Journal: Advanced Science

Article Title: Oncogenic State and Cell Identity Combinatorially Dictate the Susceptibility of Cells within Glioma Development Hierarchy to IGF1R Targeting

doi: 10.1002/advs.202001724

Figure Lengend Snippet: The role of IGF1R in the initiation and progression of the OPC‐derived glioma model. A,B) Representative images (A) and quantification (B) of sphere formation by tumor OPCs in the presence of different growth factors (GFs) as indicated. EGF, epithelial growth factor; FGF, basic fibroblast growth factor; HGF, hepatocyte growth factor; NT‐3, neurotrophin‐3; BDNF, brain‐derived neurotrophic factor; GH, growth hormone. All growth factors were provided as 10 ng mL −1 . Scale bar: 100 µm. C) The projection of indicated genes on the tSNE map of a tumor OPC cell line that is also shown in Figure . D) The representative FACS plot and Western blots showing the expression of IGF1R on mouse OPC‐like glioma cells. E) In vitro sphere assay of OPC‐like glioma cells based on their surface IGF1R expression sorted by FACS. Scale bar: 100 µm. F,G) The survival curves of mice grafted with IGF1R high/low OPC‐like glioma cells fractions. (F) and (G) are from two independent cell lines, N = 6 mice for IGF1R high group and N = 5 mice for IGF1R low group in (F), N = 5 mice for IGF1R high group and N = 6 mice for IGF1R low group in (G). H) The genetic configuration of the CKO_NG2‐Cre ER mouse model with conditional IGF1R knockout. I) Survival curves for the three mouse models as indicated. The median survival times are indicated by the dashed line. For the repeated TAM group, the mouse model was given tamoxifen twice a month following the first round of tamoxifen treatment at postnatal (P) day 31‐P35. For the other three groups, tamoxifen was given only from P31‐P35. J) Tumor incidences in the three mouse models at the endpoint of analysis from (I). The same groups of mice were used for calculating survival curves in (I) and tumor incidence in (J), ND: Not determined. K) Representative images of brain sections from the CKO and CKO‐IGF1R (flox/flox) mice immune‐stained as indicated. The dashed line demarcates the tumor boundary. T, tumor. Scale bars: 1 mm (gross images), 100 µm (zoom‐in section). L) Genotyping data from paired normal and tumor tissues from the three mouse models indicated. N, normal. T, tumor.M) Survival curves of the NOD‐SCID mice orthotopically grafted with the tumor cells as indicated. The IGF1R KO+ rescue cells were the ones from the IGF1R KO tumor OPCs stably transfected with the lentiviral vector that overexpressed IGF1R. The expression/absence of the IGF1R protein from these cells was validated by the western blots, N = 7 mice for CKO and IGF1R KO group, N = 5 mice for IGF1R KO + rescue group. N–P) Knocking out of IGF1R by the CRISPR‐Cas9 approach. The histological analyses (N) of the tumor sections from these tumors as shown in (O). The survival curves (O) of the NOD‐SCID mice grafted with the indicated tumor cells. FACS analysis (P) confirmed the decreased expression of surface IGF1R protein. Scale bar: 100 µm in (N), N = 6 mice for sgRNA‐Ctrl group, N = 8 mice for sgRNA‐IGF1R group. Mean ± SEM. : p < 0.05, :: p <0.01, ::: p < 0.001.

Article Snippet: Dissociated cells were transferred in Neurobasal A/B27 media supplemented with 1% l ‐glutamine, 2 mg mL −1 of heparin, 20 ng mL −1 of human FGF (Peprotech, #100‐18‐B) and 20 ng mL −1 of human EGF (Peprotech, #AF‐100‐15).

Techniques: Derivative Assay, Western Blot, Expressing, In Vitro, Knock-Out, Staining, Stable Transfection, Transfection, Plasmid Preparation, CRISPR

Journal: Advanced Science

Article Title: Oncogenic State and Cell Identity Combinatorially Dictate the Susceptibility of Cells within Glioma Development Hierarchy to IGF1R Targeting

doi: 10.1002/advs.202001724

Figure Lengend Snippet: The susceptibility of human tumor OPCs toward IGF1R targeting. A) Representative images showing that IGF1R and pIGF1R are expressed in proliferating tumor OPCs from a GBM sample. The arrows indicate the cells coexpressing markers indicated. The low power images are provided at the bottom row to show no bias of imaging collection. Z ‐axis orthogonal views are provided in zoom‐in images to confirm the colocalization of the markers. Scale bars, 20 µm in the top and middle rows; 100 µm in the bottom row. B) Zoom‐in of images to show the expression of the indicated markers in one tumor OPC. The fluorescence intensity across the middle plane of the cell is shown. Scale bars, 3 µm. C) Schematic diagram showing the immunopanning method used to enrich human tumor OPCs. D) Representative images showing that PDGFR α ‐positive cells were enriched in the panned fraction. Conversely, GFAP‐positive tumor cells were largely depleted but appeared in the supernatant fraction. Scale bar: 400 µm. E–G) The MRI images (E), subtype analysis (F), and Western blots (G) of indicated proteins for a human GBM (#H5). Additional histological and pathological information of this tumor case can be found in Figures S3 and S8, Supporting Information. H,I) Representative images and the quantification from a sphere assay of a primary human GBM cell line (#H63) treated with the indicated growth factors. S: supernatant fraction, P: immunopanned tumor OPCs fraction. IGF1, 10 ng mL −1 ; EGF, 50 ng mL −1 ; FGF, 20 ng mL −1 ; PDGF, 20 ng mL −1 . OSI‐906, 0.5 µ m . Scale bar: 25 µm in (H). J) Schematic diagram showing the strategy to genetically knock down IGF1R in patient‐derived xenograft (PDX) models. K) Piggybac (PB) transposon vectors encoding multiplex miRNAs against different sites of IGF1R (Mir‐IGF1R) or against Luciferase (Mir‐Luc). L) The MRI image of the fresh tumor sample used in (P,Q). M) The knock down of IGF1R was validated by qPCR. N,O) The quantification (N) and the image (O) of tumor spheres after IGF1R was knocked down by MirRNA. Scale bar: 100 µm in (O). P,Q) The quantification results from the PDX model shown in (L), N = 3 mice for each group. R,S) The sphere assay (R) and the survival curves (S) of NOD‐SCID mice grafted with human OPC‐like glioma cells (#H5) based on their surface IGF1R expression. Scale bar: 100 µm in (R), N = 5 mice for IGF1R high group and N = 6 mice for IGF1R low group in (S). T) The survival curve of NOD‐SCID mice grafted with a human glioma stem cell line based on their surface IGF1R expression. Of note, this cell line does not express PDGFR α , N = 7 mice for IGF1R high group and N = 6 mice for IGF1R low group. Mean ± SEM. : p < 0.05, :: p < 0.01, ::: p < 0.001.

Article Snippet: Dissociated cells were transferred in Neurobasal A/B27 media supplemented with 1% l ‐glutamine, 2 mg mL −1 of heparin, 20 ng mL −1 of human FGF (Peprotech, #100‐18‐B) and 20 ng mL −1 of human EGF (Peprotech, #AF‐100‐15).

Techniques: Imaging, Expressing, Fluorescence, Western Blot, Derivative Assay, Multiplex Assay, Luciferase