Analysis of Intratumor Heterogeneity in Multiple Myeloma Using Methylcellulose Clonogenic Assays.

Abstract Multiple myeloma (MM) is a fatal disease characterized by the accumulation of malignant plasma cells in the bone marrow. Although progress has been made in better understanding growth control of this disease, effective treatment of MM patients with this disease is likely complicated by the extensive patient to patient variability that exists, as well variability within the tumor population itself. Thus, there is abundant evidence that intraclonal or intratumor heterogeneity exists in myeloma as revealed by morphologic and phenotypic heterogeneity in primary myeloma cells isolated from a single patient. We have had a long-standing interest in the growth regulation of myeloma cells and have hypothesized, along with other investigators, that there may only be a subset of myeloma cells that exhibits extensive proliferative potential. Understanding how cellular compartments within the malignant clone, as defined by identical immunoglobulin variable region sequence, may vary in growth regulation properties in isolation or in the company of less proliferative tumor cell subsets is key to understanding disease progression and how to better target the putative proliferative subset in myeloma. In this study, we have used a methylcellulose clonogenic assay to study intraclonal heterogeneity in a panel of human MM cell lines. Each of these cell lines, DP-6, KAS-6/1, KP-6, and, exhibit a variable response to IL-6 and IGF-I, and our goal was to evaluate growth responsiveness of individual subclones from each of these cell lines. Myeloma cell lines were plated at a concentration of 200-1000 cells in 1 ml Methocult H4533 in 35 mm gridded dishes with or without various cytokines. Following 3 weeks of culture, colonies were scored and those consisting of >40 cells were isolated, expanded, and studied further. Of interest, subclones isolated from each of the cell lines displayed significant differences in growth response to various cytokines in addition to specific morphologic and phenotypic differences. In this regard, results emerging from the DP-6 cell line were particularly intriguing. We have previously shown that the DP-6 cell line displays a proliferative response to both IL-6 and IGF-I and expresses autocrine IL-6 at a low level. Analysis of the growth properties of individual DP-6 clones revealed the existence of DP-6 cells (clone 1-15) that proliferate at a rapid rate in the apparent absence of exogenous growth factors. Whereas a neutralizing antibody to IL-6 did not inhibit cell growth, addition of a blocking antibody to the IGF-IR, (αIR3), completely blocked growth factor independent proliferation. Phenotypic analysis also displayed variation between the parental cell line and its subclone. For instance, the parental DP-6 cells largely expressed CD45 at a high level, whereas the clone 1-15 did not. Finally, we have also further characterized MM cell line subclones by gene profiling and FISH (fluorescence in situ hybridization) analysis to link specific phenotype and genotypes with patterns of cell growth. These results provide additional evidence that intratumor heterogeneity exists in myeloma. These studies further demonstrate how growth regulation may vary considerably among cellular subsets of the malignant population. Understanding what factors regulate the balance of specific myeloma cell subpopulations is key to an understanding of tumor progression. In summary, these studies provide a necessary foundation for future studies of the growth potential of subsets found in primary MGUS, SMM and MM patient samples.