Investigating multiple myeloma disease progression through the characterisation of CD45+/- cells
2017-02-14T01:48:58Z (GMT) by
Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy predominantly negative for the protein tyrosine phosphatase CD45. Published data show that an increased proportion of CD45-ve rather than CD45+ve MM PCs correlates with poorer patient outcomes and increased risks of extramedullary disease. Furthermore, data has also suggested that disease progression is correlated with the transition from a predominantly CD45+ve to predominantly CD45-ve disease. The aim of this study was to investigate whether characterisation of the biological differences associated with variable CD45 expression might lead to the identification of a model of disease progression that could explain this apparent adverse clinical impact. Although MM cells are typically considered to be CD45-ve, variable CD45 expression has been observed in patients and a hierarchy of expression has been associated with disease progression. Therefore, before characterisation of the CD45+ve and CD45-ve cell populations could begin, representatives of each subset first needed to be identified. Several human myeloma cell lines (HMCLs) were first immunophenotyped to identify suitable candidates. Selection was primarily based on CD45 expression as well as expression of the IL-6 receptor (IL-6R) as CD45+ve cells are known to be preferentially responsive to IL-6 signalling. Four HMCLs were initially chosen to represent the CD45+ve (ANBL-6 & U266) and CD45-ve (KMS11 & KMS12BM) subgroups and the biologies of the HMCLs were characterised in terms of their signalling activity and responses to cytokines. However, due to overwhelming heterogeneity between the individual HMCLs, the selected representatives were deemed unsuitable for the characterisation of the CD45 subsets in this study. The U266 HMCL has a heterogeneous CD45 expression profile and distinct CD45+ve and CD45-ve populations that could be separated, thus providing a suitable isogenic model of the CD45 subsets, allowing the biologies of the cell populations to be characterised while minimising confounding factors due to heterogeneity between HMCLs. Following the identification of suitable representatives of the CD45+ve and CD45-ve cohorts, the differences in biological characteristics between the two subsets could be investigated. A range of parameters were examined including proliferation, signalling activity, cytokine production, response to soluble growth factors as well as response to a range of drug treatments. The CD45+ve fraction was observed to have a higher proliferative rate, responded preferentially to IL-6 treatment and was observed to be more susceptible to proteasome inhibitor treatment. In contrast, the CD45-ve subset was observed to secrete IL-6, was not found to respond to cytokines and was also more resistant to proteasome inhibitor treatment. This population also exhibited lower adhesion molecule expression as well as decreased homing capacity to stromal cells suggesting that it may be more prone to egress from the BM compared to CD45+ve counterparts. In addition to exhibiting distinct biological characteristics, the sorted CD45 populations also demonstrated properties that appeared to support the other cell population. Supernatant from the CD45+ve population promoted the proliferation of the CD45-ve subset whilst increased drug resistance was observed when the cells were in co-culture as well as when the cells were cultured in conditioned media from the other cell population. These results suggest that the CD45 subsets may cooperate together to promote tumourigenesis and overcome drug induced apoptosis. Furthermore, genetic analysis demonstrated that the 2 populations have distinct gene expression profiles and also identified the EMT and Notch pathways as possible mechanisms of disease progression that could account for the variable characteristics of the cell populations. In summary, a model of disease progression is proposed as thus – malignant PCs progress from a CD45+ve to a CD45-ve phenotype corresponding with increasing malignant and metastatic potential during MM pathogenesis. This process may be mediated by Notch-driven activation of EMT.