Journal: NPJ Precision Oncology

Article Title: Genetic and epigenetic characterization of sarcoma stem cells across subtypes identifies EZH2 as a therapeutic target

doi: 10.1038/s41698-024-00776-7

Figure Lengend Snippet: A A doxorubicin-resistant (doxo R ) derivative line of WT GCT (undifferentiated pleomorphic sarcoma) cells was generated through serial passage in increasing concentrations of doxorubicin. H&E stains of representative subcutaneous tumors generated from WT and Doxo R SW872 cells. B H&E stains of representative subcutaneous tumors generated from WT and Doxo R cells SW872 cells (dedifferentiated liposarcoma). C Cell-titer glo viability assays for doxorubicin dose-response in WT and doxoR derivative GCT cells. Data points are the mean ± SD of triplicate wells and are representative of three different experiments. D A doxorubicin-resistant (doxo R ) derivative line of WT SW872 cells was generated through serial passage in doxorubicin and exhibited an increased IC 50. Data points are the mean ± SD of triplicate wells and are representative of three different experiments. E Representative photographs of SW872 WT and Doxo R cells after 48 hours of treatment with either DMSO (vehicle) or 300 nM doxorubicin. bars = 250 nm. F WT and Doxo R GCT cells were subjected to soft-colony formation assays and the abundance and size of colonies was counted with imageJ software. Each column is a biological replicate. Colony size is shown in units as pixels (px 2 ). G Aldefluor assay showing increased bright population in doxo R (right) cells vs WT GCT cells (left). The DEAB controls are shown in red ( n = 3). H WT and Doxo R SW872 cells were subjected to soft-colony formation assays and the abundance and size of colonies was counted with imageJ software. Colony size is shown in arbitrary units as pixels 2 . I Aldefluor assay showing increased bright population in doxo R (right) cells vs WT SW872 cells (left) ( n = 3). * P < 0.05, ** P < 0.05.

Article Snippet: Xenograft tumors of SW872 and GCT cells (and their doxorubicin-resistant derivatives) were established in immunocompromised NSG mice (Jackson Labs Cat#005557) after injection of a 1:1 mixture of cells in PBS and Matrigel (1×10^6 cells/injection).

Techniques: Generated, Software

Journal: NPJ Precision Oncology

Article Title: Genetic and epigenetic characterization of sarcoma stem cells across subtypes identifies EZH2 as a therapeutic target

doi: 10.1038/s41698-024-00776-7

Figure Lengend Snippet: A Sorting scheme of Aldefluor bright and dim sorted cells among five soft tissue sarcoma cell lines. A representative soft-agar colony forming assay from sorted GCT cells shows the increased propensity for anchorage-independent growth in Aldefluor bright cells. B Table showing the percent Aldefluor positive population among the five different cell lines. LMS=leiomyosarcoma (SKLMS1), RH=rhabdomyosarcoma, MFS=myxofibrosarcoma, UPS=undifferentiated pleomorphic sarcoma (GCT), LPS=dedifferentiated liposarcoma (SW872). B Percent Aldefluor positive (bright) cells for each of the different lines tested. C Venn diagram showing the overlap of differentially expressed genes meeting a significance of p < 0.05. 36 genes were upregulated and 1 gene (BRCA1) was downregulated. D Table of the commonly upregulated genes shown in panel C subdivided according to fold change in expression. E Gene ontology analysis of the 37 shared, differentially regulated genes across the five sarcoma types.

Article Snippet: Xenograft tumors of SW872 and GCT cells (and their doxorubicin-resistant derivatives) were established in immunocompromised NSG mice (Jackson Labs Cat#005557) after injection of a 1:1 mixture of cells in PBS and Matrigel (1×10^6 cells/injection).

Techniques: Expressing

Journal: NPJ Precision Oncology

Article Title: Genetic and epigenetic characterization of sarcoma stem cells across subtypes identifies EZH2 as a therapeutic target

doi: 10.1038/s41698-024-00776-7

Figure Lengend Snippet: A Tazemetostat does not significantly inhibit cell viability during short term treatment. Data are the mean ± SD of three biological replicates, and are representative of three independent experiments. There is no statistically significant difference between the groups. B Representative soft-agar assay of WT GCT cells treated with vehicle or Tazemetostat (25 uM) (right) and quantification with imageJ (left). C Treatment schema of WT GCT cells subjected to long-term culture with vehicle ( A, B ) or Tazemetostat for continuous exposure ( C, E ) or on-off drug exposure ( D, F ). D Aldefluor assay of WT GCT cells treated with vehicle or Tazemetostat over a 3 week period. DEAB controls for each experimental arm are shown in the top row, for which the corresponding gates are directly maintained in the bottom row. The percentage of Aldefluor bright cells relative to DEAB controls is shown in brackets. E Continuous treatment of GCT cells with either Tazemetostat or two additional EZH2 inhibitors EI1 and PF-06726304 decreases the abundance of bright cells in the Aldefluor assay. * p < 0.001 for all three treatment groups compared to control. F Western blot of H3K27me3 expression in GCT cells following treatment with vehicle or the EZH2 inhibitors Tazemetostat, EI1 and PF-06726304. Beta actin is shown as a loading control.

Article Snippet: Xenograft tumors of SW872 and GCT cells (and their doxorubicin-resistant derivatives) were established in immunocompromised NSG mice (Jackson Labs Cat#005557) after injection of a 1:1 mixture of cells in PBS and Matrigel (1×10^6 cells/injection).

Techniques: Soft Agar Assay, Control, Western Blot, Expressing

Journal: NPJ Precision Oncology

Article Title: Genetic and epigenetic characterization of sarcoma stem cells across subtypes identifies EZH2 as a therapeutic target

doi: 10.1038/s41698-024-00776-7

Figure Lengend Snippet: A SW872 (LPS) and derivative doxo R cells treated for 96 hours with doxorubicin and Tazemetostat alone or in combination. B GCT (UPS) and derivative doxo R cells treated for 96 hours with doxorubicin and Tazemetostat alone or in combination. Data for A , B are the mean ± SD of three biological replicates analyzed by ANOVA, * p < 0.05, *** p < 0.001 **** p < 0.0001. C Viability assays of unique, independently generated soft tissue sarcoma cell lines from oncogene-mediated forward transformation of mesenchymal stem cells in vivo treated with doxorubin at the indicated concentrations alone or in combination with 25 uM Tazemetostat. Data for A-C are the mean ± SD of three biological replicates analyzed by ANOVA, * p < 0.05, *** p < 0.001 **** p < 0.0001.

Article Snippet: Xenograft tumors of SW872 and GCT cells (and their doxorubicin-resistant derivatives) were established in immunocompromised NSG mice (Jackson Labs Cat#005557) after injection of a 1:1 mixture of cells in PBS and Matrigel (1×10^6 cells/injection).

Techniques: Generated, Transformation Assay, In Vivo