Role Of Bone Marrow-Derived Cells In The Origin Of Liver Cancer Using Single-Cell Sequencing

Background and Objective Hepatocellular carcinoma（HCC）is a highly lethal malignant tumor,and its mortality rate ranks third in cancer^-related deaths worldwide.The existing methods of diagnosis and treatment fail to improve the prognosis of HCC patients.Therefore,we intend to explore the cell types initiating HCC,so as to provide a new theoretical basis for clinical diagnosis and treatment of HCC.Studies have shown that HCC may originate from mature hepatocytes or liver stem/progenitor cells that take part in liver parenchyma remodeling.Besides,bone marrow^-derived cells（BMDCs）are also proven to home to the damaged liver and participate in hepatocyte regeneration.It has been reported that BMDCs are involved in the initiation of various cancers,including gastric cancer,skin cancer and lung adenocarcinoma.However,its role in the origin of HCC remains unclear.Thus,this study aims to explore the involvement of BMDCs in the origin of HCC,reveal the occurrence and evolution of HCC at the single cell level,and provide new ideas for clinical diagnosis and treatment of HCC through the following research: establishing a diethylnitrosamine（DEN）^-induced mouse model of liver cancer after lethal irradiation and allogeneic GFP⁺ bone marrow transplantation,isolating single cells from the tumor tissue,and analyzing their copy number alteration（CNA）patterns to identify the origin of tumor cells;constructing phylogenetic trees based on CNAs to explore the evolution model of HCC;evaluating the similarity between DEN^-induced mouse HCC and human HCC through bioinformatics analysis to provide new opportunities for clinical transformation of basic liver cancer research.Methods 1.The DEN-induced mouse model of liver cancer after allogeneic whole bone marrow transplantation was established^---all bone marrow cells of male GFP transgenic C57BL/6 mice was transplanted into male wild^-type C57BL/6 mice via tail vein following elimination of their own bone marrow cells by lethal irradiation,then DEN was used to induce the formation of HCC.H&E stainingand immunohistochemistry were used to confirm the identity of the liver tumor.2.The expression pattern of GFP and a specific marker of HCC,glypican 3（GPC3）,in the tumor tissue was examined by frozen tissue immunofluorescence.3.Single cell suspension of tumor tissue was prepared,single cells were acquired under microscope,whole genome amplification was conducted using multiple annealing and looping^-based amplification cycles（MALBAC）technology,DNA library was constructed,and whole genome sequencing at low^-depth（⁰.3×）was performed.4.The patterns of single cell CNAs were analyzed using bioinformatics methods,a CNA event matrix was constructed,and a phylogenetic tree was built based on this event matrix.5.The presence of GFP sequence in the genome of tumor cells was detected by nested PCR.6.The transplantation model of bone marrow(CD45⁺ bone marrow cells)without bone marrow mesenchymal stem cells(BM^-MSCs)was constructed,followed by inducement of mouse liver cancer using DEN.The effect of BM^-MSCs on hepatocarcinogenesis was explored at the single cell level.7.Flow cytometry was performed to evaluate the specificity of CD45 microbeads enriching CD45⁺ bone marrow cells.8.The DEN^-induced mouse model of liver cancer after gender^-mismatched allogeneic bone marrow transplantation was established.Immunofluorescence combined with fluorescence in situ hybridization was used to detect sex chromosomes of HCC cells for further verifying the origination of HCC cells.9.Phylogenetic trees of six sequenced mice were constructed to explore the model of copy number evolution in mouse HCC.10.The orthologs of mutant genes related to human HCC in CNA^-positive regions shared by multiple mice were searched using bioinformatics analysis to assess the similarity between mouse HCC and human HCC.Results 1.The DEN^-induced mouse model of liver cancer after allogeneic whole bonemarrow transplantation was successfully established.The tumor in the liver was HCC confirmed by histopathological examination.2.Frozen tissue immunofluorescence revealed the co^-localization of GPC3 and GFP in some tumor cells of the liver cancer tissue,preliminarily suggesting that BMDCs might participate in the origination of HCC.3.Analysis of single cell CNAs showed that there were both GFP⁺ and GFP^-tumor cells in the liver cancer tissue.4.GFP⁺ and GFP^-tumor cells shared some CNA^-positive regions,indicating that they were likely to be of the same origin,that is,from the bone marrow.Besides,there was also significant heterogeneity among tumor cells,since they had their own unique CNAs.5.The results of nested PCR confirmed that GFP sequence was present in the genome of both GFP⁺ and GFP^-tumor cells,further indicating that BMDCs may be the major origin of HCC in mice.6.The DEN^-induced mouse model of liver cancer after transplantation of bone marrow(CD45⁺ bone marrow cells)without BM^-MSCs was successfully constructed.The tumor incidence of mice in CD45⁺ bone marrow transplantation group was significantly reduced,indicating that BM^-MSCs were vital for the occurrence of HCC.7.GFP⁺ and GFP^-tumor cells still existed in liver cancer tissues of mice in CD45⁺ bone marrow transplantation group.Single^-cell CNA analysis showed that these two groups of cells had highly similar CNA patterns,and nested PCR also confirmed that their genomes contained GFP sequence.Flow cytometry revealed that a small amount of CD45^-bone marrow cells were mixed in CD45⁺ donor bone marrow,suggesting that BM^-MSCs,which had not been completely removed,might initiate HCC.Sequencing results also provide another possibility that BM^-MSCs may not be the only subset of bone marrow cells involved in the origin of HCC.8.The DEN^-induced mouse model of liver cancer after gender^-mismatched allogeneic bone marrow transplantation was successfully established.By detecting sex chromosomes of GPC3⁺ cells,i.e.HCC cells,it was shown that allHCC cells were derived from donor bone marrow,while there was no stable fusion occurred between donor BMDCs and HCC cells.This result suggested that BMDCs directly participated in the origin of HCC in this mouse model.9.In this mouse model of liver cancer,the evolution pattern of CNAs was not unitary.Instead,punctuated and gradual evolution models coexisted.10.Bioinformatics analysis revealed that there were orthologous genes corresponding to human HCC mutant genes in CNA^-positive regions shared by multiple mice.These findings suggested that there was a certain similarity between mouse HCC formed in this experimental model and human HCC.Conclusions In this study,we successfully established a DEN^-induced mouse model of liver cancer after bone marrow transplantation.By analyzing CNA patterns of single liver cancer cells,we found that BMDCs were the main cells originating HCC.We speculated that BMDCs could be recruited to sites of liver injury caused by a chemical carcinogen DEN and participate in liver regeneration by transdifferentiation into hepatocytes.Long^-term exposure to this chemical carcinogenic environment,these cells were vulnerable to malignant transformation and thereby initiated HCC.In this model,BM^-MSCs,a subset of bone marrow cells,were critical for the occurrence of HCC.Bioinformatics analysis revealed that both gradual and punctuated copy number evolution patterns existed in this mouse model of liver cancer;mouse HCC in this model and human HCC had certain similarities.The novel finding that HCC may originate from BMDCs will promote a deeper understanding of the pathogenesis of HCC,thereby facilitating the development of new therapeutic targets or approaches.