Journal: Science Advances

Article Title: Noncanonical activation of GLI signaling in SOX2 + cells drives medulloblastoma relapse

doi: 10.1126/sciadv.abj9138

Figure Lengend Snippet: ( A ) Gene expression profiling from SOX2-eGFP + and eGFP − sorted cells isolated from tumors developed in eGFP-Sox2; Ptch1 +/− mice (RNA-seq data obtained from Dirks, 2014 dataset) was analyzed using a Kyoto Encyclopedia of Genes and Genomes (KEGG) Hedgehog signaling signature. A heatmap comparing gene expression is shown. Arrowhead indicates differentially expressed gene ( P = 0.014). ( B ) Spontaneous MB tissues developed in Ptch1-Gfap or ND2:SmoA1 mice, two independent PDOX-derived tissues [SJSHHMB-14-7196 (SJSHHMB) and RCMB18], or a human MB TMA were stained for GLI2 and SOX2. ( C ) Expression of Sox2 in a t-SNE projection of cells was visualized by analyzing previously run scRNA-seq data from spontaneous SmoM2 MB (sequencing data from Ocasio, 2019 dataset). ( D ) A similar scRNA-seq dataset was used to show expression of SHH biomarkers across indicated nodes. ( E ) Spontaneous MB developed in Ptch1-Gfap mice, from mice harboring an SHH subgroup PDOX (RCMB18) or from a TMA were stained for SOX2 and GFAP. ( F ) Percentages of Sox2 + cells from SmoM2 tumors treated with vehicle or vismodegib in the indicated cell clusters were obtained using Ocasio’s transcriptomic data. The numbers of Sox2 + cells were normalized to total sample cell count. Data shown represent the average percentage for each sequenced tumor. ( G ) Percentages of Gli1/Gli2 + cells in vehicle or vismodegib-treated SmoM2 tumors were similarly calculated. ( H ) A density blot showing Gli1 / Gli2 / Sox2 expression in a t-SNE projection of cells from similarly treated tumors. Square details density on astrocytic cluster. Representative IHCs, with highlighted double-labeled cells, are shown. Scale bars, 50 μm. * P ≤ 0.05.

Article Snippet: Antigen retrieval on mouse-derived tissues or an MB tissue array (Biomax, BC17012c) was performed by steaming samples in citrate buffer for 30 min before staining for GLI2 (Aviva Systems Biology), GFAP, Ki67, SOX2 (Abcam), or cleaved caspase-3 (Cell Signaling Technology) according to the manufacturer’s recommendations.

Techniques: Gene Expression, Isolation, RNA Sequencing, Derivative Assay, Staining, Expressing, Sequencing, Cell Counting, Labeling

Journal: Science Advances

Article Title: Noncanonical activation of GLI signaling in SOX2 + cells drives medulloblastoma relapse

doi: 10.1126/sciadv.abj9138

Figure Lengend Snippet: ( A ) Indicated proteins were stained in MPC1 cultures, and percentages of positive cells were determined by flow cytometry analyses. ( B ) MPC2 cultures were exposed to cyclopamine (10 μM) or its inactive analog tomatidine (10 μM), vismodegib (100 nM), or dimethyl sulfoxide (DMSO) for 16 hours before staining for bromodeoxyuridine (BrdU) and cleaved caspase-3. ( C ) MPC2 cultures were exposed to GANT-61 for 16 hours, and expression of indicated genes was determined. ( D ) Indicated cultures were exposed to GANT-61 for 72 hours before determining cell viability by MTT [3-(4,5-dimethyl-2-thiazolyl) 2,5-diphenyl-2 H- tetrazolium bromide] reduction. ( E ) MPC1 cultures were exposed to I-BET151 for 16 hours before determining the expression of indicated genes. ( F ) MPC cultures were exposed to I-BET151 for 72 hours before determining cell viability by MTT reduction. ( G ) MPC1 cultures were exposed to I-BET151 (500 nM) for 16 hours before assay BrdU incorporation and cleaved caspase-3 expression. ( H ) MPC1 cultures were exposed to I-BET151 (500 nM) for 24 hours, and percentages of positive cells were determined by flow cytometry. ( I ) Percentage of GFAP + cells in a SOX2/GLI2 + pool was similarly determined. ( J ) MPC2 cultures were transfected with pooled Genome siRNA sequences targeting the indicated genes or a scramble siRNA control ( siSC ), and expression of SHH target genes was determined 72 hours later. ( K ) MPC2 cultures were transfected with similar siRNAs or a GFP -labeled siRNA. Cell viability was determined 5 days later by MTT reduction. ( L ) MPC1 cells were similarly transfected, and numbers of SOX2 + cells were determined by flow cytometry. ( M ) MPC1 cultures were transfected with indicated vectors and, 48 hours later, exposed to I-BET151 (500 nM) for additional 72 hours. Cell viability was determined by MTT reduction. ( N ) MPC1 cultures were transfected with indicated Genome siRNA sequences. Forty-eight hours after transfection, cells were exposed to I-BET151 (500 nM) for additional 72 hours, and cell viability was similarly determined. Scale bars, 100 μm. * P ≤ 0.05.

