74829s  (Cell Signaling Technology Inc)

Journal: Science Advances

Article Title: Ara-C suppresses H3 K27–altered spinal cord diffuse midline glioma growth and enhances immune checkpoint blockade sensitivity

doi: 10.1126/sciadv.adu3956

Figure Lengend Snippet: ( A ) Representative immunohistochemistry (IHC) image of Ki67 expression in H3-SCDMG ( n = 43) versus H3-wild type-SCDMG ( n = 30). Scale bars, 100 μm. ( B ) Comparison of Ki67 index between H3-SCDMG ( n = 43) and H3-wild type-SCDMG ( n = 30). Data are presented as mean ± SD. ( C ) Patient-derived glioma cell lines (BNI2-4 and BNI3-5) were transfected with H3 K27M (H3 K27M) or empty vector (EV) lentivirus. Immunoblot analysis of histone H3 K27M and H3 K27me3. ( D and E ) Proliferative viability assay of glioma PDCs with H3 K27M (H3 K27M) or empty vector (EV), assessed by CCK8 assay. All data are mean ± SEM. n = 6 technical replicates per group. ( F ) Representative images showing EdU-positive PDCs. DAPI indicates nuclei with DNA, EdU represents cells that have incorporated EdU during the S phase, and Merge refers to the overlay of DAPI and EdU signals. The red color corresponds to EdU-positive cells. Scale bars, 50 μm. ( G ) Percentage of EdU-positive cells in PDCs with H3 K27M or EV. All data are mean ± SD. n = 7 microscopic statistical fields. ( H ) Immunofluorescence localization and expression of PDGFRA protein in PDCs with H3 K27M or EV. Scale bars, 10 μm. ( I ) Summary of IC 50 values of five drugs on three group PDC cells with different H3 backgrounds. ( J ) IC 50 curves for Ara-C in three groups of PDC cells with different H3 backgrounds as well as normal astrocyte HA; all data are mean ± SD. *0.01 ≤ P < 0.05, ***0.0001 ≤ P < 0.001, and **** P < 0.0001 for all figures.

Article Snippet: The membranes were then incubated with the following primary antibodies overnight at 4°C: Histone H3 K27M Mutant Specific (1:1000, CST, catalog no. 74829S), Tri-Methyl-Histone H3 Lys27 (1:1000, CST, catalog no. 9733S), H3 (1:5000, Proteintech, catalog no. 68345-1-Ig), Phospho-Chk1 (1:1000, CST, catalog no. 2348S), CHK1 (1:10000, abcam, catalog no. ab40866), CDK1 (1:10,000, abcam, catalog no. ab133327), CDK2 (1:5000, abcam, catalog no. ab32067), Cleaved PARP1 (1:5000, abcam, catalog no. ab32064), Cleaved Caspase-3 (1:1000, CST, catalog no. 9661S), β-actin (1:5000, Proteintech, catalog no. 66009-1), p21(1:5000, abcam, catalog no. ab109520), H3K9me3 (1:800, CST, catalog no. 13969S), PD-L1 (1:1000, Proteintech, catalog no. 17952-1-AP), PD-L2 (1:800, CST, catalog no. 82723S), anti-galectin9 (1:1000, abcam, catalog no. ab69630), PVRL2 (1:1000, Proteintech, catalog no. 27171-1-AP), and PVR (1:2000, Proteintech, catalog no. 83724-6).

Techniques: Immunohistochemistry, Expressing, Comparison, Derivative Assay, Transfection, Plasmid Preparation, Western Blot, Viability Assay, CCK-8 Assay, Immunofluorescence

