Mesenchymal stromal cells from primary osteosarcoma are non-malignant and strikingly similar to their bone marrow counterparts Jan C. Brune 1 , Ariane Tormin 1 , Maria C. Johansson 2 , Pehr Rissler 3 , Otte Brosjo ¨ 4 , Richard Lo ¨fvenberg 5 , Fredrik Vult von Steyern 6 , Fredrik Mertens 7 , Anders Rydholm 6 and Stefan Scheding 1,8 1 Lund Stem Cell Center, University of Lund, Lund, Sweden 2 Department of Oncology, Lund University Hospital, Lund, Sweden 3 Department of Pathology, Lund University Hospital, Lund, Sweden 4 Department of Orthopedics, Karolinska Hospital, Stockholm, Sweden 5 Department of Orthopedics, Umea ˚ University Hospital, Umea ˚, Sweden 6 Department of Orthopedics, Lund University Hospital, Lund, Sweden 7 Department of Clinical Genetics, Lund University Hospital, Lund, Sweden 8 Department of Hematology, Lund University Hospital, Lund, Sweden Mesenchymal stromal cells (MSC) are multipotent cells that can be isolated from a number of human tissues. In cancer, MSC have been implicated with tumor growth, invasion, metastasis, drug resistance and were even suggested as possible tumor- initiating cells in osteosarcoma (OS). However, MSC from OS and their possible tumor origin have not yet been thoroughly investigated. Therefore, primary OS mesenchymal progenitors and OS-derived MSC were studied. OS samples contained very high frequencies of mesenchymal progenitor cells as measured by the colony-forming unit fibroblast (CFU-F) assay (median: 1,117 colonies per 10 5 cells, range: 133–3,000, n 5 6). This is considerably higher compared to other human tissues such as normal bone marrow (BM) (1.3 6 0.2 colonies per 10 5 cells, n 5 8). OS-derived MSC (OS-MSC) showed normal MSC morphology and expressed the typical MSC surface marker profile (CD105/CD73/CD90/CD44/HLA-classI/CD166 positive, CD45/CD34/CD14/CD19/HLA-DR/CD31 negative). Furthermore, all OS-MSC samples could be differentiated into the osteogenic lineage, and all but one sample into adipocytes and chondrocytes. Genetic analysis of OS-MSC as well as OS-derived spheres showed no tumor-related chromosomal aberrations. OS-MSC expression of markers related to tumor-associated fibroblasts (fibroblast surface protein, alpha-smooth muscle actin, vimentin) was comparable to BM-MSC and OS-MSC growth was considerably affected by tyrosine kinase inhibitors. Taken together, our results demonstrate that normal, non-malignant mesenchymal stroma cells are isolated from OS when MSC culture techniques are applied. OS-MSC represent a major constituent of the tumor microenvironment, and they share many properties with BM-derived MSC. Stroma is a major and important constituent of solid tumors which provides the microenvironment for the malignant cells, and its essential role for tumor growth, progression, metastasis and resistance to therapy has recently been demonstrated. 1 Tumor stroma enhances tumor cell proliferation by pro- duction of cytokines (insulin-like growth factor [IGF], plate- let-derived growth factor [PDGF], epidermal growth factor [EGF], etc.), 2,3 and data from studies with human breast Key words: osteosarcoma, osteogenic sarcoma, mesenchymal stromal cells, cancer associated fibroblasts, tumor stroma Abbreviations:: MSC: Mesenchymal stromal cells; OS: osteosarcoma; CFU-F: colony forming unit-fibroblast; OS-MSC: OS-derived MSC; BM-MSC: bone marrow-derived MSC; DPBS: Dulbecco’s phosphate buffered saline; EGF: epidermal growth factor; LIF: leukemia inhibitory factor; FITC: fluorescein isothiocyanate; PE: Phycoerythrin; APC: allophycocyanin; ISCN: International system for human cytogenetic nomenclature; FISH: fluorescence in-situ hybridization; DAPI: 4 0 ,6-diamidino-2-phenylindole; 5-aza: 5-aza-2 0 -deoxycytidine; FSP: fibroblast surface protein; a-SMA: alpha-smooth muscle actin; VIM: vimentin; SEM: standard error of the mean; SAM: significance analysis of microarrays; IM: Imatinib; NI: Nilotinib. Additional Supporting Information may be found in the online version of this article Grant sponsors: Swedish Childhood Cancer Foundation, Swedish Research Council, Crafoord Foundation, Gunnar Nilsson’s Cancer Foundation, Lundgren Foundation, John and Augusta Persson Foundation, University Hospital of Lund Funds, ALF (Governmental Public Health Grant) DOI: 10.1002/ijc.25697 History: Received 21 Apr 2010; Accepted 7 Sep 2010; Online 28 Sep 2010 Correspondence to: Stefan Scheding, MD, Lund Stem Cell Center, BMC-B10, Klinikgatan 26, S-22184 Lund, Sweden, Tel: þ46 46 222 3331, Fax: þ46 46 222 3600, E-mail: stefan.scheding@med.lu.se Cancer Cell Biology Int. J. Cancer: 129, 319–330 (2011) V C 2010 UICC International Journal of Cancer IJC