Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative immunostaining for ZIKV envelope protein (ZIKV-E, green) and DAPI (blue) of GSCs and forebrain-specific NPCs 48 h post-infection (p.i.) with ZIKV. Scale bar, 50 μm. (B) Quantification of infection efficiency in four GSC and NPC lines 48 h p.i. with ZIKV. (C) Quantification of ZIKV+ cells in a panel of human GSCs and NPCs. (D) Kinetics of viral RNA copies p.i. with ZIKV by measuring viral RNA copies by qRT-PCR in NPC C4–7 and GSC3565. (E) ZIKV infection efficiency of GSCs and NPCs was measured by direct measurement of viral RNA copies. (F) Representative bright-field images 5 days p.i. with ZIKV for GSCs, NPCs, and primary astrocytes. Scale bars, 50 μm. (G) Cell viability normalized to day 5 mock, as measured 5 days p.i. with ZIKV for GSCs, NPCs, and primary astrocytes. (H) GSCs (GSC3565), differentiated GSCs, NPCs (NPC C4–7), and differentiated NPCs were assayed for cell viability 72 h p.i. with ZIKV. (I) Apoptosis of GSCs (387, 3565) and primary (NPC194, fetal human [fh] NPC) or iPSC-derived NPCs (WT83, C4–7) p.i. with ZIKV was measured by cleaved caspase-3 (CC3) staining. (J) Representative immunostaining for ZIKV-E (green), CC3 (red), and DAPI (blue) of GSCs and forebrain-specific NPCs 48 h p.i. with ZIKV. Scale bar, 50 μm. (K) Representative immunostaining for ZIKV-E (green), CC3 (red), and DAPI (blue) of GSCs and forebrain-specific NPCs 72 h p.i. with ZIKV. Scale bars, 50 μm. (L) Quantification of the percentage of CC3+ cells in DAPI+ cells for GSCs and NPCs 72 h p.i. with ZIKV. (M) Cell viability of patient-derived cultures from GBM (387 and 3565), pontine glioma (3752 and 007), meningioma (CH-157MN, IOMM-LEE), ependymoma (EP1), and medulloblastoma cell lines (DAOY, D283, HDMB03, D341) 72 h after ZIKV infection. Experiments were performed in two biological replicates with three technical repeats. Values represent mean ± SEM. NS, no significance. ****p < 0.0001 by one-way ANOVA.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Immunostaining, Infection, Quantitative RT-PCR, Derivative Assay, Staining

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative immunostaining for ZIKV-E (green), SOX2 (red), and DAPI (blue) of GSCs and forebrain-specific hiPSC-derived NPCs 48 h p.i. with ZIKV. Scale bar, 50 μm. (B) Quantification of the percentage of SOX2+ cells in DAPI+ cells for GSCs and NPCs 48 h p.i. with ZIKV. (C) Representative immunostaining for ZIKV-E (green), SOX2 or AXL (red), and DAPI (blue) of GSCs (GSC3565) without transduction (shRNA) or transduced with control shRNA (shCONT), AXL shRNA (shAXL.2), or SOX2 shRNA (shSOX2.53) for 72 h and then 48 h with ZIKV infection. Scale bars, 100 μm. (D) Quantification of the percentage of ZIKV+ cells in DAPI+ cells in GSCs 1517 and 3565 under conditions for (C), with a range of ZIKV infection. (E) Viral copy number by qRT-PCR of GSCs (GSC3565 or GSC1517) or NPC C4–7 transduced with either shCONT or SOX2 shRNA (shSOX2.52 or shSOX2.53) for 72 h and then either exposed to mock conditions or infected with ZIKV for another 72 h. All comparisons are versus shCONT. (F) Gene set enrichment (GSE) bubble plots showing pathways positively (top, r > 0.4) or negatively (bottom, r < −0.4) correlated with SOX2 expression in the TCGA GBM HG-U133A microarray dataset. Each circle represents an enriched pathway, with the border color indicating the false discovery rate (FDR)-corrected p value. (G) GSE graph showing the top pathway enrichments positively or negatively correlated with SOX2 as described in (F). (H) Correlation of mRNA levels of SOX2 with IFNAR1, IRF1, promyelocytic leukemia (PML), and IFITIM1 from the TCGA GBM HG-U133A microarray dataset. (I) Correlation between SOX2 with ISGs from the TCGA GBM HG-U133A microarray dataset. The size and color of the dots indicate the degree of correlation (p < 0.001). Blank cells indicate a non-significant correlation. (J) qPCR of ISGs (IFNAR-1, ISH20, IRF1, IFITM1, TLR3, and OAS2) in GSCs (GSC3565) transduced with either shCONT or SOX2 shRNA (shSOX2.52 or shSOX2.53). Experiments were performed in two biological replicates with three technical repeats. Values represent mean ± SEM. **p < 0.001, ****p < 0.0001 by one-way ANOVA.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Immunostaining, Derivative Assay, Transduction, shRNA, Control, Infection, Quantitative RT-PCR, Expressing, Microarray