Journal: Science Advances

Article Title: Ara-C suppresses H3 K27–altered spinal cord diffuse midline glioma growth and enhances immune checkpoint blockade sensitivity

doi: 10.1126/sciadv.adu3956

Figure Lengend Snippet: ( A ) Schematic diagram of the construction of spinal cord diffuse midline glioma PDX and PDC models. By Figdraw (ID: SSPWSb5cc5). ( B ) Photographs of subcutaneous tumors in PDX_S01 and PDX_S29 mice, results of hematoxylin and eosin (H&E) staining, and IHC images of tumor-associated markers. Black arrows point to mouse PDX tumors. ( C ) Photographs of tumor tissues after 2 weeks of Ara-C treatment for spinal glioma PDXs (PDX_S01 and PDX_S29). ( D ) Tumor volume growth curves for the control and Ara-C treatment groups. n = 8 biological replicates per group. All data are mean ± SD. **** P < 0.0001 by Student’s t test. ( E ) Two PDCs were obtained from the corresponding PDX models. Microscopic photographs were taken to demonstrate the morphology of the cells and the way they grew in suspension. Scale bars, 100 μm. Immunofluorescence staining demonstrated that both PDCs highly expressed H3 K27M, NESTIN, and PDGFRA. Nuclei labeled with DAPI. Scale bars, 10 μm. ( F ) IC 50 curves of two PDCs in the presence of different concentrations of the six antitumor drugs.

Article Snippet: The membranes were then incubated with the following primary antibodies overnight at 4°C: Histone H3 K27M Mutant Specific (1:1000, CST, catalog no. 74829S), Tri-Methyl-Histone H3 Lys27 (1:1000, CST, catalog no. 9733S), H3 (1:5000, Proteintech, catalog no. 68345-1-Ig), Phospho-Chk1 (1:1000, CST, catalog no. 2348S), CHK1 (1:10000, abcam, catalog no. ab40866), CDK1 (1:10,000, abcam, catalog no. ab133327), CDK2 (1:5000, abcam, catalog no. ab32067), Cleaved PARP1 (1:5000, abcam, catalog no. ab32064), Cleaved Caspase-3 (1:1000, CST, catalog no. 9661S), β-actin (1:5000, Proteintech, catalog no. 66009-1), p21(1:5000, abcam, catalog no. ab109520), H3K9me3 (1:800, CST, catalog no. 13969S), PD-L1 (1:1000, Proteintech, catalog no. 17952-1-AP), PD-L2 (1:800, CST, catalog no. 82723S), anti-galectin9 (1:1000, abcam, catalog no. ab69630), PVRL2 (1:1000, Proteintech, catalog no. 27171-1-AP), and PVR (1:2000, Proteintech, catalog no. 83724-6).

Techniques: Staining, Control, Suspension, Immunofluorescence, Labeling

Journal: Science Advances

Article Title: Ara-C suppresses H3 K27–altered spinal cord diffuse midline glioma growth and enhances immune checkpoint blockade sensitivity

doi: 10.1126/sciadv.adu3956

Figure Lengend Snippet: ( A ) Representative images showing EdU-positive H3-SCDMG PDCs. Scale bars, 50 μm. ( B ) Percentage of EdU-positive cells in H3-SCDMG PDCs. ( C ) Immunofluorescence image of gamma-H2AX protein in H3-SCDMG PDCs. Scale bars, 10 μm. ( D ) The number of γ-H2AX-positive foci in each PDC cell was quantified and compared. ( E and G ) Representative cell cycle graph of PDC cells treated with Ara-C (1 μM) for 48 hours. n = 3 biological replicates per group. ( F and H ) Statistical results of distributing cells at different cell cycles. Data are mean ± SD. ( I and J ) Representative images of flow apoptosis assay of PDCs (PDC_S01 and PDC_S29) after 48 hours of treatment with Ara-C (1 μM). The second quadrant corresponds to late apoptotic cells, and the fourth quadrant represents early apoptotic cells. Bar graphs analyzing the percentage of apoptotic cells. n = 3 biological replicates per group. ( K and L ) Functional analysis of genes with different RNA expression levels along with Ara-C treatment for different times. ( M ) Western blot showed the expression of cellular DNA damage repair and apoptosis-related proteins in two types of PDC cells after 2 and 5 days of Ara-C (1 μM) treatment. ( N ) IHC showed expression of H3 K27M, Ki-67, and cleaved-caspase 3 (c-caspase3) in different cells and treatments. ( O ) Percentage of Ki67 and c-caspase3–positive cells in tumors of PDX with different treatments. Data are mean ± SD. **** P < 0.0001 for all figures.

