ARC-Seq (Assay for single-nucleus RNA-seq and ATAC-seq in the same nucleus from a single cell) is a technology built upon scATAC-seq, enabling the simultaneous capture of both the single-nucleus transcriptome and single-cell ATAC information from within the same cell.
Based on 10X Genomics ChromiumTM dynamic microfluidic technology, it utilizes Tn5 transposase to digest open chromatin, forming short fragments.
Single-cell ATAC sequencing, at the epigenomic level, reveals chromatin accessibility in individual cells, distinguishes cellular heterogeneity, and obtains information such as the location of open chromatin, transcription factor binding sites, nucleosome regulatory regions, and chromatin states.
It represents a significant breakthrough in single-cell epigenetics.

Image Source: Buenrostro et al. (2013) Nature Methods,10(12), 1214. doi:10.1038/nmeth.2688

1.Breakthrough Nuclei Preparation Method:Skips the sample digestion step, performing nuclei preparation directly from frozen tissue. This method can significantly save time and resources, improve nuclei preparation efficiency, and achieve a nuclei capture rate as high as 65%-80%.
2.Elimination of Cell Dissociation Bias:Eliminates bias caused by enzymatic digestion preferences, ensuring nuclei quality far exceeds the requirements for loading onto the platform, thereby providing more accurate results for subsequent analysis.
3.Strict Quality Control:Lieven implements strict quality control throughout the entire process, providing a one-stop service workflow from experimental design to analytical output, ensuring customers obtain high-quality data and analysis results, and providing comprehensive support and solutions.
4.Lieven's Self-Developed Analysis Cloud Platform:Enables one-click data analysis, including peak calling, clustering, TF-motif analysis, cell subpopulation identification, marker gene discovery, and signaling pathway enrichment analysis.

1.Fresh/Frozen tissue, blood, cultured cell lines, etc.
2.Initial cell count greater than 500,000.
3.Fresh samples: Viability >80% after digestion.
4.Frozen tissue: Snap-frozen in liquid nitrogen and stored at -80°C.

Experimental Groups:
Cohort 1：Prostate tissues from 7 patients.
scRNA - seq: Central zone (CZ) : Transition zone (TZ) : Peripheral zone (PZ) = 10 : 11 : 12
Cohort 2：Prostate tissues from 4 patients.
1.Single - cell ARC - seq: CZ : TZ : PZ = 4 : 4 : 4
2.Spatial Transcriptome sequencing: CZ : TZ : PZ = 1 : 1 : 1

Single - Cell Capture Platforms：
BD Rhapsody, 10x Genomics

Primary Technical Methods：
Single - cell RNA sequencing (scRNA - seq), Single - cell ARC - seq (snRNA - seq + snATAC - seq), Spatial Transcriptomics (Visium), Immunohistochemistry (IHC), Multiplex Immunofluorescence (IF).

Analysis Methods：
Cell clustering analysis, Spatial feature expression analysis, Trajectory and functional enrichment analysis, Cell - cell interaction analysis, Copy number variation (CNV) analysis.

The prostate is an organ with significant spatial heterogeneity. To better understand its complex structure and cellular composition, this study constructed a comprehensive single-cell atlas of the adult human prostate. The prostate epithelium primarily consists of luminal cells, basal cells, and neuroendocrine cells. Although scRNA-seq has been widely used to study mouse prostate and malignant human prostate tissues, the characterization of the normal human prostate remains unclear. Previous studies often overlooked its regional specificity and mesenchymal heterogeneity, highlighting the need for a more comprehensive cellular atlas. Numerous studies have explored the cellular origins of prostate cancer, but the specific cell type(s) remain unclear, with multiple theories existing. Recent research suggests rare prostate stem cells might be the disease origin. This study utilized droplet-based scRNA-seq, single-nucleus multi-omics, and spatial transcriptomics to identify 126 distinct cell subpopulations, most of which are newly discovered. Through integrated multi-omics analysis, it revealed potential cellular origins of prostate cancer, significantly expanding the understanding of human prostate cellular composition.

1.Cell Types in the Adult Human Prostate:

After single-cell sequencing and rigorous quality control, transcriptome profiles of 253,381 high-quality single cells and 34,876 nuclei were obtained for downstream analysis. Initial annotation identified 15 major cell types defined by marker genes. Compared to single-nucleus strategies, single-cell methods captured more immune cells but fewer epithelial cells, indicating differential sensitivity of cell types to enzyme-based dissociation. Further subdivision of cell lineages identified 126 distinct cell types.

2.Distribution of Prostate Epithelial Cell Subpopulations:

Further clustering analysis of the two sequencing datasets yielded 35 and 19 subclusters, respectively. These cells were grouped into three main populations: traditional KRT5+ (CK5+) basal cells and DPP4+ (CD26+) luminal/acinar cells, and a unique KRT5−DPP4− epithelial population, which the authors termed "ductal Luminal (dLum) cells". Analysis showed high similarity between the same cell subpopulations across both cohorts, indicating high accuracy of cell type annotation, while cell distribution across different zones showed significant heterogeneity.

