Journal: Oncogene

Article Title: Adaptive activation of EFNB2/EPHB4 axis promotes post-metastatic growth of colorectal cancer liver metastases by LDLR-mediated cholesterol uptake

doi: 10.1038/s41388-022-02519-z

Figure Lengend Snippet: A Upregulated genes identified were GSE49355, GSE6988, and GSE35834 (CRC LM), and GSE41258 and GSE41568 (CRC PM). B KEGG pathway analysis of upregulated genes in CRC LM. C mRNA expression of EFNB2 in NC, CRC, and LM samples in GSE49355, GSE6988, and GSE35834. D mRNA expression of EFNB2 in CRC and PM samples in GSE41568. E mRNA expression of EFNB2 in CRC and PM samples in GSE41258. F Protein expression of EFNB2 in matched NC, CRC, LM tissues ( n = 15 per group), Scale bar: 50 μm. G mRNA and protein expression of EFNB2 in NCM460 and six CRC cell lines. H Orthotopic tumor models, a liver metastasis model by spleen injection and a lung metastasis model by tail vein injection, were established by injecting SW480 cells into null mice. I mRNA and protein expression of EFNB2 in orthotopic tumor tissue at different time points (1 week, 2 weeks, and 3 weeks) ( n = 6). J mRNA and protein expression of EFNB2 in lung metastasis tumor tissue at different time points (1 week, 2 weeks, and 3 weeks) ( n = 6). K mRNA and protein expression of EFNB2 in liver metastasis tumor tissue at different time points (1 week, 2 weeks, and 3 weeks) ( n = 6). L Imaging of animal model of liver metastasis by spleen injection with sh-EFNB2 or sh-NC SW620 Luc cells ( n = 6). Scale color bar: 9.74 × 10 7 –4.19 × 10 8 . M Imaging of animal intrahepatic model by liver injection with sh-EFNB2 or sh-NC SW620 Luc cells ( n = 5 per group). Scale color bar: 3.75 × 10 6 –4.36 × 10 7 . N LM tumor tissues from sh-EFNB2 or sh-NC mice were digested and LM cells were cultured. O The viability of SW620 cells from LM tumor tissues in sh-EFNB2 and sh-NC groups, as analyzed using CCK-8 assays. P Proliferation of SW620 cells from LM tumor tissues in sh-EFNB2 and sh-NC groups, as analyzed using EdU assays. Scale bar: 50 μm. All experiments were performed in triplicate. Measurement data were presented as the mean ± SD. Student’s t -tests were used for statistical analysis. ns. represents no statistical difference; * p < 0.05; *** p < 0.001.

Article Snippet: All the lentivirus-EFNB2 cDNAs were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Techniques: Expressing, Injection, Imaging, Animal Model, Cell Culture, CCK-8 Assay

Journal: Oncogene

Article Title: Adaptive activation of EFNB2/EPHB4 axis promotes post-metastatic growth of colorectal cancer liver metastases by LDLR-mediated cholesterol uptake

doi: 10.1038/s41388-022-02519-z

Figure Lengend Snippet: A Schematic diagram of signal conduction direction. B Protein structure of EFNB2. C Construction of four types of EFNB2 overexpression plasmids, including EFNB2 full length (FL), EFNB2 ΔC, EFNB2 ΔC + H, and EFNB2 Δ E . D Liver metastasis model by spleen injection with vector, EFNB2 FL, EFNB2 ΔC, EFNB2 ΔC + H, and EFNB2 Δ E SW480 Luc cells ( n = 6 per group) and animal imaging to monitor tumor growth and staining PCNA in LM tumor tissues (Purple: PCNA, Blue: DAPI). Scale bar: 25 μm. E Viability of SW480 and HT29 cells transfected with vector, EFNB2 FL, EFNB2 ΔC, EFNB2 ΔC + H, and EFNB2 ΔE, as analyzed using CCK-8 assays. F Viability of SW480 and HT29 cells transfected with vector, EFNB2 ΔE, Vector + rEFNB2 ΔC + H, and EFNB2 ΔE + rEFNB2 ΔC + H, as analyzed using CCK-8 assays. G EdU assays of SW480 and HT29 cells transfected with vector, EFNB2 FL, EFNB2 ΔC, EFNB2 ΔC + H, and EFNB2 ΔE. H EdU assays of SW480 and HT29 cells transfected with vector, EFNB2 ΔE, Vector + rEFNB2 ΔC + H, and EFNB2 ΔE + rEFNB2 ΔC + H. Scale bar: 25 μm. All experiments were performed in triplicate. Measurement data are presented as the mean ± SD. Student’s t -tests were used for statistical analysis. ns. represents no statistical difference; * p < 0.05; *** p < 0.001.