Article Snippet: The membranes were then incubated with the following primary antibodies overnight at 4°C: Histone H3 K27M Mutant Specific (1:1000, CST, catalog no. 74829S), Tri-Methyl-Histone H3 Lys27 (1:1000, CST, catalog no. 9733S), H3 (1:5000, Proteintech, catalog no. 68345-1-Ig), Phospho-Chk1 (1:1000, CST, catalog no. 2348S), CHK1 (1:10000, abcam, catalog no. ab40866), CDK1 (1:10,000, abcam, catalog no. ab133327), CDK2 (1:5000, abcam, catalog no. ab32067), Cleaved PARP1 (1:5000, abcam, catalog no. ab32064), Cleaved Caspase-3 (1:1000, CST, catalog no. 9661S), β-actin (1:5000, Proteintech, catalog no. 66009-1), p21(1:5000, abcam, catalog no. ab109520), H3K9me3 (1:800, CST, catalog no. 13969S), PD-L1 (1:1000, Proteintech, catalog no. 17952-1-AP), PD-L2 (1:800, CST, catalog no. 82723S), anti-galectin9 (1:1000, abcam, catalog no. ab69630), PVRL2 (1:1000, Proteintech, catalog no. 27171-1-AP), and PVR (1:2000, Proteintech, catalog no. 83724-6).

Techniques: Immunofluorescence, Apoptosis Assay, Functional Assay, RNA Expression, Western Blot, Expressing

Journal: Science Advances

Article Title: Ara-C suppresses H3 K27–altered spinal cord diffuse midline glioma growth and enhances immune checkpoint blockade sensitivity

doi: 10.1126/sciadv.adu3956

Figure Lengend Snippet: ( A ) The schematic diagram delineates the experimental protocol for the orthotopic implantation of spinal cord PDX models and subsequent treatment in immunodeficient mice. ( B ) Representative in vivo imaging of luciferase-tagged spinal cord tumor orthotopic xenograft models in the treatment group. ( C ) Tumor burden in luciferase-tagged PDX tumor models was quantified by measuring the region of interest (ROI) values obtained from in vivo imaging. ( D ) Kaplan-Meier curves illustrate the survival of mice. Each group consisted of eight biological replicates. ( E ) Representative images of IHC for tumor-associated markers (H3 K27M, H3 K27me3, Ki67, and cleaved-caspase3) in PDX tumors with different treatments for 2 weeks. ( F ) The schematic diagram shows the treatment for patients in investigator-initiated clinical trial. Patients were enrolled in the treatment after being diagnosed with recurrence via magnetic resonance imaging (MRI). Following the implantation surgery of an Ommaya reservoir into the intracranial ventricle, patients began to receive six cycles of intrathecal Ara-C chemotherapy. The treatment was administered monthly, with three times given in the first week of each cycle, at an intrathecal dose of 30 mg/m 2 body surface area. Patients underwent MRI scans every 3 months for follow-up. ( G ) Statistical summary of the assessment of treatment outcomes in four patients. ( H to J ) Pre- and posttreatment MRI images of three patients. The tumor lesions are marked with red. The nonneoplastic lesion below the red-marked area in patient P3 may be a syrinx in the spinal cord caused by the compression of the central canal due to the recurrent tumor. The schematic diagrams of (A) and (F) were drawn by Figdraw (ID: OPSWY8c6c8 and RYSPR13933).

Article Snippet: The membranes were then incubated with the following primary antibodies overnight at 4°C: Histone H3 K27M Mutant Specific (1:1000, CST, catalog no. 74829S), Tri-Methyl-Histone H3 Lys27 (1:1000, CST, catalog no. 9733S), H3 (1:5000, Proteintech, catalog no. 68345-1-Ig), Phospho-Chk1 (1:1000, CST, catalog no. 2348S), CHK1 (1:10000, abcam, catalog no. ab40866), CDK1 (1:10,000, abcam, catalog no. ab133327), CDK2 (1:5000, abcam, catalog no. ab32067), Cleaved PARP1 (1:5000, abcam, catalog no. ab32064), Cleaved Caspase-3 (1:1000, CST, catalog no. 9661S), β-actin (1:5000, Proteintech, catalog no. 66009-1), p21(1:5000, abcam, catalog no. ab109520), H3K9me3 (1:800, CST, catalog no. 13969S), PD-L1 (1:1000, Proteintech, catalog no. 17952-1-AP), PD-L2 (1:800, CST, catalog no. 82723S), anti-galectin9 (1:1000, abcam, catalog no. ab69630), PVRL2 (1:1000, Proteintech, catalog no. 27171-1-AP), and PVR (1:2000, Proteintech, catalog no. 83724-6).

