Journal: eBioMedicine

Article Title: DNA methylation mediates the immunosuppressive tumour microenvironment in metastatic endometrial clear cell carcinoma

doi: 10.1016/j.ebiom.2025.105954

Figure Lengend Snippet: Epigenetic silencing of the ETS1 regulon compromises anti-tumour immunity . a–g. Analysis of the ECCC sequencing cohort with paired transcriptomic and methylation data (Pn, n = 16; Pm, n = 14). a. Motif enrichment analysis of immune-related hypermethylated DMRs. The y-axis shows the enrichment significance (–log10 (p-value)); the x-axis indicates the motif rank. Significant enrichment was observed for transcription factor binding motifs from the ETS (left) and ZF (right) families. p-Values were determined using a hypergeometric test. b–d. Reduced regulon activity of ETS1 (b, p = 0.002), GATA6 (c, p = 0.038), and PRDM1 (d, p = 0.034) in Pm versus Pn tumours. e. Correlation between ETS1 expression and its target genes. The x-axis represents the Spearman correlation coefficient; the y-axis shows the corresponding p-value. p-Values were calculated by Spearman correlation test. f. Functional enrichment analysis of ETS1 target genes across the GO database. The y-axis shows the enrichment significance (–log10 (q-value)), derived from a hypergeometric test with Benjamini–Hochberg adjustment. The x-axis lists significantly enriched terms. Numbers above bars indicate the count of enriched genes per term. g. Regulatory network of the ETS1 regulon. Transcription factors are represented as ovals and target genes as rectangles; connecting lines indicate regulatory interactions. Oval size corresponds to interaction strength; blue borders denote targets overlapping with downregulated DEGs. h–j. Correlation of ETS1 regulon activity with TME features: TIL density (h, ρ = 0.885, p < 0.001), effector cell signature score (i, ρ = 0.692, p < 0.001), and TLS signature score (j, ρ = 0.726, p < 0.001). p-Values were calculated by Spearman correlation test. k–m, Analyses were performed on the overall ECCC cohort (Pn, n = 27; Pm, n = 24). k. Representative immunohistochemical staining of ETS1. Scale bar = 100 μm. l-m. ETS1 protein expression (H-score) in tumour cells (l, p = 0.248) and immune cells (m, p = 0.002) in Pm versus Pn tumours. All p-values were determined using the Mann–Whitney U test unless otherwise specified. ECCC, endometrial clear cell carcinoma; DMR, differentially methylated region; DMG, differentially methylated gene; Pn, non-metastatic primary tumours; Pm, metastatic primary tumours; DEG, differentially expressed gene; TME, tumour microenvironment; TIL, tumour-infiltrating lymphocyte; TLS, tertiary lymphoid structure.

Article Snippet: Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min, followed by incubation with primary ETS1 antibody (Cat#66598-1-Ig, RRID: AB_2881958 , Proteintech, China) overnight at 4 °C.

Techniques: Sequencing, Methylation, Binding Assay, Activity Assay, Expressing, Functional Assay, Derivative Assay, Immunohistochemical staining, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

doi: 10.1101/2025.07.17.665404

Figure Lengend Snippet: (A-D) Scatter plots showing correlation between ETS1 expression and the hEMT score in (A) human HNSCC scRNA-seq cohort, (B) UASCC cell lines, (C) TCGA HNSCC tumors, (D) HNSCC PDO samples. (E) Stacked plot illustrating ETS1 ChIP-Seq peaks on hEMT genes from two cell lines. (F) A Venn diagram showing shared ETS1 ChIP-Seq peaks between the KYSE150 and SCC25 cell lines, with representative peaks shown in (G). (H-K) GSEA line plots showing enrichment of the hEMT gene signature following ETS1 overexpression (H) or knockdown (I), or in ETS1-high tumors within TCGA ESCC (J) and HNSCC (K) cohorts. (L) An IGV plot showing H3K27ac ChIP-Seq profiles in nonmalignant esophageal tissues, primary ESCC, and lymph node (LN) metastases (PRJNA665151), with quantification shown in panel (M). (N) ETS1 mRNA expression in the same samples as panel (L) and in GSE9349 (P) dataset. (O) Motif sequence enrichment analysis of gained H3K27ac peaks in LN metastases from PRJNA665151.

