PhD Candidate University of Adelaide, SAHMRI ADELAIDE, South Australia, Australia
Introduction: Bone marrow (BM) adipocytes (BMADs) make up 70% of the BM microenvironment and can promote multiple myeloma (MM) plasma cell proliferation and survival in vitro. The phenotypes of BMADs in MM and monoclonal gammopathy of undetermined significance (MGUS), and how BMADs support MM progression remain largely unknown. Here, we aimed to assess BMAD phenotype in differentiating and mature BMADs in BM samples from MGUS and MM patients and analyse their gene expression profile, to identify potential secreted factors which may play a role in supporting MMPC growth and survival.
Methods: BMAD quantitation was conducted on CD138-stained trephines from age-matched controls (n=4), MGUS (n=9), and MM (n=14) patients via Osteomeasure and NDP.view2. Mesenchymal stromal cells (MSCs) isolated from control (n=7), MGUS (n=14), and MM (n=14) trephines were cultured in adipogenic media for 21 days, stained with Nile Red/DAPI and ImageJ used to quantitate BMAD number and lipid droplets/BMAD. For single cell RNA sequencing (scRNAseq) cells were enzymatically digested from 5 MM patient trephines (n=2 newly diagnosed, n=3 treated) and 1 control, viable GLYA- cells FACs sorted and scRNAseq conducted (10x Genomics Chromium V3.1). To identify novel microenvironmental factors that may interact with PCs, gene expression of secreted or cell surface ligands (ConnectomeDB2020) was assessed in sequenced cells, with cognate receptor expression in MM PCs confirmed in RNA sequencing data from 764 newly diagnosed MM patients (CoMMpass). Statistical analyses included Kruskal-Wallis, log-rank tests, and Pearson’s correlation.
Results: Histomorphometric analysis revealed MM patients with larger mean BMAD size (>2100um2) had significantly poorer overall survival, compared with patients with smaller BMADs (median survival: 113.3 and 231.1 weeks; p=0.045, log-rank test). In vitro, there was two-fold reduction in BMAD number and 2.6-fold reduction in lipid droplets/BMAD in MM (p=0.0048), compared with MGUS. scRNAseq of 13683 BM cells, identified 81 mesenchymal lineage cells, characterised by CXCL12, FABP4, and SPP1 expression. 32% of the cluster were FABP4+ adipocyte-lineage cells, and uniquely expressed genes for 14 secreted or cell surface ligands, when compared with other MSC lineages. Furthermore, the receptor for 10/14 of these ligands were expressed by MMPCs in most of newly diagnosed MM patients, suggesting the potential for crosstalk between BMADs and MMPCs.
Conclusions: Decreases in MM BMADs and lipid droplets demonstrated altered morphology and differentiation capacity in vitro, whilst histomorphometric analyses suggest association between BMAD phenotype and MM progression. For the first time, scRNAseq on MM trephines identified FABP4+ adipocyte-lineage cells express factors that directly interact with MMPCs. These findings help characterise BMADs as sources of MM-supportive factors, with further studies focussing on the identified factors and potential roles in MM progression.