Techniques: In Vivo Imaging, Luciferase, Magnetic Resonance Imaging

Journal: Science Advances

Article Title: Ara-C suppresses H3 K27–altered spinal cord diffuse midline glioma growth and enhances immune checkpoint blockade sensitivity

doi: 10.1126/sciadv.adu3956

Figure Lengend Snippet: ( A ) GSVA of RNA-seq data shows up-regulated Reactome terms in PDC_S29 after 5 days of Ara-C (1 μM) treatment. ( B ) Senescence-associated β-galactosidase staining in PDCs after Ara-C treatment (0, 1, 3, and 5 days). Scale bars, 100 μm. ( C ) SPiDER-βGal fluorescence intensity in PDCs post–Ara-C treatment. Data: mean ± SD; n = 6. ( D ) Western blot of senescence markers p21 and H3K9me3 in PDCs (PDC_S01 and PDC_S29) after Ara-C treatment (0, 2, and 5 days). ( E ) qPCR analysis of cytokine/chemokine mRNA in PDCs post–Ara-C treatment. Data: mean ± SEM; n = 3. # P < 0.05 versus control. ( F ) Workflow for tumor cell recruitment assay: PDCs treated with Ara-C or DMSO for 5 days, assessing PBMC migration using the conditioned medium. n = 3. By Figdraw (ID: ORYAP9a9fa). ( G ) Microscopic images of PBMC migration. Scale bars, 100 μm. ( H and I ) PBMC migration quantification. n = 12 fields. ( J ) H&E and IHC staining of subcutaneous tumors in C57 mice treated with PBS or Ara-C. ( K ) Quantification of marker-positive cells in tumors. Data: mean ± SD; n = 5. **** P < 0.0001. ( L ) In vivo imaging of luciferase-tagged spinal cord xenograft models. n = 6 per group. ( M ) Kaplan-Meier survival curves for different treatment groups. ( N ) Multiplex staining of H3 K27M, CD3e, and IBA1 in spinal cord xenografts with or without Ara-C. ( O ) Quantification of CD3e- and IBA1-positive cells. Data: mean ± SD; n = 3.

Article Snippet: The membranes were then incubated with the following primary antibodies overnight at 4°C: Histone H3 K27M Mutant Specific (1:1000, CST, catalog no. 74829S), Tri-Methyl-Histone H3 Lys27 (1:1000, CST, catalog no. 9733S), H3 (1:5000, Proteintech, catalog no. 68345-1-Ig), Phospho-Chk1 (1:1000, CST, catalog no. 2348S), CHK1 (1:10000, abcam, catalog no. ab40866), CDK1 (1:10,000, abcam, catalog no. ab133327), CDK2 (1:5000, abcam, catalog no. ab32067), Cleaved PARP1 (1:5000, abcam, catalog no. ab32064), Cleaved Caspase-3 (1:1000, CST, catalog no. 9661S), β-actin (1:5000, Proteintech, catalog no. 66009-1), p21(1:5000, abcam, catalog no. ab109520), H3K9me3 (1:800, CST, catalog no. 13969S), PD-L1 (1:1000, Proteintech, catalog no. 17952-1-AP), PD-L2 (1:800, CST, catalog no. 82723S), anti-galectin9 (1:1000, abcam, catalog no. ab69630), PVRL2 (1:1000, Proteintech, catalog no. 27171-1-AP), and PVR (1:2000, Proteintech, catalog no. 83724-6).

