Abstract
Malignant plasma cell (PC) disorders are clonal diseases based on their immunoglobulin variable region sequence signature. However, intratumor phenotypic, genetic, and morphologic heterogeneity is often observed, fueling the notion that a subpopulation of the malignant PC clone may have extensive self-renewal properties whereas others may not. Little information exists regarding the relationship between these putative tumor subpopulations that is instrumental to our understanding of the growth, progression and therapeutic targeting of PC disorders. We recently described establishment of two novel cell lines from one patient initially diagnosed with primary amyloidosis (ALMC-1), who eventually progressed to a combination of AL and symptomatic multiple myeloma (MM; ALMC-2). Although both cell lines are clonally related, they exhibit a number of intriguing differences, including a marked variability in their makeup of mononuclear (MoN) and multinuclear cells (2 − >10 nuclei; MuN). The ALMC-1 cell line consistently displays a much larger fraction of MuN cells (~50%) than the ALMC-2 cell line (~20%). By contrast, the growth rate of the ALMC-2 cells is significantly higher than ALMC-1 cells suggesting an inverse relationship between growth rate and fraction of MuN cells. It has been demonstrated that MoN plasmablastic morphology is a predictor of poor survival in MM, however the clinical significance of MuN PCs, which are frequently observed in bone marrow biopsies, has yet to be determined. We therefore used the ALMC-1 and ALMC-2 cell lines to begin to test the hypothesis that the MoN fraction in both cell lines contains cells with extensive self-renewal properties whereas the MuN cells are no longer capable of cell division. The central questions become by what mechanism are MuN PCs generated and how are MoN PCs maintained? Two hypotheses that can explain the dual persistence of both types of cells are:
random cell fusion; regulated or sporadic cell division of MoN cells into 2 daughter cells, one of which divides normally and the other one fails to undergo cytokinesis.
To address the first possibility, each cell line was transfected with GFP or YFP. Single positive (GFP or YFP) cells were then mixed and cocultured for assessing the frequency of dual positive cells by flow cytometry at various time points. However, studies to date suggest this mechanism does not occur. To begin to address the second possibility and determine whether extrinsic factors, e.g., cytokines, may regulate the rate of generation of MuN PCs, we cultured both cell lines +/− IL-6 and IGF-I and assessed numbers of MoN and MuN PCs. Initial analyses suggest that IL-6, but not IGF-I, increases the proportion of MoN PCs in both cell lines. To address the hypothesis that MuN PCs are no longer capable of growth, cells were labeled with bromodeoxyuridine (BrDU) to assess the capability of MuN cells to synthesize DNA. We observed that MuN PCs incorporating BrDU did so uniformly in each nucleus present in the cell. We then assessed the ability of cell division of MuN cells by quantitating normal vs abnormal metaphases in both cell lines and observed that >90% of metaphases were present in MoN PCs, suggesting that MoN PCs largely give rise to MuN PCs and the latter are likely compromised in cytokinesis. As centrosomes play a critical role in regulating cell division and centrosome abnormalities have been noted in MM by others, we next used gamma-tubulin immunofluorescence to detect centrosomes. MoN PCs exhibited uniformly normal centrosome numbers and positioning while MuN PCs showed clear centrosomal abnormalities including accumulation of centrosomes, centrosome clustering, and multiple directional centrosome polarization, suggesting a mechanism by which the MuN cells may be generated. In summary, our data suggest that MuN PCs lack growth potential and that intrinsic and/or extrinsic factors may drive accumulation of these cells. These observations support the possibility that patients with a larger proportion of MuN PCs may have differences in clinical outcome from those patients with largely MoN PCs. To test this, we have begun hematopathological analysis of a large series of patients diagnosed with various plasma cell disorders. Knowledge of the signals that regulate the balance between MoN and MuN cells could lend itself toward identification of therapeutic modalities that may maximize differentiation of tumor cells into end-stage cells.
A Millifluidic Study of Cell-to-Cell Heterogeneity in Growth-Rate and Cell-Division Capability in Populations of Isogenic Cells of Chlamydomonas reinhardtii
