Mesenchymal Stem Cells (MSC) are employed in gene and cellular therapies. Routinely MSC are isolated from bone marrow mononuclear cells (MNC) by plastic adherence. Here we compared new isolation strategies of bone marrow MSC including immunodepletion of hematopoietic cells and immunomagnetic isolation of CD105 + and CD271 + populations. Four fractions were obtained: MNC MSC, RosetteSep-isolated MSC, CD105 + and CD271 + sorted MSC. We evaluated i) number of CFU-F colonies, ii) cell phenotype, iii) in vitro differentiation of expanded cells and iv) expression of osteo/adipogenesis related genes. Results: Average number of day 9 CFU-F colonies was the highest for CD271 positive fraction. Real-Time PCR analysis revealed expression of RUNX2, PPARγ and N-cadherin in isolated cells, particularly high in CD271 + cells. Expression of CD105, CD166, CD44, CD73 antigens was comparable for all expanded populations (over 90%). We observed various levels of hematopoietic contamination with the highest numbers of CD45 + cells in MNC-MSC fraction and the lowest in CD105 + and CD271 + fractions. Cells of all the fractions were CD34 antigen negative. Expanded CD105 and CD271 populations showed higher level of RUNX2, osteocalcin, PTHR, leptin, PPARγ2 and aggrecan1 genes except for α1 collagen. After osteogenic differentiation CD105 + and CD271 + populations showed lower expression of RUNX, PPARγ2 and also lower expression of osteocalcin and PTHR than MNC, with comparable α1-collagen expression. Chondrogenic and adipogenic gene expression was higher in MNC. More clonogenic CD105 + and particularly CD271 + cells, which seem to be the most homogenous fractions based on Real-Time PCR and immunostaining data, are better suited for MSC expansion.

Rhabdomyosarcoma (RMS) is the most common type of pediatric soft tissue sarcoma. The MET receptor has an important role in the biology of RMS, and its overexpression and hyperactivation correlate with the metastatic ability of RMS. Consequently, interfering with MET expression or functionality may constitute a sound strategy for reducing the progression and metastatic potential of RMS. Our study reveals that downregulation of the MET receptor leads to changes in the morphology of ARMS cell in vivo. Tumors acquire a spindle shape that is characteristic of muscle fibers. Inhibition of MET expression or function leads to (i) a decreased expression of the early myogenic marker MyoD, (ii) a decreased ability of ARMS cells to metastasize to bone marrow cavities, (iii) downregulation of CXCR4 receptor expression and (iv) a decreased migration of MET-depleted cells towards gradients of HGF and SDF-1. Finally, we demonstrate that in vitro differentiation of alveolar RMS cells decreases their metastatic behavior by reducing both the expression of the MET and CXCR4 receptors and their migratory response to HGF and SDF-1. These findings suggest that blockers of MET receptor function and inducers of RMS cells differentiation may be clinically useful for reducing the aggressiveness and metastatic potential of RMS and may have significant implications for its treatment.

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma, which may originate from impaired differentiation of mesenchymal stem cells (MSC). Expression of MET receptor is elevated in alveolar RMS subtype (ARMS) which is associated with worse prognosis, compared to embryonal RMS (ERMS). Forced differentiation of ARMS cells diminishes MET level and, as shown previously, MET silencing induces differentiation of ARMS. In ERMS cells introduction of TPR-MET oncogene leads to an uncontrolled overstimulation of the MET receptor downstream signaling pathways. In vivo, tumors formed by those cells in NOD-SCID mice display inhibited differentiation, enhanced proliferation, diminished apoptosis and increased infiltration of neutrophils. Consequently, tumors grow significantly faster and they display enhanced ability to metastasize to lungs and to vascularize due to elevated VEGF, MMP9 and miR-378 expression. In vitro, TPR-MET ERMS cells display enhanced migration, chemotaxis and invasion toward HGF and SDF-1. Introduction of TPR-MET into MSC increases survival and may induce expression of early myogenic factors depending on the genetic background, and it blocks terminal differentiation of skeletal myoblasts. To conclude, our results suggest that activation of MET signaling may cause defects in myogenic differentiation leading to rhabdomyosarcoma development and progression.

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma usually diagnosed in children. In advanced and metastatic stages the prognosis is often poor. RMS cell lines were used for evaluation of the role of MET receptor inhibition on chemotaxis and invasion. In vivo studies were performed using NOD-SCID xenograft model. This study shows that blocking of MET expression has strong influence on metastatic behavior of RMS. MET negative cells possess a reduced potential to migrate and to invade. Downregulation of MET suppressed the ability of RMS cells to populate bone marrow. Inhibition of MET negative tumor cells engraftment into bone marrow was observed. MET negative tumors were also two to four times smaller than their wild type counterparts. Since MET receptor plays a very important role in facilitating metastasis of RMS cells, blocking of HGF-MET axis might be considered as a therapeutic option for RMS patients, at more advanced and metastatic stages.

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Introduction: The molecular basis for loss of cell differentiation in the process of carcinogenesis is not well understood but differentiation status of the given tumor strongly influences its progression and metastatic ability and therefore the overall patient survival. Rhabdomyosarcoma (RMS) is one of most frequent soft tissue tumor occurring in children and adolescent. The precise molecular mechanism responsible for the disruption of myogenesis, characteristic for RMS tumors, is not fully understood. Activation of MET receptor has been shown to influence proliferation, survival and migration of RMS cells and MET is rapidly downregulated at the onset of myogenic differentiation. However, the influence of MET receptor on differentiation status of RMS has not been studied at all. Objectives: We have focused on the induction of differentiation as a novel approach for RMS treatment and looked at the role of MET receptor in this process. Materials and Methods: The expression of muscles specific genes (MyoD, Myogenin) was evaluated using RT-PCR and western blot in differentiated RMS cells and RMS cells with downregulated MET receptor. Number of apoptotic cells was studied by Annexin V binding assay. In order to study the differentiation potential of MET negative cells, RMS animal model in NOD-SCID mice was developed. We studied tumor morphology (H&E staining), maturity (desmin staining) and proliferation rate (Ki67 staining). Results: We found that in control cells (poorly maturated RH30 cell line), the expression of MyoD (present in undifferentiated muscle cells) was high whereas during differentiation it decreased. On the other hand, Myogenin expression (present in more matured muscle cells) markedly increased at the same time. We noticed increased number of apoptotic cells in differentiation medium with 2 % of horse serum. We also obtained RMS cell lines with stable downregulation of MET receptor. The expression of differentiation related genes in MET negative cells was similar to serum differentiated RMS cells. MyoD expression was almost completely reduced in these cells. Analysis of tumor lesions formed in NOD-SCID mice showed that tumors with downregulation of MET were much more differentiated and their proliferation rate was much lower. MET negative tumors were also less metastatic in comparison to wild type controls. These data are in concordance with decreased expression of MyoD in vitro and suggest higher differentiation level of MET negative tumors in vivo. Summary: In this study, for the first time, we have shown that differentiation of RMS cells is connected to the decrease of expression and signaling of MET receptor. These findings might have the significant clinical implication for the treatment of RMS cells because they suggest that induction of differentiation of RMS cells by e.g. blocking MET receptor might have influence on the aggressiveness/metastatic potential of these tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A112.