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: Single‐cell transcriptomics and the grafting assay reveal the TIC function of tumor OPCs in adult OPC‐derived gliomas. A) The genetic configuration of the CKO_NG2‐Cre ER mouse model. B) The gross image of a tumor brain used for the scRNA‐seq in (C). C) The tSNE map of all sequenced cells from the tumor in (B). The cluster containing tumor OPCs is circled. D) The Violin plots of some marker genes from the clusters defined in (C). Same color code is used in (C) and (D). E–H) Projection of the lineage or marker genes as indicated onto the tSNE map in (C). The cluster containing tumor OPCs is circled. I) The pseudo‐time plot of all tdTomato + cells from Clusters 2, 5, 6, and 7. The presumed differentiation directions are marked as dotted arrow lines. J) The tSNE map of a mouse CKO_NG2‐Cre ER glioma cell line from the scRNA‐seq data. Distinct clustered are marked by different colors. K) Projection of the marker genes as indicated onto the tSNE map in (J). L) The representative FACS plot showing the expression of PDGFR α on mouse tumor OPCs. M) The Western blots validate the expression of FACS‐sorted glioma cells based on their surface PDGFR α expression. N) The in vitro sphere assay of tumor OPCs based on their surface PDGFR α expression sorted by FACS. Scale bar: 100 µm. O) The survival curves of mice grafted with PDGFR α high/low tumor OPC fractions, N = 4 mice for each group, :: p < 0.01.

Article Snippet: Dissociated cells were incubated with desired antibodies for 30 min at room temperature followed by FACS analysis or sorting on CytoFLEX LX or BD FACSAria II and Beckman moflo Astrios EQ cell sorter .The antibodies used for FACS are: PE‐conjugated Mouse Anti‐Human CD221 (BD, #555999), Alexa FluorR 647‐conjugated Mouse Anti‐Human CD140 α (BD, #562798), APC‐conjugated IGF1R (Abcam, #ab225298); APC‐conjugated Mouse PDGFR α (BD, #FAB1062A); PE‐conjugated anti‐human PDGFR α (BD, #FAB1264P); APC‐conjugated anti‐Human PDGFR α (BD, #FAB1264A).

Techniques: Single-cell Transcriptomics, Derivative Assay, Marker, Expressing, Western Blot, In Vitro

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 impacts of IGF1R knockout on the growth of mutant and normal OPCs at the pretransforming stage of tumor development. A,B) Schematics showing the tamoxifen (TAM) and BrdU treatment schedules for the mice analyzed either acutely (A) or long‐term (B). C,D) The quantification of BrdU + cells among all the tdTomato labeled Olig2 + cells in the brain regions of mice with the four different genotypes shown, after either the C) acute or D) long‐term experiments. Please refer to Table S7, Supporting Information, for all raw quantification data, N = 3 mice for each group. E) Representative images of the brain sections from the olfactory bulb region after the long‐term experiment. The arrows indicate cells coexpressing markers. The insets show a zoom‐in image of the indicated cells. Scale bar: 50 µm. F) Images showing the distinct cell types along the OPC lineage, where OPCs were defined as PDGFR α + and CC1 − , newly formed oligodendrocytes as PDGFR α + and CC1 + , and mature oligodendrocytes as PDGFR α − and CC1 + . Scale bar: 30 µm. G) The relative abundance of the indicated subpopulations from the corpus callosum after the acute and long‐term experiments. N = 3 mice for each group. Mean ± SEM. t ‐test, : p < 0.05, :: p < 0.01, ::: p < 0.001, :::: p < 0.0001, ns, no significance.

Article Snippet: Dissociated cells were incubated with desired antibodies for 30 min at room temperature followed by FACS analysis or sorting on CytoFLEX LX or BD FACSAria II and Beckman moflo Astrios EQ cell sorter .The antibodies used for FACS are: PE‐conjugated Mouse Anti‐Human CD221 (BD, #555999), Alexa FluorR 647‐conjugated Mouse Anti‐Human CD140 α (BD, #562798), APC‐conjugated IGF1R (Abcam, #ab225298); APC‐conjugated Mouse PDGFR α (BD, #FAB1062A); PE‐conjugated anti‐human PDGFR α (BD, #FAB1264P); APC‐conjugated anti‐Human PDGFR α (BD, #FAB1264A).

Techniques: Knock-Out, Mutagenesis, Labeling

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 incubated with desired antibodies for 30 min at room temperature followed by FACS analysis or sorting on CytoFLEX LX or BD FACSAria II and Beckman moflo Astrios EQ cell sorter .The antibodies used for FACS are: PE‐conjugated Mouse Anti‐Human CD221 (BD, #555999), Alexa FluorR 647‐conjugated Mouse Anti‐Human CD140 α (BD, #562798), APC‐conjugated IGF1R (Abcam, #ab225298); APC‐conjugated Mouse PDGFR α (BD, #FAB1062A); PE‐conjugated anti‐human PDGFR α (BD, #FAB1264P); APC‐conjugated anti‐Human PDGFR α (BD, #FAB1264A).

