Journal: Hepatology (Baltimore, Md.)

Article Title: HKDC1 promotes liver cancer stemness under hypoxia through stabilizing β-catenin

doi: 10.1097/HEP.0000000000001085

Figure Lengend Snippet: HKDC1 is upregulated in metastatic tumors and organoids in PPTR liver cancer mouse model. (A) A schematic illustration for the establishment of PPTR genetic mouse model, organoid model, and orthotopic allograft models (generated by BioRender). (B) Quantitative comparison of the gene expression of the 5 HKs in PPTR tumor tissues (n = 6, 16, 5, 13, and 9 for the 5 indicated groups, respectively) and organoids (n = 6, 7, 4, and 8 for the 4 indicated groups, respectively). One-way ANOVA test: p values: *<0.05; ****<0.0001. (C) H&E (a–c) and RNAscope staining of Hkdc1 (d–i), Hk1 (j–l), and Hk2 (m–o) on the serial sections from the indicated tumors in the PPTR genetic mouse model. Images on the same column share the same scale bar. (D) Immunoblotting of Hkdc1 and Hk1 in the normal liver and indicated PPTR tumors. (E) Immunoblotting of Hkdc1 and Hk1 in the normal liver organoids and indicated PPTR tumor organoids. Abbreviations: H&E, hematoxylin and eosin; HKDC1, hexokinase domain containing 1; PPTR, Prom1 CreERT2 ; Pten flx/flx ; Tp53 flx/flx ; Rosa-ZsGreen.

Article Snippet: RNAscope in situ hybridization of human and mouse Hkdc1 , Hk1 , Hk2 , and Hif1a mRNA transcripts was performed on freshly cut paraffin sections according to the manufacturer’s protocol (Advanced Cell Diagnostics) by Comparative Pathology Core at St. Jude’s Children’s Research Hospital.

Techniques: Generated, Comparison, Gene Expression, RNAscope, Staining, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: HKDC1 promotes liver cancer stemness under hypoxia through stabilizing β-catenin

doi: 10.1097/HEP.0000000000001085

Figure Lengend Snippet: HKDC1 upregulation is highly specific to malignant cells in patients with HCC and CCA. (A) Mean expression of HK1 , HK2 , and HKDC1 in nonmalignant cells and malignant cells from 46 published liver cancer patient samples, including 25 patients with HCC (left panel) and 12 patients with CCA (right panel). (B) tSNE plots of HKDC1 , HK1 , and HK2 expression in the malignant and nonmalignant cells in the samples in (A). (C) HKDC1 , HK1 , and HK2 expression in individual cell types using the published scRNA-seq data from 7 patients with liver cancer. Hepatocytes and cholangiocytes were derived from tumor-adjacent normal liver tissues of patients with HCC and CCA. (D) H&E and RNAscope staining of HKDC1 , HK1 , and HK2 on the serial sections from the indicated normal human liver and HCC and CCA patient tumors. Solid arrows: normal cholangiocytes; open arrows: normal hepatocytes. Inserts: boxed area in the same image. RNAscope images on the same column share the same scale bar. Abbreviations: CAFs, cancer-associated fibroblasts; CCA, cholangiocarcinoma; H&E, hematoxylin and eosin; HKDC1, hexokinase domain containing 1; scRNA-seq, single-cell RNA sequencing; TAMs, tumor-associated macrophages; TECs, tumor-associated endothelial cells.

Article Snippet: RNAscope in situ hybridization of human and mouse Hkdc1 , Hk1 , Hk2 , and Hif1a mRNA transcripts was performed on freshly cut paraffin sections according to the manufacturer’s protocol (Advanced Cell Diagnostics) by Comparative Pathology Core at St. Jude’s Children’s Research Hospital.

Techniques: Expressing, Derivative Assay, RNAscope, Staining, RNA Sequencing

Journal: Hepatology (Baltimore, Md.)

Article Title: HKDC1 promotes liver cancer stemness under hypoxia through stabilizing β-catenin

doi: 10.1097/HEP.0000000000001085

Figure Lengend Snippet: HKDC1 promotes HCC metastasis. (A) Immunoblotting of HKDC1 in the control and HKDC1-OE Huh7 cells. (B) Cell proliferation assay of the control and HKDC1-OE Huh7 cells. (C) Kaplan-Meier survival curves of mice injected with the indicated Huh7 cells. (D) HKDC1 RNAscope on the liver tumors generated by the control and HKDC1-OE Huh7 cells. (E) Immunoblotting of HKDC1 in the control, HKDC1-OK, and -OK/Res PLC/PRF/5 cells. (F) Cell proliferation assay of the control, HKDC1-OK, and -OK/Res PLC/PRF/5 cells. p values on the top of the curves: KO/Res versus KO; p values on the top of the curves: KO versus control. (G) Kaplan-Meier survival curves of mice injected with the indicated PLC/PRF/5 cells. (H) Top: whole-lung H&E images of mice injected with LX2 mixed with the HKDC1-OK and -OK/Res PLC/PRF/5 cells, respectively; bottom: high-magnification images of the boxed areas in the top image. Pie chart: The number of mice with lung metastasis (black) and with no lung metastasis (gray). Images on the same row share the same scale bar. (I) Gross lung images of the TVI mice injected with the indicated PLC/PRF/5 cells. Arrows: lung metastasis. (J) H&E images of the lung tissues in (J). Images share the same scale bar. Statistics in (B, C, F): Student t test. p values: *<0.05; **<0.01; ***<0.001; ****<0.0001. Statistics in (C) and (G): Log-rank (Mantel-Cox) test. p values: **<0.01. Abbreviations: H&E, hematoxylin and eosin; HKDC1, hexokinase domain containing 1; KO, knockout; TVI, tail veil injection.

