Myeloma Cell Interaction with Mesenchymal Stem Cells: Heterogeneity of Myeloma Cell Survival and Gene Expression Studies
Authors: Entin Igor , Herman Damir , Williams David R. , Freeman John , Shaughnessy John D , Barlogie Bart , Epstein Joshua 
Published: April 2020

Abstract
Myeloma is intimately associated with osteolytic bone disease, in part by inhibiting mesenchymal stem cell (MSC) differentiation into osteoblasts via blocking Wnt signaling (Tian, N Engl J Med2003;349:2483). Stimulation of osteoblast activity by bortezomib is associated with anti myeloma effects in patients (Zangari, Br J Haematol2005;131:71) and by MSC infusion in a model system (Yaccoby, Haematologica2006;91:192). To further evaluate the potential therapeutic utility of MSCs, we investigated the effects of MSCs on myeloma cell survival and the genomic consequences of MSC-myeloma cell interactions. CD138-purified myeloma (MM) cells from 16 consented patients were cocultured with MSCs derived from the bone marrow of 3 healthy donors (MSC 1, 2, &3, provided by Dr. D. Prockop, Tulane University). In 7 co-culture experiments, MSCs supported MM cell survival for 6–8 days compared to MM cells cultured alone, with a median viable cell count increasing by 2.3 fold v controls (1.2–4.1, p=0.003); in 4, coculture suppressed MM survival (median=0.5, range 0.3–0.7, p=0.016); and in 5 there was no effect (median=0.9, range 0.9–1.0). In 3 experiments, the effects of the different MSCs on MM cell survival varied inconsistently among the 3 MSCs, from 0.5 to 2.5 fold surviving cells. In order to understand this heterogeneity, we investigated the genomic consequences of MM-MSC interactions. RNA was extracted immediately after mixing (t=0) and following 18 hours co-culture (t=18), and changes in gene expression analyzed using the Affymetrix microarray system. Since myeloma cells adhere tightly to MSCs, we analyzed expression changes in 1,708 genes expressed only in MM cells and 4862 expressed only in MSCs. At t=18, 250 MM cell genes were changed by &gt;2 fold compared with t=0 (222 up and 28 down regulated), and 1,036 MSC genes were changed &gt;2 fold (1018 up and 18 downregulated). Each of the 3 MSCs also had unique genes expressed (40, 85 and 162 for MSC 1, 2, &3, respectively), of which expression of 12, 30, and 39, respectively, was changed by &gt;2 fold. Analysis of 776 MM- and 2398 MSC-related genes after a signal cutoff of 500 with Ingenuity Pathways Analysis software showed changes in the expression of several groups of interrelated genes following co-culture. For MM, these included ERKs, AKT, NFKB, E2F1, and FOS. The transcription regulators BTG2, ACTN2, CALR, E2F1, E2F5, and ATF7 were up regulated, while FOS and FOXM1 were down regulated. Groups changed in MSCs included IL1B, CCND1, MYC, CTBP1, EGFR, RUNX1, HRAS, and SMAD3. There were minor differences in changes of the expression patterns of some of the probesets between the three MSCs, likely related to alternative splicing or to variations in the 3′-UTR. These studies continue in order to discern possible differences in the interactions between MM cells and MSCs, and to identify whether MM cells or MSC determine the outcome of these differences.