Article Snippet: 2 μg ETS1 antibody (Cell Signaling, 14069) or HIF1A antibody (Cell Signaling, 36169) was then added and incubated at 4 °C overnight on a rotator.

Techniques: Expressing, ChIP-sequencing, Over Expression, Knockdown, Sequencing

Journal: bioRxiv

Article Title: ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

doi: 10.1101/2025.07.17.665404

Figure Lengend Snippet: (A) Heatmaps showing the relative expression of hEMT genes in UASCC cells. Data are shown as the mean of three experimental replicates. Expression is shown from high (red) to low (blue). Values expressing a fold change of 2 or greater are shown as the highest value (red). (B-C) Transwell migration assays of ETS1-overexpressing or ETS1-knockdown UASCC cells. Bar graphs show the number of migrated cells and represent the mean ± SD of three experimental replicates. ***P<0.001, ****P<0.0001; P-values were determined using an unpaired t-test or one-way ANOVA with multiple comparisons. (D) The metastatic events across various target organs in mice bearing stable control or ETS1-overexpressing TE5 cells. The number of metastatic events (grouped by location of metastasis) is shown for each mouse (horizontally) across the experimental (right) and control (left) groups. Target organs are visualized according to color; mouth (tan), stomach (orange), spleen (teal), kidney (purple), lung (red), and liver (blue). (E) Representative bioluminescence image depicting the spread of control and ETS1-overexpressing TE5 cells transfected with luciferase 21 days post-injection. (F) Kaplan-Meier plots showing mice bearing either control (red) or ETS1-ovexpressing (blue) cells. P-value was determined by the log-rank test. (G) Representative images of metastases to various organs of mice bearing control and ETS1-overexpressing TE5 cells. (H) The metastatic events across target organs in mice bearing control or stable ETS1-knockdown KYSE150 cells. (I) Representative images of the lungs of mice bearing control and stable ETS1-knockown KYSE150 cells.

Article Snippet: 2 μg ETS1 antibody (Cell Signaling, 14069) or HIF1A antibody (Cell Signaling, 36169) was then added and incubated at 4 °C overnight on a rotator.

Techniques: Expressing, Migration, Knockdown, Control, Transfection, Luciferase, Injection

Journal: bioRxiv

Article Title: ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

doi: 10.1101/2025.07.17.665404

Figure Lengend Snippet: (A) Volcano plot showing in silico drug screen results based on the PRISM Primary Repurposing dataset . (B) Cell viability (MTT) IC50 assays in both control and ETS1-overexpressing UASCC cell lines. Compounds are represented in nM using the Log10 scale. Data points represent the mean ± SD of at least two experimental replicates. (C-D) Colony formation assays upon exposure to Alvespimycin or STA9090 in control, ETS1-overexpressing, and ETS1-knockdown UASCC cell lines. Dot plots represent the mean ± SD of three experimental replicates. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; P-values were determined using an unpaired t-test. (E-F) Transwell migration assays in control and ETS1-overexpressing UASCC cell lines with or without the exposure to Alvespimycin or STA9090. Bar graphs show the number of migrated cells and represent the mean ± SD of three experimental replicates. *P<0.05, ***P<0.001, ****P<0.0001; P-values were determined using a one-way ANOVA with multiple comparisons.

Article Snippet: 2 μg ETS1 antibody (Cell Signaling, 14069) or HIF1A antibody (Cell Signaling, 36169) was then added and incubated at 4 °C overnight on a rotator.

