Multiple myeloma (MM) is characterized by a close relationship between tumor and bone microenvironment cells, which supports the survival of MM cells and determines a severe impairment of bone homeostasis.1 Mesenchymal stromal cells (MSCs) and osteoblasts (OBs) assume a critical role in this process either through the overproduction of growth factors and osteoclastogenic molecules or by undergoing alterations responsible for the suppression of bone formation that rarely repairs even after disease remission.2 This suggests that both types of cells could be primarily involved in the tumoral process. Alterations of gene expression and genomic profiles have been reported in MSCs of MM patients in comparison with healthy donors as published in Leukemia.3, 4 Moreover, we have recently shown the occurrence of different patterns of gene expression profile, not only in MSCs but also in OBs of MM patients compared with healthy individuals and in relationship to MM bone disease.5 Nevertheless, it is still unclear whether these bone microenvironment cells in MM patients are primarily tumoral. To clarify this issue, we analyzed the genomic profiles and the presence of telomere maintenance mechanisms of bone-derived MSCs and OBs obtained from a cohort of 26 MM patients at diagnosis, 7 monoclonal gammopathy of undetermined significance (MGUS) patients and 8 healthy donors (N) who underwent orthopedic surgery. Samples were checked for gene expression profiling by microarray as published earlier.5 Baseline characteristics of the MM patients together with genetic alterations of purified CD138+ MM cells are reported in Table 1.
Mesenchymal stromal cells were isolated from bone biopsies obtained from the iliac crest and were collected in V-shaped glass tube and extensively minced with surgical scissors. The supernatants were collected, and the MSCs were isolated using a Ficoll–Hypaque density gradient (Pharmacia Biotech, Uppsala, Sweden). After 1 week, the nonadherent cells were removed and the adherent cells were analyzed at different passages (p1–p3). OB cells were directly isolated from the minced bone chips as described earlier.6 The chips were digested using 1 mg/ml collagenase P (Boehringer Mannheim Corporation, Indianapolis, IN, USA) at 37 °C for 2 h under rotation, washed and placed in 100 × 20 mm dishes in Dulbecco's modified Eagle's medium/Ham's F12K without a calcium-supplemented medium (Gibco, Invitrogen, Scotland, UK) and then fed with a complete medium twice a week. After 2 weeks the chips were removed and the OBs were allowed to grow until confluence and analyzed at different passages.
The presence of the contaminating hemopoietic and MM cells was excluded by FACS analysis (BD Biosciences) in both MSCs and OBs, testing CD3, CD14, CD20 and CD138 antigens as well as CD105 and CD146 antigen expression. Moreover, the OB markers, such as osteocalcin, alkaline phosphatase, collagen I and Runx2, were evaluated in OBs compared with MSCs. As reported earlier,5 no significant difference in the immunophenotype and cell proliferation patterns of MSCs and OBs was found across MM, MGUS and N groups, whereas different patterns of gene expression profile were evidenced between MM patients and N subjects in both MSCs and OBs.
The genetic characterization of MSCs and OBs was performed by a molecular karyotyping analysis at different passages of the culture through whole-genome array-comparative genomic hybridization (array CGH) using the 105K platform (Agilent Technologies, Santa Clara, CA, USA) with an average resolution of about 60 kb. Experiments were performed according to the manufacturer's protocol v 5.0. For each patient DNA derived from CD3+ cells, isolated by an immunomagnetic method from peripheral blood (MACS; Miltenyi, Bergisch Gladbach, Germany) or saliva was used as control DNA in all experiments. Interestingly, despite the presence of genetic alterations in CD138+ MM cells in the majority of MM patients, no specific chromosomal imbalances were observed in MSCs and OBs from either MM or MGUS patients, as shown for three representative samples (Figure 1a), with the exception of OBs of two MM patients in whom we found chromosomal trisomies in the later culture passages. In addition, fluorescence in situ hybridization (FISH) analysis was performed on MSCs and OBs of selected cases in whom MM cells carried chromosomal abnormalities failed to show specific translocations or 13q deletions (data not shown).