Article Snippet: Antigen retrieval on mouse-derived tissues or an MB tissue array (Biomax, BC17012c) was performed by steaming samples in citrate buffer for 30 min before staining for GLI2 (Aviva Systems Biology), GFAP, Ki67, SOX2 (Abcam), or cleaved caspase-3 (Cell Signaling Technology) according to the manufacturer’s recommendations.

Techniques: Staining, Flow Cytometry, Expressing, BrdU Incorporation Assay, Transfection, Control, Labeling

Journal: Science Advances

Article Title: Noncanonical activation of GLI signaling in SOX2 + cells drives medulloblastoma relapse

doi: 10.1126/sciadv.abj9138

Figure Lengend Snippet: ( A ) MPC47 cultures were exposed to GANT-61 or I-BET151 for 72 hours, and cell viability was determined by MTT reduction. ( B ) MPC47 cultures were exposed to GANT-61 for 16 hours, and expression of indicated genes was determined. ( C ) MPC47 cultures were exposed to I-BET151 for 16 hours, and SHH target gene expression was determined by RT-qPCR. ( D ) MPC47 cultures were transfected with indicated pooled Genome siRNA sequences, and expression of indicated genes was similarly determined 72 hours later. ( E ) MPC47 cultures were transfected with similar siRNA sequences or a GFP-siRNA . Cell viability was determined 5 days later by MTT reduction. (F ) MPC cultures were exposed to 10058-F4 for 16 hours, and SHH target gene expression was determined. ( G ) Indicated cultures were exposed to 10058-F4 for 48 hours before determining cell viability by MTT reduction. ( H ) MPC2 cultures were transfected with pooled Genome siRNA sequences, and expression of Myc was determined 72 hours later. ( I ) MPC2 cultures were transfected with similar siRNA sequences, and expression of indicated genes was determined 72 hours later. ( J ) MPC cultures were similarly transfected, and cell viability was determined 5 days later by MTT reduction. ( K ) MPC1 cultures were transfected with pooled Genome siRNA sequences for 5 days before quantify percentages of SOX2 + cells by flow cytometry. ( L ) Indicated proteins were stained in ND2:SmoA1 brains. Square details double-positive cell in a representative image. Scale bars, 50 μm. ( M ) scRNA-seq data from SmoM2 mice exposed to vehicle or vismodegib (Ocasio’s dataset) were used to study the density of Sox2/Gli1/Gli2/Myc expression in a t-SNE plot. ( N ) Schematic of canonical and MYC-driven noncanonical SHH signaling in SOX2 + MB cells. * P ≤ 0.05.

Article Snippet: Antigen retrieval on mouse-derived tissues or an MB tissue array (Biomax, BC17012c) was performed by steaming samples in citrate buffer for 30 min before staining for GLI2 (Aviva Systems Biology), GFAP, Ki67, SOX2 (Abcam), or cleaved caspase-3 (Cell Signaling Technology) according to the manufacturer’s recommendations.

