A New Model for Studying the Dissemination of Myeloma Cells throughout the Bone Marrow Using Embryonic Zebrafish

Multiple myeloma develops from a pre-malignant clonal proliferation of plasma cells. The dissemination of myeloma cells throughout the bone marrow (BM) is an important early step in myeloma pathogenesis. Studies of myeloma cell homing in mouse models are not quantitative and cannot be used for functional genomics or drug screening. To overcome these limitations, we have developed a novel in vivo model to assess multiple myeloma (MM) cell homing, that takes advantage of the optical clarity of zebrafish (Danio rerio) embryos. We performed intra-cardiac (i.c.) injection of GFP+MM.1S cells into zebrafish embryos 48 hpf, and assessed the ability of the injected cells to enter the peripheral circulation and then traced their homing to the trunk region of zebrafish embryos, an area referred as the caudal hematopoietic tissue (CHT), by using intra-vital confocal microscopy. We next injected primary CD138+ cells derived from MM patient BM that had been stained with either DiO or DiD and demonstrated that they also homed to CHT. As a control, we injected DiO-labeled CD41-GFPlow zebrafish hematopoietic stem cells (HSCs) that are known to home to the CHT hematopoietic niche; and observed that zebrafish-derived CD41-HSCs homed to the same area as MM cells. We next analyzed changes in the transcriptome of those MM cells that homed to the CHT-niche. We dissected the zebrafish embryos to separate the CHT from other tissue containing non-adherent MM cells and performed whole human exome enrichment prior to sequencing of total RNA. We had an alignment rate of 10-15%, with a high intragenic rate an exonic rate (> 95%) and a low mismatch rate (~0.5%). RNAseq revealed that the MM cells that homed to the CHT were enriched in transcripts important for cytokine/chemokine mediated signaling, the IL-6 signalling pathway, cell-cell adhesion and angiogenesis (FDR These findings demonstrate that zebrafish can be used to study the homing of human myeloma cells to a hematopoietic niche. The rapidity of homing to CHT, which occurs within seconds of cell injection, suggests that a fraction of the CD138+ harvested from patient bone marrow already express those RNA transcripts and proteins needed for stable adhesion and residence in CHT. This hypothesis is confirmed by the RNA seq and qRT-PCR studies which directly demonstrate increased expression of relevant transcripts in adherent cells. This zebrafish model may provide new insights into the pathogenesis of MM and may be useful as a means to screen for agents which can disrupt homing and dissemination of MM cells. Disclosures No relevant conflicts of interest to declare.