1.dLum Cell Subpopulations and Spatial Localization

Further scRNA-seq analysis divided dLum cells into seven subpopulations. Among them, d1_dLum–Club cells expressed SCGB1A1 and SCGB3A1, resembling club cells; d2_dLum–KRT4 cells expressed KRT4 (CK4) and KRT13 (CK13), associated with multipotent luminal cells and hillock cells. About one-quarter of basal cells also expressed KRT13, suggesting hillock cells are a heterogeneous population.

Spatial transcriptomics analysis showed d1_dLum-Club cells located near the urethral ducts, adjacent to d2_dLum-KRT4 cells, while KRT4−KRT13+ basal cells (B1_Basal-ESR1 and B3_Basal-GPRC5A) were scattered, and the distribution of the two basal cell types was mutually exclusive, revealing a multi-layered structure around the prostate ducts.

Multiplex IF and IHC staining further validated these findings, showing club cells and CK4+ cells located on the inner side of the urethral lumen, while CK4− and CK13+ basal cells were on the basal side. Small numbers of CK4+ and CK4−CK13+ basal cells were also detected in peripheral acini, with CK4+ cells scattered between luminal and basal cells.

Furthermore, d3_dLum-LTF cells were located at the ends of the multilayered epithelium, forming columnar epithelial tubes and connecting with L1_Lum-LTF. d6_dLum-CNMD was a rare gland-distributed cell type, while d7_dLum-SPIB cells had potential antigen-presenting capacity, showing high levels of MHC molecules. These findings reveal the heterogeneity and functional diversity of dLum cell subpopulations.

2.Basal Cell Subpopulations and Spatial Localization:

Analysis revealed two KRT5+ basal cell lineages (KRT16+KRT17+ and KRT16−KRT17−), supported by snATAC-seq data. Krt4+ luminal cells have been proposed as potential stem cells capable of regenerating both prostate luminal and basal cells. Based on the transcriptomic properties and spatial distribution of club cells, CK4+ cells, and CK4−CK13+ basal cells, the study proposed a potential differentiation pathway, supported by multi-trajectory analysis. Transcription factor analysis revealed differential activation along the two differentiation trajectories, explaining the higher levels of cellular stress response factors in the KRT17+ branch.

B5_Basal-VCAN cells were the only basal subpopulation predominantly located in the peripheral and transition zones. Spatial transcriptomics showed these cells located at the base of acini but absent from the central regions of TGM4+ acini and peri-urethral ducts. These cells expressed WNT regulators (e.g., WIF1, SFRP1), potentially playing a role in the development and maintenance of prostate cell morphology.

Additionally, a small branch of the KRT16+ KRT17+ lineage, through a FOXI1+ state and expression of proton pump proteins, resembled renal intercalated cells and bronchial ionocytes, suggesting a potential function in ion reabsorption in prostate fluid. Trajectory analysis showed gradual loss of basal signaling pathways and increased ion pump expression during differentiation, suggesting basal cells might be the source of ionocytes in the prostate. IF staining further confirmed these cells were primarily located in glandular structures surrounding the urethra.

Using spatial transcriptomics, the study validated the existence of prostate glands with unique luminal features harboring CNAs and explored their relationship with prostate tumorigenesis. Analysis of TCGA-PRAD RNA-seq data found that Type 2 and SFTPA2+ signatures were common features of ETS− prostate cancer, independent of mutational driver type. Other epithelial signatures (e.g., basal, Type 1 luminal) correlated negatively with tumor purity, indicating their non-malignant identity. A similar phenomenon was observed in the Asian CPGEA cohort, suggesting ETS− tumors might originate from specific cell types. Single-cell RNA-seq data further supported these findings, with 10 out of 11 cancer cases exhibiting the Type 2 signature, 4 of which also showed the SFTPA2+ signature. The single case exhibiting a Type 1 Lum cell signature was driven by ETV4, consistent with the bulk RNA-seq results.

Spatial transcriptomics slices provided direct evidence of the transition from Type 2 preneoplastic lesions to cancer. Clone 1 (normal) and Clone 2 (tumor) both expressed the Type 2 signature, while Clone 3 had different CNAs, suggesting it might be an early branch or independent lesion. These findings support the hypothesis that Type 2 prostate cancer originates from Type 2 acini.

Furthermore, the study found multiple independent Type 2 tumors and SFTPA2+ tumors, with distinct CNA patterns and clonal origins. Each lesion was surrounded by normal Type 1 luminal cells.

By employing single-cell sequencing, spatial transcriptomics, and integrated multi-omics analysis, this study constructed a high-resolution atlas of the adult human prostate, revealing its cellular composition, spatial heterogeneity, and potential cellular origins of prostate cancer. Key findings include:
1.Identification of 126 unique cell subtypes, including multiple novel ductal luminal and basal cell subtypes.
2.Discovery that Type 2 and SFTPA2+ luminal cells may be the common cell of origin for ETS fusion-negative prostate cancer.
3.Revelation of the regional specificity and function of fibroblasts, particularly their role in prostate epithelial cell differentiation.

Link to Original Article:https://doi.org/10.1038/s41588-025-02139-9