Abstract
To identify new potential therapeutical targets in multiple myeloma (MM), we have defined the phenotype of the subset of proliferative myeloma cells (n=66) in comparison with that of normal PC (n=25). Proliferation was evaluated by ex vivo incorporation of BrdU (labeling index, LI). Surface PC phenotype was performed in a four-color assay with CD38, CD45, CD138 and the mAb indicated. For intracellular BrdU staining, cells were first labeled with CD38, CD45 and CD138, fixed and permeabilized before BdrU staining. At least 1000 normal PC and 10000 myeloma cells were analyzed. We show that all bone marrow PC, either malignant or normal, always included a subset of proliferative PC (BrdU+) that was always located within the CD45++subpopulation. Indeed, CD45++ myeloma cells (median 12%) had a labeling index 7.5-fold higher of that of CD45+/− myeloma cells (7.1% versus 0.94%). Actually, in all cases of MM, CD45++ myeloma cells were always the most proliferative myeloma cells. As observed for myeloma cells, LI of normal PC was heterogeneous i.e., higher in the CD45++ population of PC: CD45++ PC (median 65%) had a LI 5.7-fold higher of that of CD45+/− PC. Compared to myeloma cells, LI of PC were higher in both subsets, of 20.5% and 3.6% for CD45++ and CD45+/−, respectively. Non-malignant PC from blood or tonsil were homogeneously CD45++ and did proliferate (LI> 10% and up to 45% for reactive PC). In all PC (normal, reactive, malignant), we found an inverse correlation between CD45 and Bcl-2, confirming a known inverse correlation between proliferation and Bcl-2 expression. Our data suggest that a minor cycling Bcl2lowCD45++ population of myeloma cells differentiate into a no more cycling major Bcl2high CD45+/− population of myelom a cells that accumulates.
We further characterized the phenotype of the CD45++ myeloma cells population: we found that CD11a and to a less extend HLA-DR were expressed by CD45++ myeloma cells only in contrast to CD40 and CXCR4 that were expressed by all myeloma cells. Moreover, all CD45++ myeloma cells coexpressed CD11a. Thus, the-to-be-killed population of myeloma cells could be targeted through CD45 or CD11a.
Whole-mount BrdU staining of proliferating cells by DNase treatment: application to postnatal mammalian retina