Cell

Spatially exploring RNA biology in archival formalin-fixed paraffin-embedded tissues

Zhiliang Bai1,11 zhiliang.bai@yale.edu ∙ Dingyao Zhang2,3,11 ∙ Yan Gao4,11 ∙ Bo Tao5,11 ∙ Daiwei Zhang6,11 ∙ Shuozhen Bao1,11 ∙ Archibald Enninful1 ∙ Yadong Wang2 ∙ Haikuo Li1 ∙ Graham Su1 ∙ Xiaolong Tian1 ∙ Ningning Zhang2 ∙ Yang Xiao7 ∙ Yang Liu5 ∙ Mark Gerstein3 ∙ Mingyao Li6,8 mingyao@pennmedicine.upenn.edu ∙ Yi Xing4,8 xingyi@chop.edu ∙ Jun Lu2,9 jun.lu@yale.edu ∙ Mina L. Xu5 mina.xu@yale.edu ∙ Rong Fan

Abstract

The capability to spatially explore RNA biology in formalin-fixed paraffin-embedded (FFPE) tissues holds transformative potential for histopathology research. Here, we present pathology-compatible deterministic barcoding in tissue (Patho-DBiT) by combining in situ polyadenylation and computational innovation for spatial whole transcriptome sequencing, tailored to probe the diverse RNA species in clinically archived FFPE samples. It permits spatial co-profiling of gene expression and RNA processing, unveiling region-specific splicing isoforms, and high-sensitivity transcriptomic mapping of clinical tumor FFPE tissues stored for 5 years. Furthermore, genome-wide single-nucleotide RNA variants can be captured to distinguish malignant subclones from non-malignant cells in human lymphomas. Patho-DBiT also maps microRNA regulatory networks and RNA splicing dynamics, decoding their roles in spatial tumorigenesis. Single-cell level Patho-DBiT dissects the spatiotemporal cellular dynamics driving tumor clonal architecture and progression. Patho-DBiT stands poised as a valuable platform to unravel rich RNA biology in FFPE tissues to aid in clinical pathology evaluation.