Techniques: In Silico, Control, Knockdown, Migration

Journal: bioRxiv

Article Title: ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

doi: 10.1101/2025.07.17.665404

Figure Lengend Snippet: (A-B) The protein (A) and mRNA (B) levels of ETS1 in UASCC cell lines treated with Alvespimycin and STA9090 for 24 hours. Bar graphs represent the mean ± SD of three experimental replicates. **P<0.01, ***P<0.001, ****P<0.0001; P-values were determined using a one-way ANOVA with multiple comparisons. Western blot images are representative of three experimental replicates. (C) IGV plots of HIF1A ChIP-Seq in indicated samples at the ETS1 promoter region. Signal values of normalized peak intensity are shown in the upper left corner. (D) ChIP-qPCR assays measuring ETS1 and HIF1A binding on the promoter of ETS1 in TE5 control (black) and ETS1 overexpressing (blue) cells. Data is represented as fold change relative to IgG control. Individual data points from two replicates are shown. ****P<0.0001; P-values were determined using a one-way ANOVA with multiple comparisons. (E-F) HIF1A and ETS1 mRNA expression (E) and protein (F) levels in UASCC parental cell lines treated with siNC or siHIF1A for 48 hours. Bar graphs represent the mean ± SD of three experimental replicates. ****P<0.0001; P-values were determined using a one-way ANOVA with multiple comparisons. Western blot images are representative of three experimental replicates. (G-H) The protein levels of ETS1 in control and ETS1-overexpressing UASCC cell lines with or without the exposure to Alvespimycin or STA9090 for 24 hours. Western blot images are representative of three experimental replicates.

Article Snippet: 2 μg ETS1 antibody (Cell Signaling, 14069) or HIF1A antibody (Cell Signaling, 36169) was then added and incubated at 4 °C overnight on a rotator.

Techniques: Western Blot, ChIP-sequencing, ChIP-qPCR, Binding Assay, Control, Expressing

Journal: bioRxiv

Article Title: ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

doi: 10.1101/2025.07.17.665404

Figure Lengend Snippet: (A–B) Volcano plots showing GSEA results of enriched Hallmark pathways in (A) ETS1-overexpressing vs. control TE5 cells and (B) ETS1-high vs. ETS1-low tumor samples from TCGA HNSCC and ESCC cohorts. Significantly enriched pathways are highlighted in color. (C) Heatmap showing the fold change in expression of selected leading-edge genes in ETS1-overexpressing vs. control UASCC cells. Data represent the mean of three experimental replicates. Expression levels range from high (red) to low (blue), with values at or above a fold change of 2 displayed as the maximum (red). (D) Representative western blot images showing protein levels of STAT1, PDL1, and ETS1 in UASCC cells under the indicated conditions. Images are representative of three independent experiments. (E) Quantification of flow cytometry analysis of PDL1 cell surface expression. **P<0.01, ****P<0.0001; P-values were determined using an unpaired t-test. (F) IGV plot depicting ETS1 ChIP-Seq signal at the STAT1 promoter region in KYSE150 cells. Normalized peak intensity values are indicated in the upper left corner. (G–I) ChIP-qPCR analysis of ETS1 binding at the STAT1 promoter in the indicated cell lines. Data are presented as fold enrichment relative to IgG control. Individual data points from two replicates are shown. ***P<0.001, ****P<0.0001; P-values were determined using an unpaired t-test. (J) Volcano plot showing proteins correlated with ETS1 in TCGA HNSCC proteomics data. Significantly correlated proteins are highlighted in color.

Article Snippet: 2 μg ETS1 antibody (Cell Signaling, 14069) or HIF1A antibody (Cell Signaling, 36169) was then added and incubated at 4 °C overnight on a rotator.

Techniques: Control, Expressing, Western Blot, Flow Cytometry, ChIP-sequencing, ChIP-qPCR, Binding Assay

Journal: bioRxiv

Article Title: ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

doi: 10.1101/2025.07.17.665404

Figure Lengend Snippet: (A–D) The proportion of CD3 + , CD4 + and CD8 + T cells out of total live cells from control or ETS1-overexpressing (A-C) allografts, or (D) cervical lymph nodes, measured by flow cytometry analysis. Bars represent mean ± SD of independent samples. *P<0.05, **P<0.01, ***P<0.001; P-values were determined using an unpaired t-test. (E-F) Allografts growth curves and survival curves of control or ETS1-overexpressing tumor-bearing mice. *P<0.05, P-values were determined using a two-way ANOVA (E). P-value was determined by the log-rank test (F). (G-I) IHC of control and ETS1-overexpressing tumors stained with CD3 + T cells (red) and CD8 + T cells (blue). Quantification of CD3 + T cells (G) and CD8 + T cells (H) are provided. (I) Images are representative of 10 tumors, 5 control and 5 ETS1-overexpressing. Scale bar = 0.1mm.