Article Snippet: All the lentivirus-EFNB2 cDNAs were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Techniques: Over Expression, Injection, Plasmid Preparation, Imaging, Staining, Transfection, CCK-8 Assay

Journal: Oncogene

Article Title: Adaptive activation of EFNB2/EPHB4 axis promotes post-metastatic growth of colorectal cancer liver metastases by LDLR-mediated cholesterol uptake

doi: 10.1038/s41388-022-02519-z

Figure Lengend Snippet: A mRNA expression of EPHB4 and other EPHs in CRC and LM samples in GSE6988. B Viability of SW480 and HT29 cells transfected with vector, EFNB2-OE, and EFNB2-OE + siEPHB4, as analyzed using CCK-8 assays. C EdU assays of SW480 and HT29 cells transfected with vector, EFNB2-OE, EFNB2-OE + siEPHB4, and EFNB2-OE + NVP-BHG712. Scale bar: 25 μm. D Liver metastasis model created by spleen injection with SW480 luc cells transfected with vector, EFNB2-OE, EFNB2-OE + siEPHB4, and EFNB2-OE + NVP-BHG712 ( n = 6 per group), and animal imaging to monitor tumor growth. Scale color bar: 2.81 × 10 6 –4.40 × 10 7 . E Liver infection model with SW480 luc cells transfected with vector, EFNB2-OE, EFNB2-OE + siEPHB4, and EFNB2-OE + NVP-BHG712 ( n = 6 per group), and animal imaging to monitor tumor growth. Scale color bar: 2.50 × 10 7 –4.55 × 10 8 . F PCNA staining by IHC in LM tumor tissues (vector, EFNB2-OE, EFNB2-OE + siEPHB4, and EFNB2-OE + NVP-BHG712). Scale bar: 50 μm. G PCNA staining by IHC in tumor tissues (vector, EFNB2-OE, EFNB2-OE + siEPHB4, and EFNB2-OE + NVP-BHG712). Scale bar: 50 μm. H LM tumor tissues from vector or EFNB2-OE mice were digested and LM cells were cultured. I The viability of SW480 cells from LM tumor tissues in the vector and EFNB2-OE groups, as analyzed using CCK-8 assays. J The proliferation of SW480 cells from LM tumor tissues in the vector and EFNB2-OE groups, as analyzed using EdU assays. Scale bar: 50 μm. All experiments were performed in triplicate. Measurement data are presented as the mean ± SD. Student’s t -tests were used for statistical analysis. ns. represents no statistical difference; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: All the lentivirus-EFNB2 cDNAs were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Techniques: Expressing, Transfection, Plasmid Preparation, CCK-8 Assay, Injection, Imaging, Infection, Staining, Cell Culture

Journal: Oncogene

Article Title: Adaptive activation of EFNB2/EPHB4 axis promotes post-metastatic growth of colorectal cancer liver metastases by LDLR-mediated cholesterol uptake

