The Plasticity of Myeloma Plasma Cells as Expressed by Dedifferentiation into an Immature, Resilient, Apoptosis-Resistant Phenotype.

Progression of myeloma (MM) is considered to be a multistage and dynamic process of cell differentiation, survival, proliferation and dissemination. We have previously demonstrated the proliferative potential of purified CD45^lowCD38^high mature MM cells in SCID-hu mice (Yaccoby & Epstein, Blood, 1999), the ability of CD138-selected MM cells to produce myeloma in our novel SCID-rabbit model (Yata et al., ASH, 2003), and the interdependence of MM bone disease and tumor growth whereby MM cells induce osteoclast activity and are dependent on osteoclasts in vivo (Yaccoby et al., BJH, 2002) and ex vivo (Yaccoby et al., Cancer Res., 2004). The aim of this study was to determine the osteoclast-induced phenotypic changes associated with survival of MM cells in long term co-culture. CD138-selected (>95% purity) MM cells from 16 patients were co-cultured with human osteoclasts for up to 20 weeks. The pre-cultured baseline cells were typically CD45^{low/inermediate}CD38^high, CD19⁻CD34⁻. At the end of long term co-culture (>6 weeks) MM cells had BrdU labeling index (LI) of 2.5±2.0 and their viability was 97%±1%. The phenotype of co-cultured MM cells consistently shifted to a less mature phenotype, with CD45 expression increasing from CD45^low to CD45^{intermmediata/high} and reduced expression of CD38 from CD38^high to subpopulations with CD38^intermediate levels, as determined by flow cytometry and confirmed by qRT-PCR. Further flow analysis revealed that co cultured MM cells also expressed low levels of CD19 and CD34, and identified a small subpopulation of CD138^lowCD45^high MM cells. Morphologically, the co-cultured MM cells uniformly gained plasmablastic characteristics when compared to pre-cultured cells. Previous reports suggested that IL-6 was important for maintaining subpopulation of CD45-expressing MM cells. However, blocking IL-6 activity in co-cultures with anti-IL6 and anti-IL6R neutralizing antibodies (5 μg/ml, each) did not affect the immature phenotype of MM cells. Intriguingly, long term co-culture of normal CD34⁺ hematopoietic stem cells (HSCs) with osteoclasts results in loss of CD34 expression, suggesting a common mechanism for osteoclast-induced MM PC and HSC plasticity. To investigate if the observed phonotypic changes are associated with apoptosis resistance, we determined the effects of 3 days exposure to the pro-apoptotic agent dexamethasone (DEX, 10⁻⁷ M) on MM cells cultured alone or in co-cultures (n=5), at initiation (baseline) and after 6 weeks of co-cultures. The percent apoptotic cells was determined by trypan blue exclusion and annexin V flow cytometry. When baseline MM cells were cultured alone, DEX significantly increased the percent of apoptotic cells over that spontaneous rate (p<0.01). In contrast, when MM cells recovered from co-cultures after 6 weeks they survived and were resistant to DEX-induced apoptosis (16%±11% and 24%±21% apoptotic cells in the absence and presence of DEX, respectively). As reported, osteoclasts supported survival of MM cells at baseline and after 6 weeks of co-culture (p<0.01), and protected MM PCs from DEX-induced apoptosis. Our data demonstrate the phenotypic plasticity of primary myeloma cells, whereby mature MM cells are reprogrammed and acquire autonomous survival properties after co-culture with osteoclasts. We hypothesize that in vivo these cells are dormant, resistant to spontaneous and drug-induced apoptosis, and could be responsible for relapse.