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

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 crosstalk of IGF1R and PDGFR α pathways in tumor OPCs. A) Activation of signaling pathways in tumor OPCs after stimulation with IGF1 (20 ng mL −1 ) or PDGFAA (20 ng mL −1 ) for the indicated periods. OE, overexposed. The red asterisk indicates the presence of pIGF1R after PDGFAA stimulation for 120 min. B) Sphere assay for mouse tumor OPCs treated with the indicated inhibitors. Spheres were formed in the presence of 10 ng mL −1 IGF1. Mean ± SEM. ::: p < 0.001. C) Representative images showing purified mutant and WT OPCs used for the biochemical analysis in (D). Scale bar: 50 µm. D) The response of WT and Trp53/NF1 double mutant OPCs to IGF1 in the presence or absence of OSI‐906. E) Activation of indicated RTKs in mouse tumor OPCs after stimulation with IGF1 for the indicated periods. F) Activation of indicated RTKs in human tumor OPCs after stimulation with IGF1 for the indicated periods. G,H) Co‐IP assay to determine the interaction between mouse PDGFR α and IGF1R. Exogeneous expression of mouse PDGFR α and IGF1R in either HEK293 cells (G) or the endogenous PDGFR α and IGF1R in mouse tumor OPCs (H) was performed separately. I) Co‐IP assay to determine the interaction between exogenously expressed human PDGFR α and IGF1R in HEK293 cells. J) The effect of OSI‐906 on IGF1 (20 ng mL −1 ) and/or PDGFAA (20 ng mL −1 ) signal transduction. The cells were analyzed 2 h after OSI‐906 (0.5 µ m ) treatment. K) IGF1R‐null mouse tumor OPCs responded to the stimulation of growth factors (20 ng mL −1 ) as indicated. OE, overexposed. L) Schematic showing the hypothetical crosstalk between IGF1R and PDGFR α in tumor OPCs.

Article Snippet: Dissociated cells were incubated with desired antibodies for 30 min at room temperature followed by FACS analysis or sorting on CytoFLEX LX or BD FACSAria II and Beckman moflo Astrios EQ cell sorter .The antibodies used for FACS are: PE‐conjugated Mouse Anti‐Human CD221 (BD, #555999), Alexa FluorR 647‐conjugated Mouse Anti‐Human CD140 α (BD, #562798), APC‐conjugated IGF1R (Abcam, #ab225298); APC‐conjugated Mouse PDGFR α (BD, #FAB1062A); PE‐conjugated anti‐human PDGFR α (BD, #FAB1264P); APC‐conjugated anti‐Human PDGFR α (BD, #FAB1264A).

Techniques: Activation Assay, Purification, Mutagenesis, Co-Immunoprecipitation Assay, Expressing, Transduction

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: A new generation of oral IGF1R inhibitor that can penetrate the blood–brain barrier. A) Chemical evolution of PB‐020. B) The Western blots of the major pathways in the brain tissues from the mice that orally treated with the vehicle or the inhibitors as indicated. C) HPLC analyses of the brain extracts from the mice orally treated with the compound as indicated. Mice were dosed by gavage with 50 mg kg −1 . At predosing (0 h) and different post‐dosing time points, the mice were euthanized, and their blood and brain samples were processed for HPLC analysis. D) IC 50 of five lines of adherent glioma cell lines as indicated ( N = 3, mean ± SD). Among them, U87MG, DBTRG, and KNS‐81 lines were maintained in 10% serum. Mouse glioma cell line Ms1483 and human GBM cell line #H5 were maintained in the stem cell media. E) The sphere assay of mouse tumor OPCs treated with the indicated inhibitors (0.5 µ m ) and 20 ng mL −1 IGF1. Western blots showing that PB‐020 also decreased the total level of both IGF1R and PDGFR α . F) Survival curves of NOD‐SCID mice orthotopically grafted with mouse tumor OPCs and orally treated with vehicle, PPP or PB‐020. N = 5 mice for vehicle and PPP group, N = 6 for PB‐020 group. G) Representative images of the brain sections from (F). Scale bar: 100 µm. H,I) Photos of mice and the H&E staining of the brain sections (including the hippocampus) treated with the vehicles or inhibitors as indicated. Scale bar: 400 µm in (I). J) The percentage tumor OPCs (Olig2 + ) among proliferating (top) or all tumor cells (bottom) in the tumor mice after treated with vehicle or PB‐020. Mean ± SEM, N = 6 for each group. K) The docking model of PB‐020 in the ligand‐binding site 1 of apo IGF1R. The zoom‐in view of PB‐020 bound in the binding site 1 of IGF1R. L) The comparison of the docked binding modes of PPP and PB‐020 in the ligand‐binding site 1 of apo IGF1R. Comparing to PPP, PB‐020 is pushed out a little bit further to the entrance of the IGF1 binding pocket (marked as dark gray shade) because of two fluorine atoms. All oxygen atoms are colored in red. : p < 0.05, :: p < 0.01, ns, no significance.

Article Snippet: Dissociated cells were incubated with desired antibodies for 30 min at room temperature followed by FACS analysis or sorting on CytoFLEX LX or BD FACSAria II and Beckman moflo Astrios EQ cell sorter .The antibodies used for FACS are: PE‐conjugated Mouse Anti‐Human CD221 (BD, #555999), Alexa FluorR 647‐conjugated Mouse Anti‐Human CD140 α (BD, #562798), APC‐conjugated IGF1R (Abcam, #ab225298); APC‐conjugated Mouse PDGFR α (BD, #FAB1062A); PE‐conjugated anti‐human PDGFR α (BD, #FAB1264P); APC‐conjugated anti‐Human PDGFR α (BD, #FAB1264A).

Techniques: Western Blot, Staining, Ligand Binding Assay, Binding Assay