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Introduction Sarcomas often show a limited response to current treatments. A hypothesis to explain this resistance relies on the existence of subpopulations of drug-resistant cancer stem cells (CSCs) responsible for relapses and metastasis. Therefore, there is a need for patient-close disease models amenable for testing the effect of anti-cancer therapies on CSCs. As a proof-of principle of their possible utility in future personalise medicine strategies, we have established a panel of patient-derived sarcoma primary cell lines, characterised their CSC subpopulations and tested the anti-tumour activity of the mithramycin analogue EC-8042 on these cell lines. Material and methods Fresh surgical samples of sarcomas were disaggregated and put in culture. In those cell lines adapted to grow in vitro, their proliferation ability and their potential to growth tumour xenografts were evaluated. CSCs subpopulations were characterised according to their ability to grow as tumorspheres, and the presence of cells presenting ALDH1 activity (Aldefluor© activity) or transcriptional activity due to SOX2/OCT4 in cells expressing the reporter system Sore6 (Sore6 activity). Cell survival, apoptotic induction and modulation of Sore6 activity was analysed after drug treatments. Finally, the expression/phosphorylation of signalling proteins was analysed by Western blotting. Results and discussions We have established a panel of patient-derived primary cell lines able to reproduce in vivo the histopathological features of their tumours of origin. Most of these cell lines were able to grow as serially passaged tumorspheres and showed between 1% and 15% of Aldefluor© positive cells. In addition, we detected Sore6 activity in 25% of cells in a cell line expressing the Sore6 reporter. Finally, these cell lines showed a distinct sensitivity to EC8042, with IC50 values ranging from 0.1 to 1 µM. EC-8042 was able to inhibit the expression of CSC-related factors such as SOX2, NOTCH1 and C-MYC in a dose-dependent fashion and moreover, this drug efficiently targeted Sore6 positive cells. Importantly, we found a strong correlation between the presence of intra-cellular NOTCH1 and the cytotoxic response to EC-8042. Conclusion This panel of sarcoma primary cell lines with preserved CSC properties constitutes a valuable patient-close platform to test anti-cancer drugs. We found that EC-8042 could act as an anti-pluripotency agent in sarcomas and we identified intra-cellular NOTCH1 as a possible marker of response for this drug.

Introduction Sarcomas often show a limited response to cytotoxic drugs which still remain as the most utilised agents for the treatment of these diseases. This lack of response could be explained by the existence of drug-resistant Cancer Stem Cells (CSCs), which are responsible of tumour relapse. It has been established that transformed human mesenchymal stromal/stem cell (MSCs) are the most likely cell-of-origin for many types of sarcomas. Therefore, we previously developed a model of sarcomagenesis based on transformed Bone Marrow Mesenchymal Stromal/Stem Cells (BM-MSCs). This model has proven very useful to explore the evolution of CSCs subpopulations and to search for CSC-specific markers and therapies. Here we use this model to analyse the role of SOX2 as a CSC-specific marker by isolating subpopulations presenting high SOX2-mediated transcriptional activity. Material and methods We depleted or overexpressed SOX2 in a xenograft-derived cell line (T5H-O) generated by a cell-origin-model of undifferentiated pleomorphic sarcoma developed from transformed BM-MSC. In addition, these cells were transfected with a lentiviral-based reporter system in which a composite SOX2/OCT4 response element (SORE6) coupled to a minimal cytomegalovirus (CMV) promoter controls the expression of fluorescent reporter genes. T5H-O cells expressing this system allowed us to detect and isolate viable cells expressing transcriptionally active SOX2 and/or OCT4 by flow cytometry. Results and discussions The depletion of SOX2 protein inhibited CSC related-properties of T5H-O cells (e.g. tumorsphere forming potential) whereas T5H-O cells expressing high levels of SOX2 dramatically increased their invasive properties and in vivotumour growth potential. We found that SORE6 activity in our sarcoma model was mainly due to SOX2, rather than OCT4. Comparing to SORE6- population, SORE6 +positive cells showed enhanced tumorsphere-forming potential and increased invasion ability. Importantly, SORE6 +cells were significantly more tumorigenic than the SORE6- population, thus indicating that SOX2 activity marks a subpopulation with increased tumor-promoting ability in sarcomas. Finally, we found that EC-8042 and trabectedin, anti-tumour drugs previously reported to target CSCs in sarcoma, were able to eliminate the SORE6 +CSC subpopulation in sarcomas. Conclusion Our results indicate that SOX2 activity is a bona fide CSC-marker in sarcoma, and that SORE6 reporter system is an excellent approach for testing the effectiveness of CSC-specific treatments.

Introduction Cytotoxic drugs like doxorubicin remain as the most utilised agents in sarcoma treatment. However, advanced sarcomas often show resistance to these drugs, mainly through the overexpression of members of the ATP binding cassette (ABC) family which act as efflux pumps for drugs. Therefore, the development more effective treatments able to prevent drug resistance would improve sarcoma treatments. Multi-kinase inhibitors provided an efficient way to target several pro-tumorigenic pathways using a single agent and have shown anti-tumour activity in a range of tumours. Aberrant activation of pro-tumoral kinase is common in sarcoma, however the effect of multikinase inhibitors in sarcoma has been barely tested. Here, we aimed to study the anti-tumour effect of one of such inhibitors in cell-of-origin sarcoma models and sarcoma patient-derived primary cell lines, as well as its ability to prevent drug resistance and synergize with doxorubicin. Material and methods Cell survival, apoptotic induction, cell cycle progression and DNA damage were analysed after drug treatments. The existence of synergism between drugs was evaluated using statistic tools. Drug effect on signalling proteins was studied using phospho-antibody arrays and Western-blotting analysis. Interaction between drugs and ABC transporters were characterised using substrate and inhibition assays. Finally, in vivo tumour growth and pharmacodynamic response after drug treatments were evaluated in xenografts models. Results and discussions Sarcoma cells were sensitive to sub-micromolar concentrations of the multikinase inhibitor, which induced cell cycle arrest, DNA damage and apoptosis. Evaluation of the phosphorylation status of signalling kinases evidenced that PI3K/AKT/mTOR and ERK1/2 were the most highly activated pathways in sarcoma cells and that the drug efficiently inhibited them in vitro and in vivo . By using specific mTOR inhibitors and agonists, we confirmed that the inhibition of this pathway contributed to the cytotoxic effect of the drug. In addition, this drug inhibited the expression and activity of ABC transporters and was not a substrate for them. In line with this ability, we found a synergistic cytotoxic effect when sarcoma cells were treated with combinations of the kinase inhibitor and doxorubicin both in vitro and in vivo. Conclusion A novel multikinase inhibitor induced a consistent cytotoxic effect and was able to counteract drug resistance in sarcoma cells, thus highlighting its therapeutic potential when combined with current treatments.