Article Snippet: 2 μg ETS1 antibody (Cell Signaling, 14069) or HIF1A antibody (Cell Signaling, 36169) was then added and incubated at 4 °C overnight on a rotator.

Techniques: Control, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

doi: 10.1101/2025.07.17.665404

Figure Lengend Snippet: (A) A schematic graph showing experimental design of ex vivo CD8 + T cell co-culture assay. (B) Bar graphs showing relative cell viability of MOC2 cells co-cultured with CD8 + T cells as described in (A). Bars show the means ± SD representative of two 4 replicates. *P < 0.01, **P < 0.01, ***P < 0.001. P-values were determined using unpaired t-tests. (C-D) UMAP plots showing the expression levels of GZMB and (D) quantification of T cell cytotoxic or exhaustive marker genes of CD8 + T cells from ETS1-high vs. -low HNSCC tumors. The average ETS1 expression in cancer cells in each tumor was calculated and used for sample classification. (E-F) Patient survival curves analyzed using the pan-cancer ICB-treated cohort (n=976 patients across multiple cancer types ) via the KM plotter , . Patients were stratified using either the mean hEMT score (E) or mean ETS1 score (F). (G-H) Patient survival curves analyzed using a lung squamous carcinoma cohort treated with anti-PDL1 antibody (n=87) . Patients were stratified using either the mean hEMT score (G) or mean ETS1 score (H).

Article Snippet: 2 μg ETS1 antibody (Cell Signaling, 14069) or HIF1A antibody (Cell Signaling, 36169) was then added and incubated at 4 °C overnight on a rotator.

Techniques: Ex Vivo, Co-culture Assay, Cell Culture, Expressing, Marker

Journal: bioRxiv

Article Title: ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

doi: 10.1101/2025.07.17.665404

Figure Lengend Snippet: (A-D) Scatter plots showing correlation between ETS1 expression and the hEMT score in (A) human HNSCC scRNA-seq cohort, (B) UASCC cell lines, (C) TCGA HNSCC tumors, (D) HNSCC PDO samples. (E) Stacked plot illustrating ETS1 ChIP-Seq peaks on hEMT genes from two cell lines. (F) A Venn diagram showing shared ETS1 ChIP-Seq peaks between the KYSE150 and SCC25 cell lines, with representative peaks shown in (G). (H-K) GSEA line plots showing enrichment of the hEMT gene signature following ETS1 overexpression (H) or knockdown (I), or in ETS1-high tumors within TCGA ESCC (J) and HNSCC (K) cohorts. (L) An IGV plot showing H3K27ac ChIP-Seq profiles in nonmalignant esophageal tissues, primary ESCC, and lymph node (LN) metastases (PRJNA665151), with quantification shown in panel (M). (N) ETS1 mRNA expression in the same samples as panel (L) and in GSE9349 (P) dataset. (O) Motif sequence enrichment analysis of gained H3K27ac peaks in LN metastases from PRJNA665151.

Article Snippet: Primary antibodies used were ETS1 (1:1,000, Cell Signaling, 14069), HIF1A (1:1,000) (Cell Signaling, 36169), STAT1 (1:1,000, Cell Signaling, 14994), PD-L1 (1:1000, Proteintech, #66248-1-Ig) and Actin (1:3,000, Developmental Studies Hybridoma Bank, JLA20).

