). These included up-regulation of miRNA-222, -181, -146, and -382; together with down-regulation of miRNA-15a, -143, and -199a, in MM-derived hMSCs, compared to ND-MSCs (p<0.05). Moreover, gene expression profiling showed higher expression of CDKN1A, CDKN2A, STAT1, FOSL2, and BCL6 mRNAs, in MM- versus ND- MSCs (fold changes of 3.94, 2.3207, 2.3207, 1.735 and 1.933 respectively, all p<0.05 value).
MM1S-GFP+/Luc+ cells were seeded onto scaffolds in presence of either normal- or MM-derived MSCs, in the presence or absence of bortezomib (5nM), and cultured for up to 1 month. Importantly, the 3D model demonstrated stromal-induced protection of MM1S, thus allowing for a more realistic culture system. MM1S cells were protected from bortezomib-induced death by stroma in both 2D and 3D culture over a short time period (48 hours), but, after a 2 week period, this protection was only found in 3D, compared to the 2D model, where no MM1S remained (with or without stroma). Stroma-seeded scaffolds also showed evidence of enhancing culture longevity of primary patient MM cells.
MiRNA, mRNA, and functional differences between ND- and MM- MSCs demonstrate changes induced in local stroma in response to MM cells that may encourage tumor growth and require further investigation. The different growth patterns and drug responses of MM.1S to therapies in 2D vs 3D culture suggests that a 3D co-culture environment may represent a more realistic model of drug resistance and minimal residual disease than 2D or monoculture models. Both MM and ND- MSCs were able to induce protection of MM.1S cells in the 3D environment; the mechanisms behind this protection need to be characterized to increase efficacy of current therapeutics within the bone marrow.
Ghobrial:Millennium: Advisory Board Other; Novartis: Advisory Board, Advisory Board Other.
Asterisk with author names denotes non-ASH members.