doi: 10.1038/s41388-022-02519-z

Figure Lengend Snippet: A GSEA analysis of EFNB2 expression in CRC, as evaluated using GSE6988. A total of 24 CRC LM samples were divided into two groups, including a high EFNB2 expression group (12 samples) and a low EFNB2 expression group (12 samples). B Total cholesterol levels were measured in the CRC LM tumor tissues, including the vector, EFNB2-OE, EFNB2-OE + siEPHB4, and EFNB2-OE + NVP-BHG712 groups ( n = 6 per group). C Total cholesterol levels in the tumor tissues of the liver injection model, including the vector, EFNB2-OE, EFNB2-OE + siEPHB4, and EFNB2-OE + NVP-BHG712 groups ( n = 6 per group). D Cholesterol levels were measured in CRC cells. E Cholesterol levels were measured in SW480 and HT29 cells, including the vector, EFNB2-OE and EFNB2-OE + siEPHB4, EFNB2-OE + NVP-BHG712 groups, with or without 10% FBS treatment. F Expression of cholesterol uptake related genes (LDLR, VLDLR, and SCARB1), and cholesterol synthesis related genes (HMGCS1, HMGCR, NSDHL, MSMO1, and DHCR24) in CRC LM tumor tissues, including the vector, EFNB2-OE, EFNB2-OE + siEPHB4, and EFNB2-OE + NVP-BHG712 groups ( n = 6 per group). G Expression of cholesterol uptake related genes (LDLR, VLDLR, and SCARB1), and cholesterol synthesis related genes (HMGCS1, HMGCR, NSDHL, MSMO1, and DHCR24) in SW480 cells in FBS-free culture, including the vector, EFNB2-OE, EFNB2-OE + siEPHB4, and EFNB2-OE + NVP-BHG712 groups. H Total cholesterol levels in SW620 and LoVo cells in FBS-free culture, including sh-NC, sh-EFNB2, sh-NC + 5 μg/ml cholesterol (Cho), and sh-EFNB2 + 5 μg/ml Cho. I Total cholesterol levels in SW480 and HT29 cells in FBS-free culture, including vector + 5 μg/ml Cho, EFNB2-OE + 5 μg/ml Cho, and EFNB2-OE + 5 μg/ml Cho + NVP-BHG712. All experiments were performed in triplicate. Measurement data are presented as the mean ± SD. Student’s t -tests were used for statistical analysis. ns. indicated no statistical difference; *** p < 0.001.

Article Snippet: All the lentivirus-EFNB2 cDNAs were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Techniques: Expressing, Plasmid Preparation, Injection

Journal: Oncogene

Article Title: Adaptive activation of EFNB2/EPHB4 axis promotes post-metastatic growth of colorectal cancer liver metastases by LDLR-mediated cholesterol uptake

doi: 10.1038/s41388-022-02519-z

Figure Lengend Snippet: A Protein expression of total SREBP2 and activated SREBP2 in CRC LM tissues transfected with vector and EFNB2-OE ( n = 6). B Transcription factor analysis of LDLR. C STAT3 binding to the promoter region of LDLR in SW480 and SW620 cells, as assessed using ChIP assay. D Role of STAT3 in promoting LDLR transcription, according to a dual luciferase report assay. E Protein expression of LDLR, STAT3, and p-STAT3 in SW620 and LoVo cells, including si-NC, siSTAT3, and SH-4-54 groups. F Protein expression of JAK2, p-JAK2, STAT3, and p-STAT3 in SW480 and HT29 cells, including the vector, EFNB2-OE, EFNB2-OE + NVP-BHG712, and EFNB2-OE + siEPHB4 groups. G Protein expression of JAK2, p-JAK2, STAT3, and p-STAT3 in SW620 and LoVo cells, including the sh-NC, sh-EFNB2, and sh-EFNB2 + rEFNB2 groups. H mRNA and protein expression of LDLR, VLDLR, and SCARB1 in SW480 and HT29 cells, including the vector, EFNB2-OE, EFNB2-OE + SH-4-54, and EFNB2-OE + siSTAT3 groups. All experiments were performed in triplicate. Measurement data are presented as the mean ± SD. Student’s t -tests were used for statistical analysis. ns. indicates no statistical difference; *** p < 0.001.