Techniques: Expressing, ChIP-sequencing, Over Expression, Knockdown, Sequencing

Journal: bioRxiv

Article Title: ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

doi: 10.1101/2025.07.17.665404

Figure Lengend Snippet: (A) Heatmaps showing the relative expression of hEMT genes in UASCC cells. Data are shown as the mean of three experimental replicates. Expression is shown from high (red) to low (blue). Values expressing a fold change of 2 or greater are shown as the highest value (red). (B-C) Transwell migration assays of ETS1-overexpressing or ETS1-knockdown UASCC cells. Bar graphs show the number of migrated cells and represent the mean ± SD of three experimental replicates. ***P<0.001, ****P<0.0001; P-values were determined using an unpaired t-test or one-way ANOVA with multiple comparisons. (D) The metastatic events across various target organs in mice bearing stable control or ETS1-overexpressing TE5 cells. The number of metastatic events (grouped by location of metastasis) is shown for each mouse (horizontally) across the experimental (right) and control (left) groups. Target organs are visualized according to color; mouth (tan), stomach (orange), spleen (teal), kidney (purple), lung (red), and liver (blue). (E) Representative bioluminescence image depicting the spread of control and ETS1-overexpressing TE5 cells transfected with luciferase 21 days post-injection. (F) Kaplan-Meier plots showing mice bearing either control (red) or ETS1-ovexpressing (blue) cells. P-value was determined by the log-rank test. (G) Representative images of metastases to various organs of mice bearing control and ETS1-overexpressing TE5 cells. (H) The metastatic events across target organs in mice bearing control or stable ETS1-knockdown KYSE150 cells. (I) Representative images of the lungs of mice bearing control and stable ETS1-knockown KYSE150 cells.

Article Snippet: Primary antibodies used were ETS1 (1:1,000, Cell Signaling, 14069), HIF1A (1:1,000) (Cell Signaling, 36169), STAT1 (1:1,000, Cell Signaling, 14994), PD-L1 (1:1000, Proteintech, #66248-1-Ig) and Actin (1:3,000, Developmental Studies Hybridoma Bank, JLA20).

Techniques: Expressing, Migration, Knockdown, Control, Transfection, Luciferase, Injection

Journal: bioRxiv

Article Title: ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

doi: 10.1101/2025.07.17.665404

Figure Lengend Snippet: (A) Volcano plot showing in silico drug screen results based on the PRISM Primary Repurposing dataset . (B) Cell viability (MTT) IC50 assays in both control and ETS1-overexpressing UASCC cell lines. Compounds are represented in nM using the Log10 scale. Data points represent the mean ± SD of at least two experimental replicates. (C-D) Colony formation assays upon exposure to Alvespimycin or STA9090 in control, ETS1-overexpressing, and ETS1-knockdown UASCC cell lines. Dot plots represent the mean ± SD of three experimental replicates. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; P-values were determined using an unpaired t-test. (E-F) Transwell migration assays in control and ETS1-overexpressing UASCC cell lines with or without the exposure to Alvespimycin or STA9090. Bar graphs show the number of migrated cells and represent the mean ± SD of three experimental replicates. *P<0.05, ***P<0.001, ****P<0.0001; P-values were determined using a one-way ANOVA with multiple comparisons.

Article Snippet: Primary antibodies used were ETS1 (1:1,000, Cell Signaling, 14069), HIF1A (1:1,000) (Cell Signaling, 36169), STAT1 (1:1,000, Cell Signaling, 14994), PD-L1 (1:1000, Proteintech, #66248-1-Ig) and Actin (1:3,000, Developmental Studies Hybridoma Bank, JLA20).

