Fig 1: Knockdown of EphB4 in cancer cells combined with radiation treatment promotes suppressive intratumoral immune populations.A Experimental design for C57BL/6 J mice implanted with MOC2 cancer cells. The buccal tumor received single dose of radiation treatment (10 Gy) on day 7 after implantation. B–G Flow cytometric quantification of CD4 + T cells (B), regulatory T cells (Tregs) defined by CD4 + CD25+ (C) and CD4 + CD25+ Foxp3+ expression (D), myeloid-derived suppressor cells (MDSCs) defined by CD45 + Ly6G + Ly6C+ (E), CD45 + CD11b+ Ly6Ghigh Ly6Clow (F), and CD45 + CD11b+ Ly6Glow Ly6Chigh (G) in the tumor microenvironment (TME) (Ctrl sh, n = 5; EphB4 sh n = 7). H Experimental design for coculture of LY2 Ctrl or EphB4 shRNA cancer cells with CD4 + T cells. Cancer cells were incubated with interferon-gamma (IFNg) for 48 h, then OVA peptide overnight. CD4 + T cells from DO11.10 BALB/c mice were then cocultured with the cancer cells for 72 hours and subsequently harvested for flow cytometry. I Flow cytometry quantification of Tregs defined by CD4+ Foxp3+ and CD4 + CD25+ Foxp3+ expression (n = 6 per group). J Flow cytometry quantification of Treg immunosuppressive function defined by IL-10 expression (n = 6 per group). K Flow cytometry quantification of CD4 + T cells treated with conditioned media from MOC2 control or EphB4 shRNA cells showing PD-1, CD69, and CD25 expression (n = 5 per group). The experiments were performed once with their own biological replicates. Comparison between control and experimental groups was done using a Mann Whitney test for the in vivo experiment and a two-sided student’s t-test for the in vitro experiments. Significance was determined if the p-value was < 0.05*, < 0.01**, < 0.001***, and < 0.0001****. p-values are indicated for the figures (B) ns p = 0.3636, (C) **p = 0.0025, (D) **p = 0.0051, (E) *p = 0.0177, (F) *p = 0.0101, (G) *p = 0.0101, (I) CD3 + CD44+Foxp3+ ****p < 0.0001; CD3 + CD4 + CD25+Foxp3+ *p = 0.0394, (J) **p = 0.0027, (K) ****p < 0.0001. The error bars represent the standard error of the mean ( ± SEM).
Fig 2: Knockdown of EphB4 in HNSCC cancer cells promotes distant metastasis.A Schematic showing experimental design for C57BL/6 J mice implanted with 100k MOC2 cancer cells. 3 fractions of 8 Gray (Gy) radiation therapy (RT) were given as indicated. B Kaplan-Meier curves showing lung metastasis free survival of MOC2 control (Ctrl) shRNA (sh) (n = 18) versus EphB4 shRNA(n = 20) tumors implanted in C57BL/6 J mice. Numbers at risk indicate mice that were alive without metastases at specified timepoints. C Contingency table quantifying the incidence of lung metastasis detected by computed tomography (CT) scans in C57BL/6 mice implanted with MOC2 cancer cells by 36 days post-implantation (DPI). D Schematic showing experimental design for BALB/c mice implanted with 100k LY2 cancer cells. One fraction of 8 Gy RT was given as indicated. E Kaplan-Meier curves showing distant metastasis free survival of LY2 Ctrl shRNA (n = 10) vs LY2 EphB4 (n = 10) shRNA tumors implanted in BALB/c mice. Numbers at risk indicate mice that were alive without metastases at specified timepoints. F Contingency table quantifying the incidence of distant metastases detected by CT scans in BALB/c mice implanted with LY2 Ctrl shRNA or EphB4 shRNA tumors by 35 DPI. The experiments were replicated twice. For Kaplan-Meier survival curves, significance was determined by a log-rank Mantel-Cox test. For contingency tables indicating the incidence of metastases, significance was determined by a Chi-square test. Significance was determined if the p-value was < 0.05*, < 0.01**, and < 0.001***. p-values are indicated for the figures (B) ***p = 0.0009, (C) **p = 0.0044, (E) *p = 0.0200, (F) **p = 0.0073. The error bars represent the standard error of the mean ( ± SEM).