Article Snippet: All the lentivirus-EFNB2 cDNAs were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Techniques: Expressing, Transfection, Plasmid Preparation, Binding Assay, Luciferase

Journal: Oncogene

Article Title: Adaptive activation of EFNB2/EPHB4 axis promotes post-metastatic growth of colorectal cancer liver metastases by LDLR-mediated cholesterol uptake

doi: 10.1038/s41388-022-02519-z

Figure Lengend Snippet: A LM model created by spleen injection with SW480 luc cells transfected with vector, EFNB2-OE, EFNB2-OE + siLDLR #1, or EFNB2-OE + siLDLR #2 ( n = 6 per group) and animal imaging to monitor tumor growth and PCNA staining by IHC in tumor tissues. Scale bar: 50μm. B Liver injection model created by spleen injection with SW480 luc cells transfected with vector, EFNB2-OE, EFNB2-OE + siLDLR #1, or EFNB2-OE + siLDLR #2 ( n = 6 per group) and animal imaging to monitor tumor growth and PCNA staining by IHC in tumor tissues. Scale bar: 50 μm. C Cholesterol detection in an LM model transfected with vector, EFNB2-OE, EFNB2-OE + siLDLR #1, or EFNB2-OE + siLDLR #2 ( n = 6 per group). D Total cholesterol levels in tumor tissues in an LM model and a liver injection model transfected with vector, EFNB2-OE, EFNB2-OE + siLDLR #1, or EFNB2-OE + siLDLR #2 ( n = 6 per group). E Total cholesterol levels in SW480 CRC cells in FBS-free and cholesterol culture (5 μg/ml Cho), including vector, EFNB2-OE, EFNB2-OE + siLDLR #1, and EFNB2-OE + siLDLR #2 groups. F Total cholesterol levels in HT29 CRC cells in FBS-free and cholesterol culture (5 μg/ml Cho), including vector, EFNB2-OE, EFNB2-OE + siLDLR #1, and EFNB2-OE + siLDLR #2 groups. G The viability of SW620 and LoVo cells in FBS-free culture, including the siNC, siNC + 5 μg/ml Cho, siLDLR #1 + 5 μg/ml Cho, and siLDLR #2 + 5 μg/ml Cho groups, as analyzed using CCK-8 assays. H Viability of SW480 and HT29 cells in FBS-free culture, including vector + 5 μg/ml Cho, EFNB2-OE + 5 μg/ml Cho, EFNB2-OE + NVP-BHG712 + 5 μg/ml Cho, EFNB2-OE + siLDLR #1 + 5 μg/ml Cho, and EFNB2-OE + siLDLR #2 + 5 μg/ml Cho groups, as analyzed using CCK-8 assays. All experiments were performed in triplicate. Measurement data are presented as the mean ± SD. Student’s t -tests were used for statistical analysis. ns. indicates no statistical difference; ** p < 0.01, *** p < 0.001.

Article Snippet: All the lentivirus-EFNB2 cDNAs were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Techniques: Injection, Transfection, Plasmid Preparation, Imaging, Staining, CCK-8 Assay

Journal: Oncogene

Article Title: Adaptive activation of EFNB2/EPHB4 axis promotes post-metastatic growth of colorectal cancer liver metastases by LDLR-mediated cholesterol uptake

doi: 10.1038/s41388-022-02519-z

Figure Lengend Snippet: A Overall survival analysis of patients with CRC LM, based on the protein expression of EFNB2 ( n = 30). B A high cholesterol model was established by providing a high cholesterol diet to BALB/c nude mice. C Total cholesterol levels in the liver tissues of mice with high cholesterol and normal control feeding ( n = 6 per group). D CRC LM models were established using SW620 cells in mice with high cholesterol and normal control feeding, including the sh-NC, sh-EFNB2, and sh-NC + NVP-BHG712 groups ( n = 6 per group). E Overall survival analysis of CRC LM models in mice with high cholesterol and normal control feeding, including the sh-NC, sh-EFNB2, and sh-NC + NVP-BHG712 groups ( n = 6 per group). F Adaptive activation of the EFNB2/EPHB4 axis promoted CRC LM by LDLR-mediated cholesterol uptake. All experiments were performed in triplicate. Measurement data are presented as the mean ± SD. Student’s t -tests were used for statistical analysis. *** p < 0.001.

Article Snippet: All the lentivirus-EFNB2 cDNAs were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Techniques: Expressing, Control, Activation Assay