Techniques: RNA Sequencing, Staining, Fluorescence, Western Blot, Control, Migration, Immunohistochemistry, Marker, In Vivo Imaging, Luciferase, Multiplex Assay

Journal: Science Advances

Article Title: Ara-C suppresses H3 K27–altered spinal cord diffuse midline glioma growth and enhances immune checkpoint blockade sensitivity

doi: 10.1126/sciadv.adu3956

Figure Lengend Snippet: ( A ) The design and groups to evaluate the treatment efficacy of Ara-C and Ara-C in combination with immune checkpoint inhibitors in a mouse spinal cord orthotopic H3 K27M-mutant glioma model. The schematic diagram was drawn by Figdraw (ID: URROU785a8). ( B ) Representative in vivo imaging of luciferase-tagged spinal cord H3 K27M CT-2A orthotopic xenograft models in different treatment groups. Each group comprised eight biological replicates. ( C ) Kaplan-Meier curves illustrate the overall survival of mice in different treatment groups. ( D ) Representative images of H&E staining and IHC of different markers on day 10 post-treatment in five treatment groups. ( E ) Statistical comparison of different marker-positive cells between control and other different treatment groups. *0.01 ≤ P < 0.05, **0.001 ≤ P < 0.01, ***0.0001 ≤ P < 0.001, and **** P < 0.0001 for all figures. n.s., not significant.

Article Snippet: The membranes were then incubated with the following primary antibodies overnight at 4°C: Histone H3 K27M Mutant Specific (1:1000, CST, catalog no. 74829S), Tri-Methyl-Histone H3 Lys27 (1:1000, CST, catalog no. 9733S), H3 (1:5000, Proteintech, catalog no. 68345-1-Ig), Phospho-Chk1 (1:1000, CST, catalog no. 2348S), CHK1 (1:10000, abcam, catalog no. ab40866), CDK1 (1:10,000, abcam, catalog no. ab133327), CDK2 (1:5000, abcam, catalog no. ab32067), Cleaved PARP1 (1:5000, abcam, catalog no. ab32064), Cleaved Caspase-3 (1:1000, CST, catalog no. 9661S), β-actin (1:5000, Proteintech, catalog no. 66009-1), p21(1:5000, abcam, catalog no. ab109520), H3K9me3 (1:800, CST, catalog no. 13969S), PD-L1 (1:1000, Proteintech, catalog no. 17952-1-AP), PD-L2 (1:800, CST, catalog no. 82723S), anti-galectin9 (1:1000, abcam, catalog no. ab69630), PVRL2 (1:1000, Proteintech, catalog no. 27171-1-AP), and PVR (1:2000, Proteintech, catalog no. 83724-6).

Techniques: Mutagenesis, In Vivo Imaging, Luciferase, Staining, Comparison, Marker, Control

Journal: Science Advances

Article Title: Ara-C suppresses H3 K27–altered spinal cord diffuse midline glioma growth and enhances immune checkpoint blockade sensitivity

doi: 10.1126/sciadv.adu3956

Figure Lengend Snippet: ( A ) Representative immunohistochemistry (IHC) image of Ki67 expression in H3-SCDMG ( n = 43) versus H3-wild type-SCDMG ( n = 30). Scale bars, 100 μm. ( B ) Comparison of Ki67 index between H3-SCDMG ( n = 43) and H3-wild type-SCDMG ( n = 30). Data are presented as mean ± SD. ( C ) Patient-derived glioma cell lines (BNI2-4 and BNI3-5) were transfected with H3 K27M (H3 K27M) or empty vector (EV) lentivirus. Immunoblot analysis of histone H3 K27M and H3 K27me3. ( D and E ) Proliferative viability assay of glioma PDCs with H3 K27M (H3 K27M) or empty vector (EV), assessed by CCK8 assay. All data are mean ± SEM. n = 6 technical replicates per group. ( F ) Representative images showing EdU-positive PDCs. DAPI indicates nuclei with DNA, EdU represents cells that have incorporated EdU during the S phase, and Merge refers to the overlay of DAPI and EdU signals. The red color corresponds to EdU-positive cells. Scale bars, 50 μm. ( G ) Percentage of EdU-positive cells in PDCs with H3 K27M or EV. All data are mean ± SD. n = 7 microscopic statistical fields. ( H ) Immunofluorescence localization and expression of PDGFRA protein in PDCs with H3 K27M or EV. Scale bars, 10 μm. ( I ) Summary of IC 50 values of five drugs on three group PDC cells with different H3 backgrounds. ( J ) IC 50 curves for Ara-C in three groups of PDC cells with different H3 backgrounds as well as normal astrocyte HA; all data are mean ± SD. *0.01 ≤ P < 0.05, ***0.0001 ≤ P < 0.001, and **** P < 0.0001 for all figures.