Earlier evidence indicated that long-term cultures of murine bone-marrow-derived MSCs increased telomerase activity (TA) and proceeded to malignant transformation.7 Here we evaluated the occurrence of the two known telomere maintenance mechanisms, TA and alternative lengthening of telomere (ALT), in cultures in early passages of MSCs and OBs in an attempt to investigate the potential susceptibility of MSCs to undergoing malignant transformation. TA was determined using the telomeric repeat amplification protocol, a PCR-based method in which telomerase extends a radiolabeled synthetic primer resembling telomeric DNA,8 in isolated cells. ALT was detected by assaying ALT-associated promyelocytic leukemia (PML) nuclear bodies, using a combined technique of PML immunofluorescence and telomere FISH,8 in fresh cultures obtained from MM and MGUS patients. Notably, no alteration was found in all tested MSCs and OBs regarding either TA (Figure 1b) or ALT (data not shown), suggesting that both cell types do not have an immortalized phenotype.
Our study data indicate that bone OBs and MSCs do not seem to be part of the tumor clone. Accordingly, it has been previously reported that MSCs are cytogenetically normal by FISH analysis, supporting the notion that MM cells and MSCs have no common progenitor.4 Clearly, our study data should be confirmed in a larger set of patient-derived samples. At present, there are no published studies of isolated OBs in MM patients. In our study, we isolated both MSCs and OBs directly from the bone at the first passage rather than from the previously used bone marrow aspirate.3, 4 Overall, our study data indicate that in MM patients both MSCs and OBs do not express tumoral features, suggesting that the previously reported transcriptional alterations of MSCs and OBs6 in MM patients do not depend on primary karyotype or telomerase alterations, prompting further investigations including studies of DNA methylation profile and analysis of miRNA expression in MSCs and OBs.
Podar K, Richardson PG, Hideshima T, Chauhan D, Anderson KC . The malignant clone and the bone-marrow environment. Best Pract Res Clin Haematol 2007; 20: 597–612.
Giuliani N, Rizzoli V, Roodman GD . Multiple myeloma bone disease: pathophysiology of osteoblast inhibition. Blood 2006; 108: 3992–3996.
Corre J, Mahtouk K, Attal M, Gadelorge M, Huynh A, Fleury-Cappellesso S et al. Bone marrow mesenchymal stem cells are abnormal in multiple myeloma. Leukemia 2007; 21: 1079–1088.
Garayoa M, Garcia JL, Santamaria C, Garcia-Gomez A, Blanco JF, Pandiella A et al. Mesenchymal stem cells from multiple myeloma patients display distinct genomic profile as compared with those from normal donors. Leukemia 2009; 23: 1515–1527.
Todoerti K, Lisignoli G, Storti P, Agnelli L, Novara F, Manferdini C et al. Distinct transcriptional profiles characterize bone microenvironment mesenchymal cells rather than osteoblasts in relationship with multiple myeloma bone disease. Exp Hematol 2009; 38: 141–153.
Lisignoli G, Piacentini A, Toneguzzi S, Grassi F, Cocchini B, Ferruzzi A et al. Osteoblasts and stromal cells isolated from femora in rheumatoid arthritis (RA) and osteoarthritis (OA) patients express IL-11, leukaemia inhibitory factor and oncostatin M. Clin Exp Immunol 2000; 119: 346–353.
Miura M, Miura Y, Padilla-Nash HM, Molinolo AA, Fu B, Patel V et al. Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation. Stem Cells 2006; 24: 1095–1103.
Villa R, Daidone MG, Motta R, Venturini L, De Marco C, Vannelli A et al. Multiple mechanisms of telomere maintenance exist and differentially affect clinical outcome in diffuse malignant mesothelioma. Clin Cancer Res 2008; 13: 4134–4140.
This study was supported by grants from Regione Emilia-Romagna (NG, VR), Associazione Italiana Ricerca sul Cancro (AIRC) (NG, AN), the International Myeloma Foundation Award 2009 (NG) and a fellowship from Fondazione Italiana Ricerca sul Cancro (FIRC) (LA).
The authors declare no conflict of interest.
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Giuliani, N., Lisignoli, G., Novara, F. et al. Bone osteoblastic and mesenchymal stromal cells lack primarily tumoral features in multiple myeloma patients. Leukemia 24, 1368–1370 (2010) doi:10.1038/leu.2010.96
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