Additional file 10 of Liver cancer stem cell dissemination and metastasis: uncovering the role of NRCAM in hepatocellular carcinoma
Creators: name: Zhou, Lingyun
name: He, Linye
name: Liu, Chang-Hai
name: Qiu, Huandi
name: Zheng, Li
name: Sample, Klarke Michael
name: Wu, Qin
name: Li, Jiaxin
name: Xie, Kunlin
name: Ampuero, Javier
name: Li, Zhihui
name: Lv, Duoduo
name: Liu, Miao
name: Romero-Gómez, Manuel
name: Hu, Yiguo
name: Tang, Hong
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 10: Supplementary Materials and Methods. Supplementary Text. Fig. S1. The use of NRCAM for HCC diagnosis and metastasis prediction. (A) Flowchart. HCC: Hepatocellular carcinoma, ICC: Intrahepatic cholangiocarcinoma, TCGA: Cancer Genome Atlas, ROC: receiver operating curve. (B) A ROC was used to evaluate the predictive value of serum AFP, PIVKA-II, and NRCAM for HCC diagnosis. (C) IHC staining for ARG1 and Hep Par 1 in HCC without metastasis. (D) IHC staining of ARG1 and Hep Par 1 for HCC with metastasis. Fig. S2. Single-cell profile and evolution trajectory of metastasis HCC. (A) Experiment procedure overview. Five scRNA-seq datasets were generated from HCC tumor tissues to provide transcriptomes for 36,085 individual cells. H&amp;E, IHC, and IF were performed on FFPE tissue in parallel. (B) A UMAP demonstrating the 23 main cell clusters. (C) Violin plots showing the normalized expression for cell type-specific markers. (D) Cell type assignment to clusters. (E) Potential hepatocyte trajectory with color scales representing pseudotime. Epcam expression across the trajectory with color scales representing expression. Potential hepatocyte trajectory for seven distinct cell states. State 1-2 represented LCSCs, and state 3-7 mature HCC. The red arrows demonstrate the potential cell-level evolutionary trajectory. Fig. S3. Marker expression in HCC. (A) KRT19 expression trajectory, color scale represents expression. (B) trajectory of adult liver markers (TAT, TDO2, SSTR2, CYP7A1, CYP3A4 and CYP2B6), color scale represents expression. (C) large-scale copy number variation (CNV) identified in hepatocytes from the five patients using scRNA-seq data, normal hepatocytes were used as a reference; amplification is represented using red and blue for deletion. (D) Cell ratio of aneuploid in clusters. (E) Epithelial scores (consisting of SFN, EPCAM, KRT17, KRT86, KRT81, KRT18, KRT222, KRT10, KRT23, KRT19, KRT80, KRT36, KRT17 and KRT27) for the hepatocyte clusters. (F) UMAP demonstrating malignant cells (red) identified by CNV and epithelial scoring. (G) GSEA plots showing stemness gene enrichment patterns and marker expression according to NRCAM status (high to low) in TCGA samples. (H) MYC expression according to NRCAM status (high to low) in TCGA samples. (I) TCGA NRCAM and MYC expression correlation. P=&lt;0.05 (*). Fig. S4. Regulation of NRCAM in mouse LCSCs. (A) MIG-Vec, MIG-MYC, MIG-NRCAM, MIG-MYC-NRCAM, and MIG-MYC-shNRCAM plasmid structures. (B) qPCR and (C) western blotting for shRNA-mediated NRCAM inhibition, shRNA1 had a good inhibitory effect on NRCAM and was used in subsequent experiments. (D, E) The GFP positive rate of MIG-Vec and MIG-MYC organoids two and 14 days after transduction. (F) MYC and Nrcam levels in MIG-Vec and MIG-MYC organoids. (G) Plate clone formation assay for MIG-Vec and MIG-MYC organoids. (H, I) GFP positive rate of MIG-Vec and MIG-NRCAM organoids two and 14 days after transduction. (J) NRCAM and Myc levels in MIG-Vec and MIG-NRCAM organoids. (K) plate clone formation assay. The data was displayed using the mean ± SD where applicable. P=&lt;0.05 (*), P=&lt;0.01 (**), P=&lt;0.001 (***). Fig. S5. Identification of HCC metastasis in the murine oncogene-driven HCC allograft model. (A) MYC levels in MIG-MYC, MIG-MYC-NRCAM, and MIG-MYC-shNRCAM organoids. (B) Intrahepatic injection of MIG-Vec, MIG-MYC, MIG-MYC-NRCAM, and MIG-MYC-shNRCAM organoids into irradiation-induced transient immune deficient C57BL/6 mice. (C) Mouse lungs and livers 28 days after allografting for the MIG-Vec, MIG-MYC, MIG-MYC-shNRCAM, and MIG-MYC-NRCAM groups. (D) Tissue