Article Snippet: RNAscope in situ hybridization of human and mouse Hkdc1 , Hk1 , Hk2 , and Hif1a mRNA transcripts was performed on freshly cut paraffin sections according to the manufacturer’s protocol (Advanced Cell Diagnostics) by Comparative Pathology Core at St. Jude’s Children’s Research Hospital.

Techniques: Western Blot, Control, Proliferation Assay, Injection, RNAscope, Generated, Knock-Out

Journal: Hepatology (Baltimore, Md.)

Article Title: HKDC1 promotes liver cancer stemness under hypoxia through stabilizing β-catenin

doi: 10.1097/HEP.0000000000001085

Figure Lengend Snippet: HKDC1 is induced by hypoxia to support HCC cell growth under hypoxia. (A) GSEA analysis found the enrichment of HALLMARK_HYPOXIA gene signature in HKDC1-KO versus control and KO/Res versus KO PLC/PRF/5 cells. (B) Spearman correlation analysis of HKDC1 and HIF1A gene expression using RNA-seq data on HCC and CCA patient tumors in TCGA. (C) Spearman correlation analysis of Hkdc1 and Hif1a gene expression in PPTR tumors and organoids. (D) Hkdc1 and Hif1a RNAscope staining on HCC and PPTR tumor serial sections. Images on the same column share the same scale bar. (E) Immunoblotting of HKDC1 in the indicated cells cultivated under normoxia (N, 21% O 2 ) and hypoxia (H, 1% O 2 ) for 24 hours. (F) Cell proliferation assay of the control and HKDC1-KO PLC/PRF/5 cells under normoxia and hypoxia. p values on the top of the curves: control cells, hypoxia versus normoxia; p values on the top of the curves: KO cells, hypoxia versus normoxia. Statistics in (F) Student t test of the control versus KO1 cells. p value, **<0.01; ***<0.001; ****<0.0001. Abbreviations: CCA, cholangiocarcinoma; GSEA, gene set enrichment analysis; HKDC1, hexokinase domain containing 1; PPTR, Prom1 CreERT2 ; Pten flx/flx ; Tp53 flx/flx ; Rosa-ZsGreen; TCGA, The Cancer Genome Atlas.

Article Snippet: RNAscope in situ hybridization of human and mouse Hkdc1 , Hk1 , Hk2 , and Hif1a mRNA transcripts was performed on freshly cut paraffin sections according to the manufacturer’s protocol (Advanced Cell Diagnostics) by Comparative Pathology Core at St. Jude’s Children’s Research Hospital.

Techniques: Control, Gene Expression, RNA Sequencing, RNAscope, Staining, Western Blot, Proliferation Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: HKDC1 promotes liver cancer stemness under hypoxia through stabilizing β-catenin

doi: 10.1097/HEP.0000000000001085

Figure Lengend Snippet: HKDC1 promotes HCC stemness by binding to GSK3β and stabilizing β-catenin. (A) RNAscope staining of Hkdc1 and IHC staining of CK19, EpCAM, and β-catenin on serial sections of tumors from the PPTR genetic model and patients with HCC and CCA. All images share the same scale bar. (B) Spearman correlation analysis of HKDC1 and β-catenin protein levels in HCC and CCA patient tumors. (C) Immunoblotting of the indicated proteins in the control, HKDC1-OK, and -OK/Res PLC/PRF/5 cells. (D) GSK3β IP from HKDC1-FLAG-OE Huh7 cells using FLAG antibody. (E) GSK3β IP from PLC/PRF/5 cells using HKDC1 antibody. (F) Colony formation assay of the PCL/PFR/5 cells transfected with scrambled or CTNNB1 siRNA. (G) Image-based quantification of the total colony area in (F). (H) Colony formation assay of the indicated PCL/PFR/5 cells cultured under normoxia and hypoxia. (I) Image-based quantification of the total colony area in (H). Statistics in (A) and (G): Student t test. p values: *<0.05; **<0.01; ***<0.001; ****<0.0001. Abbreviations: CCA, cholangiocarcinoma; HKDC1, hexokinase domain containing 1; IHC, immunohistochemistry; PPTR, Prom1 CreERT2 ; Pten flx/flx ; Tp53 flx/flx ; Rosa-ZsGreen.

Article Snippet: RNAscope in situ hybridization of human and mouse Hkdc1 , Hk1 , Hk2 , and Hif1a mRNA transcripts was performed on freshly cut paraffin sections according to the manufacturer’s protocol (Advanced Cell Diagnostics) by Comparative Pathology Core at St. Jude’s Children’s Research Hospital.

Techniques: Binding Assay, RNAscope, Staining, Immunohistochemistry, Western Blot, Control, Colony Assay, Transfection, Cell Culture