Abstract 6169: High throughput in-situ spatial sequencing of proteins and RNA in FFPE tissue
Authors: Lawson Michael , Ishitsuka Yuji , Pawlowski Andrew , Hong Zhenmin , Koh Jake , Gouin Kenneth , Ing Nathan , Que Richard , Youn Yeoan , Ding Sara , Facchini Tony , Costello Ryan , Nelson Katelyn , Stover Jamie , Lee Howon , Duggal Sarthak , Plaziak Taylor , Cameron Kaitlin , Abdu Mimi , Witters Daan , Fabani Martin , Glezer Eli 
Published: March 2024

Abstract
We have developed an high throughput high resolution in situ multi-omic platform to enable studies in the rapidly evolving landscape of oncology and immunology research.
Despite the potential of existing tools, the speed and throughput of current methodologies pose significant limitations. Here, we introduce a novel spatial sequencing platform employing a rapid 4-color SBS chemistry with sub-micron resolution imaging and an ultra-high throughput capacity. The platform simultaneously profiles RNA transcripts and proteins within formalin-fixed paraffin-embedded (FFPE) tissues. With up to 40 cm^2 of tissue area imaging within a 24-hour timeframe, the platform can power extensive, multi-omic studies in cancer research.
To evaluate the performance of the platform, we prepared 5 um serial sections from healthy and tumor kidney FFPE block and used a novel tool to precisely position multiple tissue sections on a slide. Following probe binding and amplification, we used SBS readout to profile 105 transcripts and 6 proteins (via DNA-conjugated antibodies). Images were analyzed using a custom pipeline for feature detection and decoding, and cell segmentation using Cellpose. Results showed strong correlation to single-cell transcription profiles and cell type signatures defined by scRNA-Seq. Additionally, we see good agreement between single cell transcript and protein profiles. Existing spatial biology platforms are known to struggle with degraded and challenging clinical sample types (e.g. decalcified bone marrow), due to RNA fragmentation. We successfully profiled immune cells in FFPE-embedded bone marrow biopsies, measuring 143 transcript targets and 8 proteins. We believe this represents a significant step forward for in-situ analysis of bone marrow samples, and could be valuable for studying the pathology and response to treatment in blood cancers such as AML and CML.
In-situ sequencing of antibody genes and T-cell receptors provides a deeper view of cellular immune response. As a step towards this goal, we demonstrated in-situ sequencing of antibody heavy and light chains within a B cell line, resulting in accurate readout of 28 bases within the CDR3 regions.
In conclusion, we present an SBS-based approach for in-situ detection of targeted gene transcripts and proteins at an unprecedented throughput, and its application to the analysis of human kidney and bone marrow FFPE tissue samples. The capability to deeply profile tissues at high throughput could enable larger translational studies, and assembly of 3D maps of gene and protein expression at sub-cellular resolution.
Citation Format: Michael Lawson, Yuji Ishitsuka, Andrew Pawlowski, Zhenmin Hong, Jake Koh, Kenneth Gouin, Nathan Ing, Richard Que, Yeoan Youn, Sara Ding, Tony Facchini, Ryan Costello, Katelyn Nelson, Jamie Stover, Howon Lee, Sarthak Duggal, Taylor Plaziak, Kaitlin Cameron, Mimi Abdu, Daan Witters, Martin Fabani, Eli Glezer. High throughput in-situ spatial sequencing of proteins and RNA in FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6169.