Journal: Scientific Reports

Article Title: Transcriptomic analysis identifies a potential target for bevacizumab resistant glioblastoma

doi: 10.1038/s41598-025-20847-4

Figure Lengend Snippet: Elevated expression of nuclear EGR1 in tumors that were poor-responders to bevacizumab therapy. ( a , b ) Sections were reacted with rabbit anti-EGR1 antibody, as described in the Methods. ( a ) Box and dot plot depicting nuclear EGR1 expression in tumors that were poor-responders to bevacizumab (five mouse tumors from two PDXs-G64 and G108), and in tumors that were good-responders to bevacizumab (five mouse tumors from two PDXs-G39 and G59). Mouse ids are color-labeled. H-scores were determined for each sample as described in the Methods. Statistical analysis: linear mixed model ( p = 0.02). ( b ) A representative image is shown of immunohistochemical staining for EGR1 in a tumor that was a poor-responder to bevacizumab and in a tumor that was a good-responder to bevacizumab. Arrows denote EGR1 + nuclei. Scale bar denotes 40 μm. ( c , d ) Sections were reacted with rat mAb anti-EGR1 antibody, as described in the Methods. ( c ) Box and dot plot depicts nuclear EGR1 expression in tumors that were poor-responders to bevacizumab (five mouse tumors from two PDXs-G64 and G108), and nuclear EGR1 expression in tumors that were good-responders to bevacizumab (five mouse tumors from two PDXs-G39 and G59). Mouse ids are color-labeled. Statistical analysis: linear mixed model ( p = 0.03). ( d ) A representative image is shown of immunofluorescence for nuclear EGR1 in a tumor that was a poor-responder to bevacizumab and in a tumor that was a good-responder to bevacizumab. Arrows denote EGR1 + nuclei. Scale bar denotes 20 μm. ( e ) Primary cells from PDX tumors were propagated as neurospheres in neural basal media with EGF and bFGF and harvested for flow cytometry. Cells were stained with a live/dead marker (LIVE/DEAD™ Fixable Violet Kit, Thermo # L34955 ) and then fixed and permeabilized (True Nuclear Buffer Set, BioLegend # 424401), and stained with Alexa Fluor ® 647 anti-EGR1 Antibody (BioLegend # 943906), followed by analysis on a BD Fortessa (BD Biosciences) flow cytometer. A violin plot is shown. Data were analyzed using the FlowJo (v10.10.0) software, as described in the Methods. Mean EGR1 fluorescence intensity in PDX tumors that were poor-responders (897.9) as compared to the mean fluorescent intensity in PDX tumors that were good-responders (673.0) to bevacizumab therapy. Statistical analysis: Student’s two-sided T-Test ( p < 0.001).

Article Snippet: Triple-labeling for EGR1, Ki67 and human nuclei on frozen sections utilized the following primary antibodies: rat mAb anti-EGR1 (20 μg/ml, R&D Systems MAB28181), rabbit anti-human nuclei (2 μg/ml, clone 235-1R, Neobiotechnologies RBM5-346-P1), and mouse mAb anti-Ki67 (1:2500, Cell Signaling #9449) and the first antibodies were incubated overnight at 4 °C.

Techniques: Expressing, Labeling, Immunohistochemical staining, Staining, Immunofluorescence, Flow Cytometry, Marker, Software, Fluorescence

Journal: Scientific Reports

Article Title: Transcriptomic analysis identifies a potential target for bevacizumab resistant glioblastoma

doi: 10.1038/s41598-025-20847-4

Figure Lengend Snippet: Elevated Ki67 signal in EGR1 high tumor cells in tumors that were poor-responders to bevacizumab therapy. ( a , b ) FFPE sections were triple-labeled with rat mAb anti-EGR1 antibody, rabbit anti-Ki67 antibody, and mouse anti-human nucleoli antibody, as described in the methods. Frozen sections were triple-labeled with rat mAb anti-EGR1 antibody, mouse anti-Ki67 antibody, and rabbit anti-human nuclei antibody, as described in the Methods. ( a ) Representative images are shown of immunofluorescence for nuclear EGR1, Ki67, human nucleoli and DAPI in a tumor that was a poor-responder to bevacizumab and in a tumor that was a good-responder to bevacizumab. White arrows denote nuclear EGR1 expression and Ki67 expression in human tumor nuclei. Scale bar denotes 30 μm. ( b ) Box plot of Ki67 signal in EGR1 high tumor cells in tumors that were poor-responders to bevacizumab (11 mouse tumors from two PDXs-G64 and G108), and of Ki67 signal in EGR1 high tumor cells in tumors that were good-responders to bevacizumab (8 mouse tumors from two PDXs-G39 and G59). Red line represents least squares mean per group. Statistical analysis: linear mixed model ( p = 0.026). ( c , d ) FFPE sections were double-labeled with rabbit anti-Ki67 antibody and mouse anti-human nucleoli antibody; and frozen sections were double-labeled with mouse anti-Ki67 antibody and rabbit anti-human nuclei antibody, as described in the Methods. ( c ) Representative images are shown of immunofluorescence for Ki67 (pseudo-colored red) and human nucleoli (pseudo-colored magenta) in tumors that were poor-responders to bevacizumab and in tumors that were good-responders to bevacizumab. White arrows denote nuclear Ki67 expression in human tumor nuclei. Scale bar denotes 150 μm. ( d ) Box plot of Ki67 signal in all tumor cells in tumors that were poor-responders to bevacizumab (11 mouse tumors from two PDXs-G64 and G108), and Ki67 signal in all tumor cells in tumors that were good-responders to bevacizumab (8 mouse tumors from two PDXs-G39 and G59). Red line represents least squares mean per group. Statistical analysis: linear mixed model ( p = 0.53). ( e ) Fraction of EGR1 high tumor nuclear area to the total EGR1 + tumor nuclear area in entire tumor sections imaged with the Leica DMB6 B scanning fluorescent microscope. A significant increase in the fraction of EGR1 high tumor nuclear area to the total EGR1 + tumor nuclear area is seen in the poor-responder tumors as compared to the good-responder tumors ( p = 0.026). Statistical analysis: Wilcoxon rank-sum test.