Article Snippet: The antibodies used were as follows: H3 K27M (1:1000, CST, 74829S), NESTIN (1:200, abcam, 105389), PDGFRA (1:500, abcam, 203491), gamma-H2AX (1:250, abcam, 81299), PDL1 (1:100, Proteintech, 17952-1-AP), PDL2 (1:200, CST, 82723S), and PVR (1:500, Proteintech, 27486-1), each diluted to a concentration of 1:200.

Techniques: Immunohistochemistry, Expressing, Comparison, Derivative Assay, Transfection, Plasmid Preparation, Western Blot, Viability Assay, CCK-8 Assay, Immunofluorescence

Journal: Science Advances

Article Title: Ara-C suppresses H3 K27–altered spinal cord diffuse midline glioma growth and enhances immune checkpoint blockade sensitivity

doi: 10.1126/sciadv.adu3956

Figure Lengend Snippet: ( A ) Schematic diagram of the construction of spinal cord diffuse midline glioma PDX and PDC models. By Figdraw (ID: SSPWSb5cc5). ( B ) Photographs of subcutaneous tumors in PDX_S01 and PDX_S29 mice, results of hematoxylin and eosin (H&E) staining, and IHC images of tumor-associated markers. Black arrows point to mouse PDX tumors. ( C ) Photographs of tumor tissues after 2 weeks of Ara-C treatment for spinal glioma PDXs (PDX_S01 and PDX_S29). ( D ) Tumor volume growth curves for the control and Ara-C treatment groups. n = 8 biological replicates per group. All data are mean ± SD. **** P < 0.0001 by Student’s t test. ( E ) Two PDCs were obtained from the corresponding PDX models. Microscopic photographs were taken to demonstrate the morphology of the cells and the way they grew in suspension. Scale bars, 100 μm. Immunofluorescence staining demonstrated that both PDCs highly expressed H3 K27M, NESTIN, and PDGFRA. Nuclei labeled with DAPI. Scale bars, 10 μm. ( F ) IC 50 curves of two PDCs in the presence of different concentrations of the six antitumor drugs.

Article Snippet: The antibodies used were as follows: H3 K27M (1:1000, CST, 74829S), NESTIN (1:200, abcam, 105389), PDGFRA (1:500, abcam, 203491), gamma-H2AX (1:250, abcam, 81299), PDL1 (1:100, Proteintech, 17952-1-AP), PDL2 (1:200, CST, 82723S), and PVR (1:500, Proteintech, 27486-1), each diluted to a concentration of 1:200.

Techniques: Staining, Control, Suspension, Immunofluorescence, Labeling

Journal: Science Advances

Article Title: Ara-C suppresses H3 K27–altered spinal cord diffuse midline glioma growth and enhances immune checkpoint blockade sensitivity

