The role of bone marrow-derived cells in the origin of liver cancer revealed by single-cell sequencing

Abstract

Objective: Epithelial cancers often originate from progenitor cells, while the origin of hepatocellular carcinoma (HCC) is still controversial. HCC, one of the deadliest cancers, is closely linked with liver injuries and chronic inflammation, which trigger massive infiltration of bone marrow-derived cells (BMDCs) during liver repair. Methods: To address the possible roles of BMDCs in HCC origination, we established a diethylnitrosamine (DEN)-induced HCC model in bone marrow transplanted mice. Immunohistochemistry and frozen tissue immunofluorescence were used to verify DEN-induced HCC in the pathology of the disease. The cellular origin of DEN-induced HCC was further studied by single cell sequencing, single-cell nested PCR, and immunofluorescence-fluorescence in situ hybridization. Results: Studies by using single cell sequencing and biochemical analysis revealed that HCC cells in these mice were coming from donor mice BMDCs, and not from recipient mice. Furthermore, the copy numbers of mouse orthologs of several HCC-related genes previously reported in human HCC were also altered in our mouse model. DEN-induced HCCs exhibited a similar histological phenotype and genomic profile as human HCCs. Conclusions: These results suggested that BMDCs are an important origin of HCC, which provide important clues to HCC prevention, detection, and treatments.

Keywords: Hepatocellular carcinoma; bone marrow-derived cells (BMDCs); copy number alteration; genome sequencing; origination.

Figure 1

Histopathology revealed the potential origin of hepatocellular carcinoma (HCC). (A) Schematic diagram of the experimental design. (B) The efficiency of bone marrow transplantation was assessed by analysis of green fluorescent protein (GFP) in peripheral leukocytes using flow cytometry. (C) Representative pictures of the mouse liver. Arrows indicate hepatocarcinogenic nodules. (D) Diethylnitrosamine (DEN)-induced HCC was verified by hematoxylin and eosin (H&E) staining and immunohistochemistry, scale bar = 1.0 mm. (E) Colocalization of GFP and glypican 3 (GPC3) in the experimental group (DEN induction group-tumor tissue and cirrhosis tissue) and the control group (normal liver without DEN induction and normal liver without transplantation), as determined by means of immunofluorescence.

Figure 2

Single-cell sequencing revealed the origination and evolution of hepatocellular carcinoma (HCC) in the whole bone marrow transplantation mice. (A) Representative copy number alteration (CNA) patterns of single tumor cells from diethylnitrosamine (DEN)-induced HCC in Mouse #5. The copy number profile (blue and red dots) indicated by log2 (copy number ratio) is plotted along the genome with a bin size of 500 kb. (M5: Mouse #5, B: blood cell, G: green cell, NG: non-green cell, Bulk: include 100 cells). (B) CNA patterns of single tumor cells from DEN-induced HCC in Mouse #5. The copy number profile indicated by log2 (copy number ratio) is plotted along the genome with a bin size of 500 kb. (Green: green cell, gray: non-green cell). (C) Multiple-cell segmentation (top) and trinary event matrices (bottom) for HCC from whole bone marrow transplantation mice. (D) A phylogenetic tree was constructed based on the CNA trinary event matrices of single cells. (E) Nested polymerase chain reaction (PCR) was used for detecting GFP at the single-cell level (P control: positive control from the genomic DNA of GFP mouse liver tissue. N control: negative control from wild-type mouse peripheral lymphocytes).

Figure 3

Following validation of the origination and evolution of DEN-induced HCC in mice as revealed by single-cell sequencing. (A) CNA patterns of single tumor cells from a DEN-induced HCC in whole bone marrow transplantation mice (Mouse #10). The copy number profile indicated by log2 (copy number ratio) is plotted along the genome with a bin size of 500 kb. (Green: green cell, gray: non-green cell). (B) Multiple-cell segmentation (top) and trinary event matrices (bottom) for HCC in Mouse #10. (C) The phylogenetic tree was constructed based on the CNA trinary event matrices of single cells of HCC in Mouse #10. (D) Nested PCR for detecting GFP at the single-cell level in Mouse #10. P control: positive control from the genomic DNA of GFP mouse liver tissue. N control: negative control from wild-type mouse peripheral lymphocytes. Abbreviation are defined in Figures 1 and 2.

Figure 4

HCC cells from male mice transplanted with female mouse bone marrow contained only X chromosomes, without Y chromosomes. (A) Construction of the experimental animal model and schematic diagram of the FISH process. (B) X (red) and Y (green) fluorescence in situ hybridization (FISH) confirmed that all GPC3⁺ HCC cells originated from bone marrow (Group a: nontransplantation, non-DEN induction; Group b: Bone marrow (transplantation, non-DEN induction; Group c: nontransplantation, DEN induction; Group d: whole bone marrow transplantation, DEN induction). Abbreviations are defined in Figures 1 and 2.

Figure 5

The DEN-induced HCC model appeared to be highly relevant to human HCC. (A) Maximum-parsimony trees for HCC tumors from 6 mice. (B) Heatmap of CNAs of all 88 tumor cells in the 6 HCC mice. The color from blue to red indicates values in the form of log2 (copy number ratio) from −2 to 2. Abbreviations defined in Figures 1 and 2.