Bone Microenvironment Cells Show a Different Pattern of Gene Expression Profiling in Relationship with the Presence of Osteolytic Bone Lesions in Multiple Myeloma Patients
Authors: Todoerti Katia , Lisignoli Gina , Colla Simona , Storti Paola , Agnelli Luca , Manferdini Cristina , Verdelli Donata , Crugnola Monica , Codeluppi Katia , Deliliers Giorgio Lambertenghi , Rizzoli Vittorio , Neri Antonino , Giuliani Nicola 
Published: April 2020

Abstract
Multiple myeloma (MM) is a plasma cell malignancy with the high capacity to induce osteolytic bone lesions. Whereas previous studies identified genes overexpressed by MM cells related to the bone status, the occurrence of transcriptional alterations in the bone microenvironment cells in the relationship with the bone involvement has not yet investigated. To clarify this issue, in this study we have analyzed the gene expression profiling of mesenchymal (MSC) and osteoblastic (OB) cells obtained from MM patients (n=24; osteolytic n=10; non-osteolytic n=14) in relationship with the presence or absence of osteolytic bone lesions. MGUS subjects (n=7) and healthy donors (n=8) were also included in the study as controls. Both MSC and OB were isolated from trabecular bone biopsies without in vitro differentiation. The presence of potential contaminating cells was excluded by FACS analysis in both MCS and OB, testing CD3, CD14, CD20 and CD138 antigens, as well as the expression of CD105 and CD146; the osteoblast-related markers Osteocalcin, Alkaline Phosphatase, Collagen I and Runx2 were evaluated in OB in comparison with MSC. Thereafter a gene expression profiling analysis of isolated MSC and OB cells was performed using GeneChip® HG-U133A oligonucleotide arrays. The obtained data were validated by real time PCR. Using conventional hierarchical clustering, the unsupervised analyses performed of the whole dataset generated a dendrogram clearly distinguishing MSC and OB cellular types. When considering MSC and OB dataset separately, a preferential clustering in relation to the presence of osteolytic bone lesions was observed for MSC but not OB samples. A supervised multi class analysis identified a total of 84 probe sets differentially expressed in MSC with an intermediate transcriptional profile in MGUS-MSC between osteolytic and non osteolytic MM patients. A supervised analysis performed on MSC MM samples revealed a total of 49 probe-sets (36 up-regulated and 9 down-regulated genes) as differentially expressed in osteolytic vs. non-osteolytic patients. Specifically, genes belonging to Wnt signaling as WNT6 and extracellular matrix structure as decorin (DCN) were found to be down-regulated in osteolytic as compared to and non-osteolytic MSC. Interestingly, no significantly modulated genes were found by comparing osteolytic and non-osteolytic OB samples. Finally, we performed two distinct supervised analyses by comparing the two cellular types (MSC and OB) in the two groups of MM patients in relationship with the bone status. A distinct transcriptional pattern was observed in MSC versus OB between osteolytic and non-osteolytic MM patients (52 vs. 21 differentially expressed probe-sets, respectively), mainly involving cell-cycle realted genes. Our results highlight that in MM bone microenvironment MSC rather than OB show transcriptional alterations in relationship with the presence of osteolytic bone lesions in MM patients.