clearing and image analysis for lung and liver from the MIG-Vec (Mouse: 1-3), MIG-MYC (Mouse: 2-2), MIG-MYC-shNrcam (Mouse: 3-5), and MIG-MYC-NRCAM (Mouse: 4-3) group mice. (E) HE staining for the MIG-Vec (Mouse: 1-3), MIG-MYC (Mouse: 2-2), MIG-MYC-shNrcam (Mouse: 3-5) and MIG-MYC-NRCAM (Mouse: 4-3) groups. (F) IHC staining for NRCAM and Hep Par 1 in the lung and liver from MIG-MYC-NRCAM (Mouse: 4-3) group mice. The data was represented using the mean ± SD where applicable, P=&gt;0.05 (NS). Fig. S6. SRP278381 GEO database single-cell profile and evolution trajectory. (A) tSNE displaying the 21 main cell clusters and cell types, a differential gene expression analysis was used to identify cluster-specific markers. (B) NRCAM is predominately expressed in cluster 17, a group of bi-potent stem cells (KRT19+ and EPCAM+). (C) tSNE displaying malignant cells (red, identified using epithelial score), NRCAM+ bi-potent stem cells were malignant, and could be defined as LCSCs. NRCAM+ bi-potent stem cell distribution (Cluster 17) was mostly in adjacent normal tissue (adjacent normal: 592 from 629). (D) Hepatocyte trajectory analysis (arrow displays potential evolutionary direction): the potential trajectory of all hepatocytes, the color scale represents pseudotime; seven distinct cell states (colored by state 1-7) were identified across the potential hepatocyte trajectory. Liver stem cells were present in state 1, mature hepatocytes in state 2-6, and HCC cells in state 7. (E) Epithelial score genes, NRCAM, and MYC expression in pseudotime. (F) WNT/β-Catenin signaling activity, EMT activity, MMP3, MMP7, MMP14, and CD44 expression in pseudotime. Fig. S7. Identification of the key pathway associated with NRCAM activation in metastatic HCC. (A) Multiplex immunofluorescent (mIF) staining for NRCAM, Wnt/β-catenin signaling, and EMT in layers 2-5. Venn diagram displaying NRCAM, Wnt/β-catenin signaling, and EMT co-activation in HCC cells. (B) mIF staining for NRCAM, MMP3, MMP7, MMP14 and CD44. Venn diagram displaying MMP3, MMP7, MMP14, and CD44 co-activation in HCC cells. (C) mIF staining for NRCAM, Wnt/β-catenin signaling, and EMT in layer 1-5. Venn diagram displaying NRCAM, Wnt/β-catenin signaling, and EMT co-activation in HCC cells. (D) mIF staining for NRCAM, MMP3, MMP7, MMP14 and CD44. Venn diagram displaying MMP3, MMP7, MMP14, and CD44 co-activation in HCC cells. (E) Notch signaling activity on the trajectory (color scale representing activity level). (F) Notch signaling activity changes in pseudotime. (G) mIF staining for NRCAM and NOTCH1 in metastatic HCC tumor tissue demonstrates few cells with NRCAM and NOTCH1 co-activation. Fig. S8. NRCAM, key factors in MACF1 mediated β-catenin signaling pathway, EMT and MMPs expression in LCSCs. (A) NRCAM, Wnt7a, Ctnnb1, p-Gsk3b, Snai1, Zeb1, Vim, Cdh1, Cdh2, Mmp3, Mmp7 and Mmp14 levels. (B) Interaction Network analysis of NRCAM and MACF1 from IntAct ( https://www.ebi.ac.uk/intact/home ). (C) shRNA inhibition of MACF1. The shRNA2 had a good inhibitory effect on MACF1 and was used in subsequent experiments. (D) NRCAM, Ctnnb1, p-Gsk3b, Snai1, Zeb1, Vim, Cdh1, Cdh2, Mmp3, Mmp7 and Mmp14 levels. (E) Mouse lungs and livers 28 days after allografting for the MIG-MYC-NRCAM-Con and MIG-MYC-NRCAM-shMacf1 groups. (F) HE staining for the MIG-MYC-NRCAM-Con (Mouse: 1-1) and MIG-MYC-NRCAM-shMacf1 (Mouse: 2-1) groups. Where applicable, the data was represented using mean ± SD (n=3). P=&lt;0.05 (*), P=&lt;0.01 (**), P=&lt;0.001 (***), P=&lt;0.0001 (****). Table S1. Clinicopathological information for the two PDX HCC models. Table S2. Clinicopathological information for five patients with HCC metastasis. Table S3. shRNAs targeting Nrcam and Macf1. Table S4. The qPCR primer sequences. Table S5. Antibodies. Table S6. NrCAM mRNA in TCGA liver cancer and normal tissues. Table S7. HCC diagnostic ROCs for AFP, PIVKA-II and NRCAM. Table S8. Univariate and multivariate logistic analysis in HCC diagnosis. Table S9. Pulmonary metastasis associated with different LCSCs. Table S10. Intrahepatic metastasis associated with different LCSCs.