doi: 10.1126/sciadv.adu3956

Figure Lengend Snippet: ( A ) Representative images showing EdU-positive H3-SCDMG PDCs. Scale bars, 50 μm. ( B ) Percentage of EdU-positive cells in H3-SCDMG PDCs. ( C ) Immunofluorescence image of gamma-H2AX protein in H3-SCDMG PDCs. Scale bars, 10 μm. ( D ) The number of γ-H2AX-positive foci in each PDC cell was quantified and compared. ( E and G ) Representative cell cycle graph of PDC cells treated with Ara-C (1 μM) for 48 hours. n = 3 biological replicates per group. ( F and H ) Statistical results of distributing cells at different cell cycles. Data are mean ± SD. ( I and J ) Representative images of flow apoptosis assay of PDCs (PDC_S01 and PDC_S29) after 48 hours of treatment with Ara-C (1 μM). The second quadrant corresponds to late apoptotic cells, and the fourth quadrant represents early apoptotic cells. Bar graphs analyzing the percentage of apoptotic cells. n = 3 biological replicates per group. ( K and L ) Functional analysis of genes with different RNA expression levels along with Ara-C treatment for different times. ( M ) Western blot showed the expression of cellular DNA damage repair and apoptosis-related proteins in two types of PDC cells after 2 and 5 days of Ara-C (1 μM) treatment. ( N ) IHC showed expression of H3 K27M, Ki-67, and cleaved-caspase 3 (c-caspase3) in different cells and treatments. ( O ) Percentage of Ki67 and c-caspase3–positive cells in tumors of PDX with different treatments. Data are mean ± SD. **** P < 0.0001 for all figures.

Article Snippet: The antibodies used were as follows: H3 K27M (1:1000, CST, 74829S), NESTIN (1:200, abcam, 105389), PDGFRA (1:500, abcam, 203491), gamma-H2AX (1:250, abcam, 81299), PDL1 (1:100, Proteintech, 17952-1-AP), PDL2 (1:200, CST, 82723S), and PVR (1:500, Proteintech, 27486-1), each diluted to a concentration of 1:200.

Techniques: Immunofluorescence, Apoptosis Assay, Functional Assay, RNA Expression, Western Blot, Expressing

Journal: Science Advances

Article Title: Ara-C suppresses H3 K27–altered spinal cord diffuse midline glioma growth and enhances immune checkpoint blockade sensitivity

doi: 10.1126/sciadv.adu3956

Figure Lengend Snippet: ( A ) The schematic diagram delineates the experimental protocol for the orthotopic implantation of spinal cord PDX models and subsequent treatment in immunodeficient mice. ( B ) Representative in vivo imaging of luciferase-tagged spinal cord tumor orthotopic xenograft models in the treatment group. ( C ) Tumor burden in luciferase-tagged PDX tumor models was quantified by measuring the region of interest (ROI) values obtained from in vivo imaging. ( D ) Kaplan-Meier curves illustrate the survival of mice. Each group consisted of eight biological replicates. ( E ) Representative images of IHC for tumor-associated markers (H3 K27M, H3 K27me3, Ki67, and cleaved-caspase3) in PDX tumors with different treatments for 2 weeks. ( F ) The schematic diagram shows the treatment for patients in investigator-initiated clinical trial. Patients were enrolled in the treatment after being diagnosed with recurrence via magnetic resonance imaging (MRI). Following the implantation surgery of an Ommaya reservoir into the intracranial ventricle, patients began to receive six cycles of intrathecal Ara-C chemotherapy. The treatment was administered monthly, with three times given in the first week of each cycle, at an intrathecal dose of 30 mg/m 2 body surface area. Patients underwent MRI scans every 3 months for follow-up. ( G ) Statistical summary of the assessment of treatment outcomes in four patients. ( H to J ) Pre- and posttreatment MRI images of three patients. The tumor lesions are marked with red. The nonneoplastic lesion below the red-marked area in patient P3 may be a syrinx in the spinal cord caused by the compression of the central canal due to the recurrent tumor. The schematic diagrams of (A) and (F) were drawn by Figdraw (ID: OPSWY8c6c8 and RYSPR13933).

Article Snippet: The antibodies used were as follows: H3 K27M (1:1000, CST, 74829S), NESTIN (1:200, abcam, 105389), PDGFRA (1:500, abcam, 203491), gamma-H2AX (1:250, abcam, 81299), PDL1 (1:100, Proteintech, 17952-1-AP), PDL2 (1:200, CST, 82723S), and PVR (1:500, Proteintech, 27486-1), each diluted to a concentration of 1:200.