Techniques: In Silico, Control, Knockdown, Migration

Journal: bioRxiv

Article Title: ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

doi: 10.1101/2025.07.17.665404

Figure Lengend Snippet: (A-B) The protein (A) and mRNA (B) levels of ETS1 in UASCC cell lines treated with Alvespimycin and STA9090 for 24 hours. Bar graphs represent the mean ± SD of three experimental replicates. **P<0.01, ***P<0.001, ****P<0.0001; P-values were determined using a one-way ANOVA with multiple comparisons. Western blot images are representative of three experimental replicates. (C) IGV plots of HIF1A ChIP-Seq in indicated samples at the ETS1 promoter region. Signal values of normalized peak intensity are shown in the upper left corner. (D) ChIP-qPCR assays measuring ETS1 and HIF1A binding on the promoter of ETS1 in TE5 control (black) and ETS1 overexpressing (blue) cells. Data is represented as fold change relative to IgG control. Individual data points from two replicates are shown. ****P<0.0001; P-values were determined using a one-way ANOVA with multiple comparisons. (E-F) HIF1A and ETS1 mRNA expression (E) and protein (F) levels in UASCC parental cell lines treated with siNC or siHIF1A for 48 hours. Bar graphs represent the mean ± SD of three experimental replicates. ****P<0.0001; P-values were determined using a one-way ANOVA with multiple comparisons. Western blot images are representative of three experimental replicates. (G-H) The protein levels of ETS1 in control and ETS1-overexpressing UASCC cell lines with or without the exposure to Alvespimycin or STA9090 for 24 hours. Western blot images are representative of three experimental replicates.

Article Snippet: Primary antibodies used were ETS1 (1:1,000, Cell Signaling, 14069), HIF1A (1:1,000) (Cell Signaling, 36169), STAT1 (1:1,000, Cell Signaling, 14994), PD-L1 (1:1000, Proteintech, #66248-1-Ig) and Actin (1:3,000, Developmental Studies Hybridoma Bank, JLA20).

Techniques: Western Blot, ChIP-sequencing, ChIP-qPCR, Binding Assay, Control, Expressing

Journal: bioRxiv

Article Title: ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

doi: 10.1101/2025.07.17.665404

Figure Lengend Snippet: (A–B) Volcano plots showing GSEA results of enriched Hallmark pathways in (A) ETS1-overexpressing vs. control TE5 cells and (B) ETS1-high vs. ETS1-low tumor samples from TCGA HNSCC and ESCC cohorts. Significantly enriched pathways are highlighted in color. (C) Heatmap showing the fold change in expression of selected leading-edge genes in ETS1-overexpressing vs. control UASCC cells. Data represent the mean of three experimental replicates. Expression levels range from high (red) to low (blue), with values at or above a fold change of 2 displayed as the maximum (red). (D) Representative western blot images showing protein levels of STAT1, PDL1, and ETS1 in UASCC cells under the indicated conditions. Images are representative of three independent experiments. (E) Quantification of flow cytometry analysis of PDL1 cell surface expression. **P<0.01, ****P<0.0001; P-values were determined using an unpaired t-test. (F) IGV plot depicting ETS1 ChIP-Seq signal at the STAT1 promoter region in KYSE150 cells. Normalized peak intensity values are indicated in the upper left corner. (G–I) ChIP-qPCR analysis of ETS1 binding at the STAT1 promoter in the indicated cell lines. Data are presented as fold enrichment relative to IgG control. Individual data points from two replicates are shown. ***P<0.001, ****P<0.0001; P-values were determined using an unpaired t-test. (J) Volcano plot showing proteins correlated with ETS1 in TCGA HNSCC proteomics data. Significantly correlated proteins are highlighted in color.

Article Snippet: Primary antibodies used were ETS1 (1:1,000, Cell Signaling, 14069), HIF1A (1:1,000) (Cell Signaling, 36169), STAT1 (1:1,000, Cell Signaling, 14994), PD-L1 (1:1000, Proteintech, #66248-1-Ig) and Actin (1:3,000, Developmental Studies Hybridoma Bank, JLA20).

Techniques: Control, Expressing, Western Blot, Flow Cytometry, ChIP-sequencing, ChIP-qPCR, Binding Assay

Journal: bioRxiv

Article Title: ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

doi: 10.1101/2025.07.17.665404

Figure Lengend Snippet: (A–D) The proportion of CD3 + , CD4 + and CD8 + T cells out of total live cells from control or ETS1-overexpressing (A-C) allografts, or (D) cervical lymph nodes, measured by flow cytometry analysis. Bars represent mean ± SD of independent samples. *P<0.05, **P<0.01, ***P<0.001; P-values were determined using an unpaired t-test. (E-F) Allografts growth curves and survival curves of control or ETS1-overexpressing tumor-bearing mice. *P<0.05, P-values were determined using a two-way ANOVA (E). P-value was determined by the log-rank test (F). (G-I) IHC of control and ETS1-overexpressing tumors stained with CD3 + T cells (red) and CD8 + T cells (blue). Quantification of CD3 + T cells (G) and CD8 + T cells (H) are provided. (I) Images are representative of 10 tumors, 5 control and 5 ETS1-overexpressing. Scale bar = 0.1mm.

