Integrative single-cell multiomics analyses dissect molecular signatures of intratumoral heterogeneities and differentiation states of human gastric cancer.

Human gastric cancer is a highly lethal disease, but the underlying multiomic molecular signatures remain largely unclear. Here, we performed multi-regional sampling, parallel single-cell multiomics sequencing and integrated analyses of human gastric cancer. We identified common transcriptomic alterations of gastric cancer cells, such as aberrant down-regulation of genes associated with normal stomach function and up-regulation of KRT7, PI3, S100A4, etc. Surprisingly, aberrant and prevalent up-regulation of genes highly expressed in normal colorectal epithelial cells were also identified in cancer cells, which may be partially regulated by promoter chromatin accessibility and DNA methylation levels. We revealed the single-cell DNA methylome landscape of gastric cancer, and identified candidate DNA methylation biomarkers, such as hypermethylated promoters of TMEM240 and HAGLROS, and hypomethylated promoters of TRPM2-AS and HRH1. Additionally, the relationships between genetic lineages, DNA methylation and transcriptomic clusters were systematically revealed at single-cell level. We showed that DNA methylation heterogeneities were mainly among different genetic lineages of cancer cells. Moreover, we found that DNA methylation levels of cancer cells with poorer differentiation states tend to be higher than those of cancer cells with better differentiation states in the primary tumor within the same patient, although still lower than in normal gastric epithelial cells. Cancer cells with poorer differentiation states also prevalently down-regulated MUC1 expression and immune-related pathways, and had poor infiltration of CD8+ T cells. Our study dissected the molecular signatures of intratumoral heterogeneities and differentiation states of human gastric cancer using integrative single-cell multiomics analyses.

Integrated single-cell multiomics analysis reveals novel candidate markers for prognosis in human pancreatic ductal adenocarcinoma.

The epigenomic abnormality of pancreatic ductal adenocarcinoma (PDAC) has rarely been investigated due to its strong heterogeneity. Here, we used single-cell multiomics sequencing to simultaneously analyze the DNA methylome, chromatin accessibility and transcriptome in individual tumor cells of PDAC patients. We identified normal epithelial cells in the tumor lesion, which have euploid genomes, normal patterns of DNA methylation, and chromatin accessibility. Using all these normal epithelial cells as controls, we determined that DNA demethylation in the cancer genome was strongly enriched in heterochromatin regions but depleted in euchromatin regions. There were stronger negative correlations between RNA expression and promoter DNA methylation in cancer cells compared to those in normal epithelial cells. Through in-depth integrated analyses, a set of novel candidate biomarkers were identified, including ZNF667 and ZNF667-AS1, whose expressions were linked to a better prognosis for PDAC patients by affecting the proliferation of cancer cells. Our work systematically revealed the critical epigenomic features of cancer cells in PDAC patients at the single-cell level.

Cell-fate transition and determination analysis of mouse male germ cells throughout development.

Mammalian male germ cell development is a stepwise cell-fate transition process; however, the full-term developmental profile of male germ cells remains undefined. Here, by interrogating the high-precision transcriptome atlas of 11,598 cells covering 28 critical time-points, we demonstrate that cell-fate transition from mitotic to post-mitotic primordial germ cells is accompanied by transcriptome-scale reconfiguration and a transitional cell state. Notch signaling pathway is essential for initiating mitotic arrest and the maintenance of male germ cells' identities. Ablation of HELQ induces developmental arrest and abnormal transcriptome reprogramming of male germ cells, indicating the importance of cell cycle regulation for proper cell-fate transition. Finally, systematic human-mouse comparison reveals potential regulators whose deficiency contributed to human male infertility via mitotic arrest regulation. Collectively, our study provides an accurate and comprehensive transcriptome atlas of the male germline cycle and allows for an in-depth understanding of the cell-fate transition and determination underlying male germ cell development.

Single-Cell Multiomics Sequencing Reveals Prevalent Genomic Alterations in Tumor Stromal Cells of Human Colorectal Cancer.

To what extent stromal cells in the tumor microenvironment (TME) are transformed by colorectal cancer (CRC) cells is unexplored. To dissect alterations in these non-malignant cells, we performed single-cell multiomics sequencing of 21 patients with microsatellite-stable CRCs and 6 cancer-free, elderly individuals. Surprisingly, somatic copy number alterations (SCNAs) are prevalent in immune cells, fibroblasts, and endothelial cells in both the TME and the normal tissues of each individual. Moreover, the proportions of fibroblasts with SCNAs in tumors (11.1%-47.7%) are much higher than those in adjacent normal tissues (1.1%-10.6%), with gain of chromosome 7 strongly enriched in the TME, clearly indicating clonal expansion. Furthermore, five genes (BGN, RCN3, TAGLN, MYL9, and TPM2) are identified as fibroblast-specific biomarkers of poorer prognosis of CRC. Our study provides evidence and functional relevance of pervasive genomic alterations in the stromal cells of TME in CRC.

Single-cell multiomics sequencing and analyses of human colorectal cancer.

Although genomic instability, epigenetic abnormality, and gene expression dysregulation are hallmarks of colorectal cancer, these features have not been simultaneously analyzed at single-cell resolution. Using optimized single-cell multiomics sequencing together with multiregional sampling of the primary tumor and lymphatic and distant metastases, we developed insights beyond intratumoral heterogeneity. Genome-wide DNA methylation levels were relatively consistent within a single genetic sublineage. The genome-wide DNA demethylation patterns of cancer cells were consistent in all 10 patients whose DNA we sequenced. The cancer cells' DNA demethylation degrees clearly correlated with the densities of the heterochromatin-associated histone modification H3K9me3 of normal tissue and those of repetitive element long interspersed nuclear element 1. Our work demonstrates the feasibility of reconstructing genetic lineages and tracing their epigenomic and transcriptomic dynamics with single-cell multiomics sequencing.

Single-Cell RNA Sequencing Analysis Reveals Sequential Cell Fate Transition during Human Spermatogenesis.

Spermatogenesis generates mature male gametes and is critical for the proper transmission of genetic information between generations. However, the developmental landscapes of human spermatogenesis remain unknown. Here, we performed single-cell RNA sequencing (scRNA-seq) analysis for 2,854 testicular cells from donors with normal spermatogenesis and 174 testicular cells from one nonobstructive azoospermia (NOA) donor. A hierarchical model was established, which was characterized by the sequential and stepwise development of three spermatogonia subtypes, seven spermatocyte subtypes, and four spermatid subtypes. Further analysis identified several stage-specific marker genes of human germ cells, such as HMGA1, PIWIL4, TEX29, SCML1, and CCDC112. Moreover, we identified altered gene expression patterns in the testicular somatic cells of one NOA patient via scRNA-seq analysis, paving the way for further diagnosis of male infertility. Our work allows for the reconstruction of transcriptional programs inherent to sequential cell fate transition during human spermatogenesis and has implications for deciphering male-related reproductive disorders.