Techniques: In Vivo Imaging, Luciferase, Magnetic Resonance Imaging

Journal: Science Advances

Article Title: Ara-C suppresses H3 K27–altered spinal cord diffuse midline glioma growth and enhances immune checkpoint blockade sensitivity

doi: 10.1126/sciadv.adu3956

Figure Lengend Snippet: ( A ) GSVA of RNA-seq data shows up-regulated Reactome terms in PDC_S29 after 5 days of Ara-C (1 μM) treatment. ( B ) Senescence-associated β-galactosidase staining in PDCs after Ara-C treatment (0, 1, 3, and 5 days). Scale bars, 100 μm. ( C ) SPiDER-βGal fluorescence intensity in PDCs post–Ara-C treatment. Data: mean ± SD; n = 6. ( D ) Western blot of senescence markers p21 and H3K9me3 in PDCs (PDC_S01 and PDC_S29) after Ara-C treatment (0, 2, and 5 days). ( E ) qPCR analysis of cytokine/chemokine mRNA in PDCs post–Ara-C treatment. Data: mean ± SEM; n = 3. # P < 0.05 versus control. ( F ) Workflow for tumor cell recruitment assay: PDCs treated with Ara-C or DMSO for 5 days, assessing PBMC migration using the conditioned medium. n = 3. By Figdraw (ID: ORYAP9a9fa). ( G ) Microscopic images of PBMC migration. Scale bars, 100 μm. ( H and I ) PBMC migration quantification. n = 12 fields. ( J ) H&E and IHC staining of subcutaneous tumors in C57 mice treated with PBS or Ara-C. ( K ) Quantification of marker-positive cells in tumors. Data: mean ± SD; n = 5. **** P < 0.0001. ( L ) In vivo imaging of luciferase-tagged spinal cord xenograft models. n = 6 per group. ( M ) Kaplan-Meier survival curves for different treatment groups. ( N ) Multiplex staining of H3 K27M, CD3e, and IBA1 in spinal cord xenografts with or without Ara-C. ( O ) Quantification of CD3e- and IBA1-positive cells. Data: mean ± SD; n = 3.

Article Snippet: The antibodies used were as follows: H3 K27M (1:1000, CST, 74829S), NESTIN (1:200, abcam, 105389), PDGFRA (1:500, abcam, 203491), gamma-H2AX (1:250, abcam, 81299), PDL1 (1:100, Proteintech, 17952-1-AP), PDL2 (1:200, CST, 82723S), and PVR (1:500, Proteintech, 27486-1), each diluted to a concentration of 1:200.

Techniques: RNA Sequencing, Staining, Fluorescence, Western Blot, Control, Migration, Immunohistochemistry, Marker, In Vivo Imaging, Luciferase, Multiplex Assay

Journal: Science Advances

Article Title: Ara-C suppresses H3 K27–altered spinal cord diffuse midline glioma growth and enhances immune checkpoint blockade sensitivity

doi: 10.1126/sciadv.adu3956

Figure Lengend Snippet: ( A ) The design and groups to evaluate the treatment efficacy of Ara-C and Ara-C in combination with immune checkpoint inhibitors in a mouse spinal cord orthotopic H3 K27M-mutant glioma model. The schematic diagram was drawn by Figdraw (ID: URROU785a8). ( B ) Representative in vivo imaging of luciferase-tagged spinal cord H3 K27M CT-2A orthotopic xenograft models in different treatment groups. Each group comprised eight biological replicates. ( C ) Kaplan-Meier curves illustrate the overall survival of mice in different treatment groups. ( D ) Representative images of H&E staining and IHC of different markers on day 10 post-treatment in five treatment groups. ( E ) Statistical comparison of different marker-positive cells between control and other different treatment groups. *0.01 ≤ P < 0.05, **0.001 ≤ P < 0.01, ***0.0001 ≤ P < 0.001, and **** P < 0.0001 for all figures. n.s., not significant.

Article Snippet: The antibodies used were as follows: H3 K27M (1:1000, CST, 74829S), NESTIN (1:200, abcam, 105389), PDGFRA (1:500, abcam, 203491), gamma-H2AX (1:250, abcam, 81299), PDL1 (1:100, Proteintech, 17952-1-AP), PDL2 (1:200, CST, 82723S), and PVR (1:500, Proteintech, 27486-1), each diluted to a concentration of 1:200.

Techniques: Mutagenesis, In Vivo Imaging, Luciferase, Staining, Comparison, Marker, Control