Fig 3: EphrinB2-Fc-His activates EphB4 without concurrent activation of ephrinB2 while Fc-TNYL-RAW-GS activates both EphB4 and ephrinB2.A Experimental design for C57BL/6 J mice implanted with MOC2 WT cancer cells. Hydrodynamic tail vein injections (HTVI) and radiation therapy (8 Gy) were administered as indicated (n = 10 per group). Mice treated with RT alone served as controls. B Average tumor volume curve comparing effects of different plasmids on local tumor growth. C Dot plot showing the effects of different plasmids on tumor volume 28 DPI. D Kaplan-Meier curves showing therapeutic effects of different plasmids on overall survival in MOC2 WT implanted C57BL/6 mice. E Western blots showing protein expression of EphB4, phospho-EphB4 (pEphB4), ephrinB2 (EFNB2), phospho-ephrinB2 (pEFNB2), and beta-actin in control and experimental tumors. F Dot plots showing ratios of protein expression of pEphB4/EphB4 (n = 6 per group) and pEFNB2/EFNB2 (n = 8 per group) in control and experimental tumors. Protein bands were quantified using Image lab and ImageJ software. The experiments were performed once with their own biological replicates. For the Kaplan-Meier overall survival curves, significance was determined by a log-rank Mantel-Cox test. Comparison of tumor volume between the control and experimental groups was done using a Dunnett post hoc test after one-way ANOVA was performed. Comparison of relative protein expression between the control and experimental groups was done using Mann Whitney tests. Significance was determined if the p-value was < 0.05*, < 0.01**, < 0.001***, and < 0.0001****. p-values are indicated for the figures (C) RT vs EphrinB2-Fc ***p = 0.0002; RT vs EFNB2-Fc-His ****p < 0.0001; RT vs Fc-TNYL-RAW-GS **p = 0.0044, (D) *p = 0.0389. The error bars represent the standard error of the mean ( ± SEM).
Fig 4: Loss of EphB4 in cancer cells induces protein dysregulation concomitant with increased metastatic capacity.A Representative images and quantification for Boyden chamber invasion assay conducted on MOC2 control (n = 4) or EphB4 (n = 4) CRISPR knockout (KO) cell lines. B Variable importance in projection (VIP) score plots of mass spectrometry proteomics data conducted on MOC2 EphB4 KO (n = 5) and Ctrl (n = 5) tumors displaying upregulation of BCCIP and Ube2v1 proteins in EphB4 KO tumors compared to control tumors. C Hallmark pathways generated from RNA-sequencing of MOC2 control (n = 3) versus EphB4 (n = 3) shRNA cell lines. D RNA-sequencing of MOC2 control (n = 3) and EphB4 (n = 3) shRNA knockdown (KD) cells showing expression of genes associated with IL6-Jak-Stat3 and TNF-alpha signaling via NFκB. E Expression of genes quantified using RNA-sequencing of MOC2 control (n = 3), EphB4 (n = 3), and ephrinB2 (EFNB2) (n = 3) CRISPR KO cells. F Expression of genes associated with intermediate filament cytoskeleton quantified using RNA-sequencing of MOC2 control (n = 3), EphB4 (n = 3), and ephrinB2 (n = 3) CRISPR knockout cell lines. The experiments were performed once with their own biological replicates. For Boyden chamber quantification, comparison of invaded cells between the control and experimental group was done using a two-sided student’s t-test. Significance was determined if the p-value was < 0.05*, < 0.01**, < 0.001***, and < 0.0001****. p-values are indicated for the figures (A) ***p = 0.0002. The error bars represent the standard error of the mean ( ± SEM).
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