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Pan-cancer single-nucleus total RNA sequencing using HH-seq

ABSTRACT: Tumor heterogeneity and its drivers impair tumor progression and cancer therapy. Single-cell RNA sequencing has been used to investigate the heterogeneity of tumor ecosystems. However, most methods of scRNA-seq amplify the termini of polyadenylated transcripts, making it challenging to perform total RNA analysis and somatic mutation analysis during tumor processing. Additionally, frozen tumor samples constitute a vast and valuable material bank for cancer research. Therefore, we developed a high-throughput and high-sensitivity method called HH-seq, which combines random primers and a pre-index strategy in the droplet microfluidic platform. This innovative method allows for the detection of total RNA in single nuclei from clinically frozen samples. We also established a robust pipeline to facilitate the analysis of full-length RNA-seq data. We applied HH-seq to more than 730,000 single nuclei from 32 patients with various tumor types. The pan-cancer study enables us to comprehensively profile data on the tumor transcriptome, including expression levels, mutations, splicing patterns and clone dynamic, etc. We identified new malignant cell subclusters and explored their specific function across cancers. Furthermore, we investigated the malignant status of epithelial cells among different cancer types with respect to mutation and splicing patterns. The ability to detect full-length RNA at the single-nucleus level provides a powerful tool for studying complex biological systems and has broad implications for understanding tumor pathology.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE237166 | GEO | 2023/11/04

REPOSITORIES: GEO

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Project description:Misactivation of the Hedgehog (Hh) pathway can cause cancers such as medulloblastomas, the most common malignant brain tumor in children, and basal cell carcinomas, the most common cancer in the United States. Hedgehog signals are transmitted through primary cilia, where Hedgehog ligands bind to Patched1 and activate Smoothened through interactions with cilia-associated sterol lipids. The gene expression programs driving cellular responses to ciliary Hh signals are incompletely understood. Thus, to define Hh target genes, we performed RNA sequencing of cells after treatment with Hh ligands (Shh, Dhh, Ihh), cilia-associated lipids (7b,27-dihydroxycholesterol, 24(S),25-epoxycholesterol), or synthetic lipids or small molecules that activate Smoothened (20(S)-hydroxycholesterol, SAG). Treatment with Hh pathway agonists identified a core gene expression program comprised of 155 genes driving lipid synthesis, metabolism, signaling, adhesion, or angiogenesis. These datasets were integrated with RNA sequencing of Hh-human medulloblastomas (n=?), a Math1-Cre SmoM2 mouse genetic model of Hh-associated medulloblastoma (n=?), and human basal cell carcinomas (n=10) to ascertain how malignant Hh signaling differs from canonical Hh signaling. We discover a conserved response to ciliary Hh signals in human or mouse medulloblastomas, including known target genes such as Gli1 or Ptch1, and novel target genes such as Hsd11b1 or Retnla. Importantly, mechanistic studies reveal Hsd11b1 to be a putative negative regulator of Hh signaling that is dysregulated in malignancies. We further demonstrate Retnla to be a positive regulator of Hh signaling that drives expression of Hsd11b2, a druggable dependency underlying Hedgehog-associated medulloblastoma. Orthotopic implantation of neuroepithelial stem cells that overexpress either Hsd11b1 and Retnla demonstrate that tumors derived Hsd11ß1 overexpression are more primitive and less aggressive whereas Retnla overexpression forms tumors that are more differentiated and behave more aggressively. In sum, we illuminate the first comprehensive transcriptome of Hh signaling and highlight the intricate interplay between Hh signaling and lipid metabolism that Hh-dependent malignancies dysregulate to drive tumor progression.

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Pan-cancer single-nucleus total RNA sequencing using HH-seq

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