Techniques: Expressing, Targeted Gene Expression, Quantitative RT-PCR, Transfection, Flow Cytometry, Staining

Journal: Science Advances

Article Title: Noncanonical activation of GLI signaling in SOX2 + cells drives medulloblastoma relapse

doi: 10.1126/sciadv.abj9138

Figure Lengend Snippet: ( A ) MPC cultures were exposed to JQ-1 for 72 hours before determining cell viability by MTT reduction. ( B ) MPC47 cells were exposed to JQ-1, and expression of indicated genes was determined 16 hours later. ( C ) Mice harboring Ptch1-LacZ (MB47) subcutaneous tumors were treated with JQ-1 (30 mg/kg, i.p., q.d.) or vehicle for 8 days, before determining levels of the indicated proteins. ( D ) Expression of indicated genes was determined in similarly dosed mice. ( E ) Mice harboring similar tumors were treated with JQ-1 (30 mg/kg, i.p., q.d.), vismodegib (25 mg/kg, i.p., q.d.), or vehicle for 8 days, and the tumor volume was measured. ( F ) Mice harboring orthotopic MB47 tumors were similarly dosed, and tumor area was quantified. Scale bars, 200 μm. ( G ) Number of SOX2 + cells in brain tumors from similarly treated mice were quantified. ( H ) Mice harboring subcutaneous MB47 tumors were treated with vehicle or JQ-1 (30 mg/kg, i.p., q.d.) for 8 days, before determining percentage of positive cells by flow cytometry. ( I ) Percentages of GFAP + in SOX2/GLI2 + cells were similarly determined. ( J ) Mice harboring subcutaneous MB47 were treated with JQ-1 (30 mg/kg, i.p., q.d.), vismodegib (25 mg/kg, i.p., q.d.), or vehicle for 8 days. Equal numbers of viable cells from residual tumors were then allowed to form spheres ex vivo or to orthotopically engraft in vivo. A schematic of the procedure is shown. ( K ) Numbers of spheres grown from treated tumors described in (J) were quantified. ( L ) Tumor engraftment capability from similar residual tumors was determined. ( M ) PDOX harboring mice were dosed with vehicle, vismodegib (25 mg/kg, i.p., q.d.), or JQ-1 (30 mg/kg, i.p., q.d.) for 8 days, before staining brains for SOX2. Unless otherwise indicated, scale bars, 50 μm. * P ≤ 0.05.

Article Snippet: Antigen retrieval on mouse-derived tissues or an MB tissue array (Biomax, BC17012c) was performed by steaming samples in citrate buffer for 30 min before staining for GLI2 (Aviva Systems Biology), GFAP, Ki67, SOX2 (Abcam), or cleaved caspase-3 (Cell Signaling Technology) according to the manufacturer’s recommendations.

Techniques: Expressing, Flow Cytometry, Ex Vivo, In Vivo, Staining

Journal: Science Advances

Article Title: Noncanonical activation of GLI signaling in SOX2 + cells drives medulloblastoma relapse

doi: 10.1126/sciadv.abj9138

Figure Lengend Snippet: ( A ) Six-month-old ND2:SmoA1 mice were exposed to BMS-986158 (3 mg/kg, i.p., q.d.) for 8 days. The size of residual tumors was quantified. Scale bars, 200 μm. ( B ) Brains from similarly treated ND2:SmoA1 mice were stained for indicated proteins, and numbers of positive cells were quantified. ( C ) Two-month-old ND2:SmoA1 mice were exposed to BMS-986158 (3 mg/kg, i.p., q.d.) or vehicle, and symptom-free survival was determined. ( D ) Three weeks after orthotopically implanting SJSHHMB PDOX cells, mice were exposed to BMS-986158 (3 mg/kg, i.p., q.d.) or vehicle for 8 days before harvesting their brains, and the size residual tumors was measured. Scale bars, 200 μm. ( E ) Mice harboring SJSHHMB PDOX tissues were similarly treated, and brains were stained for indicated proteins. ( F ) Similarly implanted mice were treated with BMS-986158 (3 mg/kg, i.p., q.d.) or vehicle for 8 days before staining tissues for GLI1 and GLI2. ( G ) Mice implanted with either SJSHHMB or RCMB18 PDOX cells were similarly dosed before staining brain tissues for SOX2. ( H ) Mice orthotopically harboring a RCMB18 PDOX were dosed with BMS-986158 (3 mg/kg, i.p., q.d.) or vehicle, and symptom-free survival was determined. ( I ) Model illustrating cell diversity in SHH MB. Unless otherwise indicated, scale bars, 50 μm. * P ≤ 0.05.

Article Snippet: Antigen retrieval on mouse-derived tissues or an MB tissue array (Biomax, BC17012c) was performed by steaming samples in citrate buffer for 30 min before staining for GLI2 (Aviva Systems Biology), GFAP, Ki67, SOX2 (Abcam), or cleaved caspase-3 (Cell Signaling Technology) according to the manufacturer’s recommendations.

Techniques: Staining