Lineage tracing and single-cell analysis reveal proliferative Prom1+ tumor-propagating cells and their dynamic cellular transition during liver cancer progression

Hepatocellular carcinoma (HCC) is highly heterogeneous, and this heterogeneity contributes to therapeutic resistance and tumor recurrence. The aim of this study was to investigate the role, heterogeneity, and properties of Prom1+ cells in HCC in intact mouse models. We used Prom1C-L/+;Rosa26tdTomato/+ mice to establish two HCC mouse models representing chronic fibrotic HCC and rapid steatosis-related HCC. We performed lineage tracing at different time points post-HCC induction. We ablated the Prom1+ cell population in Prom1C-L/+;Rosa26DTA/+ mice. Single cell RNA-seq (scRNA-seq) was carried out to analyse the lineage properties, heterogeneity, and dynamics of the traced Prom1+ cells in the chronic fibrotic HCC model. Bulk RNAseq was carried out to analyse the change in transcriptome in Prom1 depleted tumor. scRNA-seq: Prom1-CreERT2-LacZ mice crossed with Rosa26-LSL-Tomato reporter mice were used (Prom1C-L/+;Rosa26tdTomato/+). HCC was induced by DEN+CCL4 treatment: mice were treated with N-nitrosodiethylamine (DEN, i.p., 1 mg/kg) at the age of 14 days; when the mice were 8 weeks of age, carbon tetrachloride (CCL4, i.p., 0.2 ml/kg) was administered twice weekly for 12-16 weeks. After tumor nodules formed (12 weeks after CCL4 treatment initiated), a single dose of 4mg tamoxifen were administrated to the mice. 3, 10 and 30 days thereafter, all viable tdTomato+ and tdTomato- tumor resident cells were sorted and sequenced by 10X Genomics to reconstruct a single-cell landscape of HCC tumors. Bulk RNA-seq: Prom1C-L/+;Rosa26DTA/+ mice and their control littermates (Prom1+/+;Rosa26DTA/+) were used. DEN+CCL4 model was established as above mentioned. N-Ras+AKT model was established by hydrodynamic tail vein injection of 20 μg of pT3-EF1a-NRasV12, 20 μg of pT3-EF1a-myr-AKT and 1.6 μg sleeping beauty transposase to the mice. Multiple doses of 4mg tamoxifen or vehicle (olive oil) were administered every other day after tumor formed. Tumor tissues were collected 16 or 6 days after the last dose of tamoxifen treatment in DEN+CCL4 or N-Ras+AKT model, respectively.