Objective: During osteoarthritis (OA), chondrocytes seem to change their spatial arrangement from single to double strings, small and big clusters. Since the pericellular matrix (PCM) appears to degrade alongside this reorganisation, it has been suggested that spatial patterns act as an image-based biomarker for OA. The aim of this study was to establish the functional relevance of spatial organisation in articular cartilage. Method: Cartilage samples were selected according to their predominant spatial cellular pattern. Young's modulus of their PCM was measured by atomic force microscopy (AFM) (~500 measurements/pattern). The distribution of two major PCM components (collagen type VI and perlecan) was analysed by immunohistochemistry (8 patients) and protein content quantified by enzyme-linked immunosorbent assay (ELISA) (58 patients). Results: PCM stiffness significantly decreased with the development from single to double strings (p ¼ 0.030), from double strings to small clusters (p ¼ 0.015), and from small clusters to big clusters (p < 0.001). At the same time, the initially compact collagen type VI and perlecan staining progressively weakened and was less focalised. The earliest point with a significant reduction in protein content as shown by ELISA was the transition from single strings to small clusters for collagen type VI (p ¼ 0.016) and from double strings to small clusters for perlecan (p ¼ 0.008), with the lowest amounts for both proteins seen in big clusters. Conclusions: This study demonstrates the functional relevance of spatial chondrocyte organisation as an image-based biomarker. At the transition from single to double strings PCM stiffness decreases, followed by protein degradation from double strings to small clusters.

Abstract. Rhabdomyosarcomas (RMS) are soft tissue tumours treated with a combination of surgery and chemotherapy. However, mortality rates remain high in case of recurrences and metastatic disease due to drug resistance and failure to undergo apoptosis. Therefore, innovative approaches targeting specific signalling pathways are urgently needed. We analysed the impact of different hedgehog (Hh) pathway inhibitors on growth and survival of six RMS cell lines using MTS assay, colony formation assay, 3D spheroid cultures, flow cytometry and western blotting. Especially the glioma-associated oncogene family (GLI) inhibitor arsenic trioxide (ATO) effectively reduced viability as well as clonal growth and induced cell death in RMS cell lines of embryonal, alveolar and sclerosing, spindle cell subtype, whereas normal skeletal muscle cells were hardly compromised by ATO. Combination of ATO with itraconazole potentiated the reduction of colony formation and spheroid size. These results show that ATO is a promising substance for treatment of relapsed and refractory RMS by directly targeting GLI transcription factors. The combination with itraconazole or other chemotherapeutic drugs has the opportunity to enforce the treatment efficiency of resistant and recurrent RMS. IntroductionRhabdomyosarcomas (RMS) are the most common paediatric soft tissue tumours, accounting for ~5% of all cancers at that age. Several histologic RMS variants can be differentiated with two thirds of RMS belonging to the embryonal subtype (ERMS) usually diagnosed in younger children below the age of 6 years. The more aggressive alveolar subtype (ARMS), which is more common in adolescents and young adults, makes up ~20% of new cases. ARMS are characterised by the reciprocal translocations t(2;13)(p35;q14) or t(1;13)(p36;q14) leading to the expression of fusion proteins consisting of the DNA-binding domains of PAX3 or PAX7 and the transactivation domain of FOXO1 (1,2). ERMS often exhibit loss of heterozygosity (LOH) at the chromosomal band 11p15.5 affecting the expression of the tumour suppressor genes H19 and CDKN1C (2). There are additional subtypes as the high grade pleomorphic RMS with a very unfavourable prognosis (3) and the sclerosing, spindle cell variant. In children this subtype often has a favourable outcome, whereas in adults it is highly aggressive (4,5).The standard therapy of RMS is surgery combined with a first line chemotherapy composed of vincristine, actinomycin D and cyclophosphamide (6). However, mortality rates remain high in case of recurrences and metastatic disease. Chemotherapy resistance and the failure of RMS cells to undergo apoptosis often occurs during disease progression (7). Therefore, new innovative approaches targeting specific signalling pathways are urgently needed.The hedgehog (Hh) pathway is involved in development, tissue regeneration but also several kinds of cancer including basal cell carcinoma, medulloblastoma, osteosarcoma, Ewing sarcoma and RMS (8-11). Ligand-dependent activation of Hh signalling occurs via ...

