Novel Model To Evaluate Changes in Gene Expression Profile of Myeloma Cells In Vivo Following Interaction with Human BM Microenvironment.

Multiple Myeloma (MM) cells interact with bone marrow (BM) microenvironment leading to induction of adhesion-mediated and cytokine mediated cell signalling which plays a critical role in promoting MM cell growth, survival, migration and development of drug resistance. We have previously evaluated gene expression changes following interaction between MM cells and BM stromal cells in vitro. However, the interaction between MM cells and microenvironment cells within the bone marrow is unique and its understanding is critical in evaluating effects of novel agents. We here describe a unique model that allows us to analyse in vivo expression changes in MM cells within the human BM milieu; and present preliminary results of expression changes following these in vivo interactions. In this model, BM stromal and IL-6-dependent human MM cell line INA-6 tranduced with GFP (green fluorescent protein) was injected in human fetal bone chip transplanted into SCID mice (SCID-hu mice). The MM cells were allowed to interact with the bone marrow for variable length of time, the bone chip was then retrieved, cells flashed out and GFP+ MM cells were separated by flow cytometry. The GFP negative fraction, containing stromal elements was also separated. Similar flow isolation process was used on INA-6^GFP+ cells cultured in vitro and used as control. Total RNA was isolated from these cells and gene expression profile analyzed using the HG-U133 array chip (Affimetrix). We report that interaction between INA-6 cells and the BM microenvironment in vivo induced significant changes in expression profile. In particular, we observed up-regulation of genes implicated in regulation of cell proliferation (RGS 1 and 2, FOS, FOSB, S100A4); DNA transcription (AP1, SWI/SNF related member 1); chromosome organization (Histone1, 2 and 3); cellular trafficking and transport (ARFGEF2, Aquarin 3 and ATPase 4B); and signal transduction (Chemokine ligand 2, 3 and 15, Chemokine receptor 1, 2 and 4, Dual specificity phosphatase 1 and 4, Protein tyrosine phosphatase 1, PIP5-kinase 1A and ZAP70). We also observed down-regulation of genes involved in apoptosis (BCL2-interacting killer, APC, E1A binding protein p300, Fas-associated via death domain, Caspase-activated Dnase, Raf1); and cell-cell adhesion molecules (Cadherin 15, Leupakin, Neurekin, CD44, ICAM2 and PECAM-1a). Although some similarities were observed in gene profile changes following in vitro and in vivo interaction with microenvironment cells, differences were also found. We are now evaluating the effects of interaction on expression profile of stromal cells as well as duration of interaction. Taken together these data confirm the ability of BM microenvironment to modulate gene expression profile of the MM cells in vivo to mediate the MM cell growth, survival and migration. This model now provides us with an opportunity to study effects of novel agents on MM cells expression profile in vivo to pre-clinically characterize their activity.