Article Snippet: Primary antibodies used were ETS1 (1:1,000, Cell Signaling, 14069), HIF1A (1:1,000) (Cell Signaling, 36169), STAT1 (1:1,000, Cell Signaling, 14994), PD-L1 (1:1000, Proteintech, #66248-1-Ig) and Actin (1:3,000, Developmental Studies Hybridoma Bank, JLA20).

Techniques: Control, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

doi: 10.1101/2025.07.17.665404

Figure Lengend Snippet: (A) A schematic graph showing experimental design of ex vivo CD8 + T cell co-culture assay. (B) Bar graphs showing relative cell viability of MOC2 cells co-cultured with CD8 + T cells as described in (A). Bars show the means ± SD representative of two 4 replicates. *P < 0.01, **P < 0.01, ***P < 0.001. P-values were determined using unpaired t-tests. (C-D) UMAP plots showing the expression levels of GZMB and (D) quantification of T cell cytotoxic or exhaustive marker genes of CD8 + T cells from ETS1-high vs. -low HNSCC tumors. The average ETS1 expression in cancer cells in each tumor was calculated and used for sample classification. (E-F) Patient survival curves analyzed using the pan-cancer ICB-treated cohort (n=976 patients across multiple cancer types ) via the KM plotter , . Patients were stratified using either the mean hEMT score (E) or mean ETS1 score (F). (G-H) Patient survival curves analyzed using a lung squamous carcinoma cohort treated with anti-PDL1 antibody (n=87) . Patients were stratified using either the mean hEMT score (G) or mean ETS1 score (H).

Article Snippet: Primary antibodies used were ETS1 (1:1,000, Cell Signaling, 14069), HIF1A (1:1,000) (Cell Signaling, 36169), STAT1 (1:1,000, Cell Signaling, 14994), PD-L1 (1:1000, Proteintech, #66248-1-Ig) and Actin (1:3,000, Developmental Studies Hybridoma Bank, JLA20).

Techniques: Ex Vivo, Co-culture Assay, Cell Culture, Expressing, Marker

Journal: bioRxiv

Article Title: ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

doi: 10.1101/2025.07.17.665404

Figure Lengend Snippet: (A-D) Scatter plots showing correlation between ETS1 expression and the hEMT score in (A) human HNSCC scRNA-seq cohort, (B) UASCC cell lines, (C) TCGA HNSCC tumors, (D) HNSCC PDO samples. (E) Stacked plot illustrating ETS1 ChIP-Seq peaks on hEMT genes from two cell lines. (F) A Venn diagram showing shared ETS1 ChIP-Seq peaks between the KYSE150 and SCC25 cell lines, with representative peaks shown in (G). (H-K) GSEA line plots showing enrichment of the hEMT gene signature following ETS1 overexpression (H) or knockdown (I), or in ETS1-high tumors within TCGA ESCC (J) and HNSCC (K) cohorts. (L) An IGV plot showing H3K27ac ChIP-Seq profiles in nonmalignant esophageal tissues, primary ESCC, and lymph node (LN) metastases (PRJNA665151), with quantification shown in panel (M). (N) ETS1 mRNA expression in the same samples as panel (L) and in GSE9349 (P) dataset. (O) Motif sequence enrichment analysis of gained H3K27ac peaks in LN metastases from PRJNA665151.

Article Snippet: For overexpression cloning, ETS1 overexpression CMV vectors were purchased from the Vectorbuilder. pLenti CMV V5-LUC Blast vector was from Addgene (#21474).

Techniques: Expressing, ChIP-sequencing, Over Expression, Knockdown, Sequencing