Ewing sarcomas (ES) are rare mesenchymal tumours, most commonly diagnosed in children and adolescents. Arsenic trioxide (ATO) has been shown to efficiently and selectively target leukaemic blasts as well as solid tumour cells. Since multidrug resistance often occurs in recurrent and metastatic ES, we tested potential additive effects of ATO in combination with the cytostatic drugs etoposide and doxorubicin. The Ewing sarcoma cell lines A673, RD-ES and SK-N-MC as well as mesenchymal stem cells (MSC) for control were treated with ATO, etoposide and doxorubicin in single and combined application. Viability and proliferation (MTS assay, colony formation, 3D spheroid culture) as well as cell death induction (western blot analysis, flow cytometry) were analysed. In the MTS viability assays ATO treatment significantly reduced the metabolic activity of all three ES cell lines (A673, RD-ES and SK-N-MC) examined. Moreover, all ES cell lines were sensitive to etoposide, whereas MSC remained unaffected by the drug concentrations used. With the exception of ATO in RD-ES cells, all drugs induced apoptosis in the ES cell lines, indicated by caspase-3 and PARP cleavage. Combination of the agents potentiated the reduction of viability as well as the inhibitory effect on clonal growth. In addition, cell death induction was obviously enhanced in RD-ES and SK-N-MC cells by a combination of ATO and etoposide compared to single application. Summarised, the combination of low dose, physiologically easily tolerable ATO with commonly used etoposide and doxorubicin concentrations efficiently and selectively suppressed viability and colony formation in ES cell lines, whereas a combination of ATO and etoposide was favourable for cell death induction. In addition to an increase of the effectiveness of the cytostatic drugs and prevention of potential drug resistance, this approach may also reduce toxicity effects, since the individual doses can be reduced.

Rhabdomyosarcomas (RMS) are the most prevalent soft tissue sarcomas affecting children and adolescents. Despite intensive treatment consisting of multimodal chemotherapy and surgery RMS patients diagnosed with metastatic disease expect long term survival rates of only 20%. Often multidrug resistance arises upon initial response emphasizing the need for new therapeutic drugs to improve treatment efficiency. Previously, we demonstrated the efficacy of the FDA approved drug arsenic trioxide (ATO) specifically inhibiting viability and clonal growth as well as inducing cell death in human RMS cell lines of different subtypes. In this study, we combined low dose ATO with lithium chloride (LiCl), which is approved as mood stabilizer for the treatment of bipolar disorder, but also inhibits growth and survival of different cancer cell types in pre-clinical research. Indeed, we could show additive effects of LiCl and ATO on viability reduction, decrease of colony formation as well as cell death induction. In the course of this, LiCl induced inhibitory glycogen synthase kinase-3β (GSK-3β) serine 9 phosphorylation, whereas glioma associated oncogene family 1 (GLI1) protein expression was particularly reduced by combined ATO and LiCl treatment in RD and RH-30 cell lines, showing high rates of apoptotic cell death. These results imply that combination of ATO with LiCl or another drug targeting GSK-3 is a promising strategy to enforce the treatment efficiency in resistant and recurrent RMS.

Sclerosing spindle cell rhabdomyosarcoma (SSRMS) is a rare rhabdomyosarcomas (RMS) subtype. Especially cases bearing a myogenic differentiation 1 (MYOD1) mutation are characterized by a high recurrence and metastasis rate, often leading to a fatal outcome. SSRMS cell lines are valuable in vitro models for studying disease mechanisms and for the preclinical evaluation of new therapeutic approaches. In this study, a cell line established from a primary SSRMS tumor of a 24-year-old female after multimodal chemotherapeutic pretreatment has been characterized in detail, including immunohistochemistry, growth characteristics, cytogenetic analysis, mutation analysis, evaluation of stem cell marker expression, differentiation potential, and tumorigenicity in mice. The cell line which was designated SRH exhibited a complex genomic profile, including several translocations and deletions. Array-comparative genomic hybridization (CGH) revealed an overall predominating loss of gene loci. The mesenchymal tumor origin was underlined by the expression of mesenchymal markers and potential to undergo adipogenic and osteogenic differentiation. Despite myogenic marker expression, terminal myogenic differentiation was inhibited, which might be elicited by the MYOD1 hotspot mutation. In vivo tumorigenicity could be confirmed after subcutaneous injection into NOD/SCID/γcnull mice. Summarized, the SRH cell line is the first adult SSRMS cell line available for preclinical research on this rare RMS subtype.