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative images of mock- or ZIKV-infected BCOs stained with neuronal markers (CTIP2 and NeuN), a neural progenitor cell marker (SOX2), and DAPI. Scale bars, 100 μm. (B) Quantification of BCO size p.i. with ZIKV. Significance was assessed by two-tailed Student’s t test, and experiments were performed in two batches with 12 organoids per group per batch. (C) BCO size fold change of ZIKV- and mock-treated groups over a period of 1 month. (D) Quantification of SOX2+ cells in ZIKV- versus mock-infected groups. *p < 0.05 by two-tailed Student’s t test. (E) Quantification of CC3+ cells in ZIKV- versus mock-infected groups. *p < 0.05 by two-tailed Student’s t test. (F) Quantification of SATB2+ cells within MAP2+ cells in ZIKV- versus mock-infected groups. **p < 0.01 by two-tailed Student’s t test. (G) Quantification of GFAP+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (H) Quantification of NeuN+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (I) Quantification of CTIP2+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (J) Bright-field images of engraftment of two patient-derived GSCs (387 and 3565) transduced with GFP into human BCOs over a time course. Scale bars, 1 mm. (K) Engrafted GSCs (GFP+) with normal BCO immunostained for integrin αvβ5 (red), GFP (green), and DAPI (blue). Scale bars, 200 μm. (L) Quantification of integrin αvβ5+ cells in normal BCOs or GSC-BCOs. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-tailed Student’s t test. (M) Representative images of GFP-labeled GSC-BCOs immunostained for integrin αvβ5 (red), GFP (green), and DAPI (blue). Scale bars, 100 μm. (N) Representative images of GFP-labeled GSC-BCOs immunostained for SOX2 (red), GFP (green), and DAPI (blue). Scale bars, 100 μm. (O) Images of GFP-labeled GSC-GFP BCOs 13 days p.i. with ZIKV. Scale bars, 1 mm. (P) Representative images of residual GSCs (green) and DAPI staining (blue) of GFP-labeled GSC-GFP BCOs cultured under mock conditions or with ZIKV for 2–4 weeks. Scale bars, 200 μm. The percentage of GFP+ cells among DAPI+ cells was quantified. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-way ANOVA. (Q) Representative immunostaining for integrin αvβ5 (red), GFP (green), ZIKV-E (white), and DAPI (blue) of GFP-labeled GSC-GFP BCOs mock- or ZIKV-infected for 2–4 weeks. Scale bars, 200 μm (left) and 100 μm (center). The percentage of ZIKV-E+ cells among integrin αvβ5 cells was quantified. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-tailed Student’s t test. (R) Representative images of 387 and 3565 GSC-BCOs with or without ZIKV, respectively, stained with SOX2, ZIKV-E, and DAPI. GFP shows the presence of GSCs (scale bars, 50 μm). ZIKV-E+, GFP+, and ZIKV-E+ cells among GFP+ cells were quantified by counting (two GSCs cell lines, two repeats, n = 12 organoids/group); *p < 0.05 by two-tailed Student’s t test. (S) Schematic of the experiment design. (T) Volcano plot showing differences between GSC-BCO ZIKV versus GSC-BCO mock. 113 genes were differentially expressed (greater than 1.5-fold) between these two groups (*p < 0.05). (U) Network analysis of genes differentially expressed upon ZIKV infection, represented as a bubble plot.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Infection, Staining, Marker, Two Tailed Test, Derivative Assay, Transduction, Labeling, Cell Culture, Immunostaining

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Immunostaining of the subventricular zone (SVZ) of mice 72 h following ZIKV infection ZIKV-E (green), SOX2 (red, top panels), and integrin αvβ5 (red, bottom panels). Scale bars, 50 μm. (B) Higher magnification of images from (A), demonstrating ZIKV infection of SOX2+ (top panels) and integrin αvβ5+ cells. Scale bars, 10 μm. (C) Survival of ZIKV-infected NSG mice from (A) was plotted by the Kaplan-Meier method. (D) ZIKV-infected brains from the mice in (A) were collected upon death, and histology was assessed by H&E staining. Scale bars, 20 μm. (E) Survival of NSG mice following implantation of GSCs treated with isotype control, P1F6 antibody, ZIKV, combined P1F6 and ZIKV, combined CRISPR knockout (KO) of integrin β5 (sgRNA1 sgRNA2) with ZIKV inoculation, analyzed by log rank test; p < 0.01. (F) H&E staining of tumor-bearing brains from (E). Scale bars, 50 μm. (G) Intraoperative brain slices from GBM patients were pre-incubated with an IgG control antibody or an integrin-blocking antibody under mock conditions or upon ZIKV infection (10e3 FFU). Slices then underwent immunofluorescence staining for ZIKV-E (green), integrin αvβ5 (red), and DAPI (blue). Scale bars, 10 μm. (H) Intraoperative brain slices from GBM patients were pre-incubated with an IgG control antibody or an integrin-blocking antibody under mock conditions or upon ZIKV infection. Slices then underwent a viral RNA copy assay by qRT-PCR. Experiments were performed in two biological replicates with three technical repeats. Values represent mean ± SEM. ****p < 0.0001 by one-way ANOVA.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Immunostaining, Infection, Staining, Control, CRISPR, Knock-Out, Incubation, Blocking Assay, Immunofluorescence, Quantitative RT-PCR