Single-cell analysis reveals cancer stem cell heterogeneity in hepatocellular carcinoma

Abstract

Intratumor molecular heterogeneity of hepatocellular carcinoma is partly attributed to the presence of hepatic cancer stem cells (CSCs). Different CSC populations defined by various cell surface markers may contain different oncogenic drivers, posing a challenge in defining molecularly targeted therapeutics. We combined transcriptomic and functional analyses of hepatocellular carcinoma cells at the single-cell level to assess the degree of CSC heterogeneity. We provide evidence that hepatic CSCs at the single-cell level are phenotypically, functionally, and transcriptomically heterogeneous. We found that different CSC subpopulations contain distinct molecular signatures. Interestingly, distinct genes within different CSC subpopulations are independently associated with hepatocellular carcinoma prognosis, suggesting that a diverse hepatic CSC transcriptome affects intratumor heterogeneity and tumor progression.

Conclusion: Our work provides unique perspectives into the biodiversity of CSC subpopulations, whose molecular heterogeneity further highlights their role in tumor heterogeneity, prognosis, and hepatic CSC therapy. (Hepatology 2018;68:127-140).

Figure 1. Heterogeneous expression patterns of hepatic CSC surface markers CD133, CD24 and EpCAM, in spheroid and monolayer HCC cells shown by immunofluorescence

(A) HuH1 and HuH7 cells were grown as monolayers and immunostaining was performed for CD133, CD24 and EpCAM as indicated. Nuclear DNA was counterstained by DAPI. Shown are representative confocal images of each surface marker, a merged image and the corresponding image of surface rendering (SR). Scale bars are 50 μm. (B) HuH1 and HuH7 cells were grown as spheroids and immunostaining was performed for CD133, CD24 and EpCAM as indicated. Nuclear DNA was counterstained by DAPI. Shown are representative confocal images of each surface marker, a merged image and the corresponding image of SR. Scale bars are 50 μm.

Figure 2. Single-cell functional analysis reveals distinct self-renewal and differentiation capacities of HCC cells defined by hepatic CSC surface markers

(A and B) Barplots showing the percent of single cells with >2 cell divisions during 2-week culture after flow sorting. Single cells from HuH1 (A) or HuH7 (B) were stained and cells were sorted based on surface marker (CD133, CD24 and EpCAM) status, and further cultured individually in normoxia or hypoxia conditions for 2 weeks. Experiments were performed in triplicate and data are shown as mean ± SEM. (C and D) Violin plots showing the cell number (log2) of single cell-derived clones. The single cells from HuH1 (C) and HuH7 (D) were sorted and cultured as in (A and B), and the surface marker status is shown as indicated. (E) Barplots showing the percentage of CSC surface marker positive cells (CD133+, EpCAM+ and CD24+; bottom) within the single-cell derived cell populations. The single cells from HuH1 and HuH7 were defined and sorted as in (A and B) and cultured for one month. The derived cell population was analyzed by flow cytometry. The surface marker status of the original single cells is shown on the top panel as CD133+, EpCAM+ CD24+, Triple+ and Triple−. “n” indicates the number of the original cultured single cells. Unsorted cells were used as a control and in this instance, “n” indicates replicate analyses.

Figure 3. Single-Cell transcriptomic analysis reveals heterogeneity of marker-defined CSCs

(A) Multidimensional scaling analysis illustrates the relative similarity between all 118 single HCC cells, cell pools (10cell [n=8], 100cells [n=12]) and population controls. The small-size solid circles represent single cells, the medium-size shaded circles with a black outline represent 10 cells while the large-size open circles represent 100 cells. The distance between any two cells reflects the similarity of their transcriptomic profiles. Cells group mainly by their origin (color code) as indicated, i.e., HuH1, HuH7 cells, one primary HCC fresh tissue (P1 [n=20]), mouse hematopoietic stem cell (mHSC [n=7]), mouse embryonic cell (mEC [n=7]) and human embryonic stem cell (hESC [n=2]). All data were median normalized. (B) Multidimensional scaling illustrates the relative similarity between all 55 single cells from the HuH7 cell line with defined surface marker status. Cell cluster by marker expression status as indicated, but each subpopulation also contains outliers that are more similar to cells in other subpopulations. (C, D and E) Survival Risk Predictions of 240 tumor cases (C) and 231 non-tumor cases (D) in the LCI cohort, and 250 cases of tumor in TCGA with available survival data were performed using the Triple+ vs Triple− differential expressed gene set from HuH7 cells. The corresponding Kaplan-Meier survival curves are shown for high-risk and low-risk survival groups with the log-rank p value and the permuted p value.

Figure 4. Distinct molecular signaling of marker-defined CSCs and their associations with HCC prognosis

(A) A Venn diagram is shown of genes with ≥5-fold alteration in the comparison of surface marker positive (Triple+, CD133+, EpCAM+ or CD24+) and Triple− HuH7 cells. (B) A Forest plot shows the hazard ratios and 95% confidence intervals (CI) for overall survival in various HCC cohorts (LCI, TCGA and LEC). The gene sets used for analyses are indicated in (A). (C–F) The top networks associated with CD133+, CD24+, EpCAM+ and triple+ HuH7 cells defined by single-cell transcriptome and IPA analyses are shown. Genes with shaded shapes are included in the signatures and are highlighted in colors, i.e., red (up-regulated), green (down-regulated). Open shapes represent genes that are not on the list of significant genes but are reported to be associated with the network. Arrows represent positive regulation of gene expression, with solid arrows indicating direct regulation and broken arrows indirect regulation.

Figure 5. Transcriptomic patterns of HuH1, HuH7 and P1 based on 10X genomics single cell analysis

(A) 2D visualization of (10X Genomics) data from 899 HuH1 cells, 2088 HuH7 cells and 860 freshly isolated primary HCC cells by t-SNE. (B) t-SNE plots showing expression and distribution of hepatic CSC related genes. (C) HCC associated genes. (D) stemness-associated genes or immune cell genes. For panels B–D, each cell was colored based on their normalized expression of indicated genes, i.e., a gradient of gray, yellow, and red indicating low to high expression.