Abstract A022: Patho-DBiT: Spatially exploring RNA biology in archival formalin-fixed paraffin- embedded tissues
Authors: Bai Zhiliang , Zhang Dingyao , Gao Yan , Tao Bo , Li Mingyao , Xing Yi , Lu Jun , Xu Mina , Fan Rong 
Published: November 2024

Despite recent breakthroughs in spatial biology, either imaging-based or sequencing-based spatial transcriptomics is largely equivalent to spatial messenger RNA (mRNA) expression quantification. An RNA molecule experiences a complex life cycle involving transcription, splicing, maturation, translation, and degradation . It is highly desirable to profile all these RNA species spanning their life cycle to explore the biology of RNA at genome scale and cellular level. Furthermore, formalin-fixed paraffin-embedded (FFPE) tissues are essential in clinical practice, being the backbone of human disease histopathological diagnoses . Pathology departments have accrued vast collections of FFPE blocks over time, creating a rich, yet underutilized compendium of materials that, accompanied by clinical data, stands as a treasure trove for human biology and translational research . In this evolving landscape, we present “Pathology-compatible Deterministic Barcoding in Tissue” (Patho-DBiT) not only enabling spatial full-coverage base-by-base whole transcriptome sequencing but also crafted to address the challenges of clinically archived FFPE tissues. Patho-DBiT integrates in situ polyadenylation, microfluidic in tissue barcoding, and computational innovations to decode rich RNA biology inherent in FFPE samples. The platform capitalizes on RNA fragmentation naturally occurring in FFPE specimens and appends poly(A) tails to a broad spectrum of RNA species, thereby overcoming traditional barriers associated with FFPE samples and even outperforming the assays conducted with frozen tissues. Patho-DBiT permits spatial co-profiling of gene expression and alternative splicing, unveiling region-specific isoforms in the mouse brain. High-sensitivity transcriptomics is constructed from 5-year archived T-cell lymphoma tissues, with cross-validation conducted using super-resolution spatial phenotyping technology (CODEX). Furthermore, genome-scale single nucleotide RNA variants (SNVs) are captured to distinguish malignant subclones from non-malignant cells in human B-cell lymphomas, dissecting spatial clonal architectures based on both SNVs and copy number variation (CNV) profiles. Patho-DBiT also enables spatially resolved co-profiling of large and small RNAs, facilitating the analysis of a microRNA-mRNA regulatory network and tRNA utilization within clinical biopsies and elucidating their roles in tumorigenesis. With superior intronic read capture efficiency, Patho-DBiT spatially mapped RNA splicing dynamics associated with the developmental trajectory of tumor B cells. High resolution Patho-DBiT with a 10-μm spot size reveals the heterogeneities of human lymphomas within a spatial neighborhood and traces the spatiotemporal molecular kinetics driving tumorprogression at the cellular level. Patho-DBiT represents a first-of-its-kind technology, enabling the spatial exploration of rich RNA biology in FFPE tissues to aid in clinical pathology research.

Citation Format: Zhiliang Bai, Dingyao Zhang, Yan Gao, Bo Tao, Mingyao Li, Yi Xing, Jun Lu, Mina Xu, Rong Fan. Patho-DBiT: Spatially exploring RNA biology in archival formalin-fixed paraffin- embedded tissues [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A022.