Article Snippet: Triple-labeling for EGR1, Ki67 and human nuclei on frozen sections utilized the following primary antibodies: rat mAb anti-EGR1 (20 μg/ml, R&D Systems MAB28181), rabbit anti-human nuclei (2 μg/ml, clone 235-1R, Neobiotechnologies RBM5-346-P1), and mouse mAb anti-Ki67 (1:2500, Cell Signaling #9449) and the first antibodies were incubated overnight at 4 °C.

Techniques: Labeling, Immunofluorescence, Expressing, Microscopy

Journal: Scientific Reports

Article Title: Transcriptomic analysis identifies a potential target for bevacizumab resistant glioblastoma

doi: 10.1038/s41598-025-20847-4

Figure Lengend Snippet: Expression of α7-nAChR protein directly correlates with nuclear EGR1 protein in poor- and good-responder tumors to bevacizumab therapy. ( a , b ) Sections were double-labeled with rat mAb anti-EGR1 antibody and rabbit anti-α7-nAChR antibody, as described in the Methods. ( a ) Representative images are shown of immunofluorescence for DAPI, α7-nAChR, and nuclear EGR1 in a tumor depicting regions of high and low EGR1 expression. Yellow lines denote regions (ROIs) of representative individual cells used for single-cell analysis. Scale bar denotes 10 μm. ( b ) Scatter plot of nuclear EGR1 expression and α7-nAChR expression in individual cells in tumors that were poor-responders to bevacizumab (5 mouse tumors from two PDXs-G64 and G108), and nuclear EGR expression and α7-nAChR expression in individual cells in tumors that were good-responders to bevacizumab (5 mouse tumors from two PDXs-G39 and G59). Mouse ids are color-labeled. Statistical analysis: Spearman correlation ( p < 0.001). ( c ) Median linear correlation R values for each PDX calculated using Prism software (2 biologic replicates, 2 technical replicates for each PDX).

Article Snippet: Triple-labeling for EGR1, Ki67 and human nuclei on frozen sections utilized the following primary antibodies: rat mAb anti-EGR1 (20 μg/ml, R&D Systems MAB28181), rabbit anti-human nuclei (2 μg/ml, clone 235-1R, Neobiotechnologies RBM5-346-P1), and mouse mAb anti-Ki67 (1:2500, Cell Signaling #9449) and the first antibodies were incubated overnight at 4 °C.

Techniques: Expressing, Labeling, Immunofluorescence, Single-cell Analysis, Software

Journal: Scientific Reports

Article Title: Transcriptomic analysis identifies a potential target for bevacizumab resistant glioblastoma

doi: 10.1038/s41598-025-20847-4

Figure Lengend Snippet: Elevated EGR1 mRNA levels were associated with a shorter survival and upregulation of angiogenesis-blood vessel genes in female recurrent GBM patients from the GLASS database. ( a , b ) The overall survival difference between low EGR1 mRNA expression (< mean) and high EGR1 mRNA expression (≥mean) in males and females with recurrent tumors is significant ( P = 0.031 and P = 0.024, respectively; statistical analysis: Log-Rank test). ( c ) When stratified by sex in recurrent tumors, higher EGR1 mRNA levels correlated with shorter overall survival in males and females ( R = −0.25, P = 0.012 and R = −0.18, P = 0.043, respectively; statistical analyses: Pearson Correlation). ( d , e ) When recurrent tumors with methylation of the MGMT promotor were stratified by sex, there was a difference in overall survival between low EGR1 mRNA expression (< mean) and high EGR1 mRNA expression (≥mean) only in females ( P = 0.0062; statistical analysis: Log-Rank test). ( f ) When tumors with methylation of the MGMT promotor were stratified by sex, EGR1 mRNA levels correlated with overall survival only in females ( P = 0.0089; statistical analyses: Pearson Correlation). ( g ) Log 2 expression of genes in the GO-Angiogenesis-Blood Vessel geneset that are downstream targets of EGR1 in recurrent GBM patients shows upregulation in patients with elevated levels of EGR1 mRNA ( p < 0.001; statistical analyses: Student T-test). ( h ) Heatmap of concordant GO-Angiogenesis-Blood Vessel genes that are downstream targets of EGR1 shows upregulation in tumors with elevated levels of EGR1 mRNA. Data gathered from GLASS database through cBioPortal and filtered for GBM tumors (247 patients with recurrent tumor information, of which 89 patients also had MGMT promoter methylation status data). Plots generated and stratified using ggsurv and ggplot2 in R.

Article Snippet: Triple-labeling for EGR1, Ki67 and human nuclei on frozen sections utilized the following primary antibodies: rat mAb anti-EGR1 (20 μg/ml, R&D Systems MAB28181), rabbit anti-human nuclei (2 μg/ml, clone 235-1R, Neobiotechnologies RBM5-346-P1), and mouse mAb anti-Ki67 (1:2500, Cell Signaling #9449) and the first antibodies were incubated overnight at 4 °C.

Techniques: Expressing, Methylation, Generated