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High resolution mapping of the breast cancer tumor microenvironment using integrated single cell, spatial and in situ analysis of FFPE tissue

View ORCID ProfileAmanda Janesick, Robert Shelansky, Andrew D. Gottscho, Florian Wagner, Morgane Rouault, Ghezal Beliakoff, Michelli Faria de Oliveira, Andrew Kohlway, Jawad Abousoud, Carolyn A. Morrison, Tingsheng Yu Drennon, Seayar H. Mohabbat, Stephen R. Williams, 10x Development Teams, Sarah E.B. Taylor
doi: https://doi.org/10.1101/2022.10.06.510405
Amanda Janesick
10x Genomics, Pleasanton, CA
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  • ORCID record for Amanda Janesick
Robert Shelansky
10x Genomics, Pleasanton, CA
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Andrew D. Gottscho
10x Genomics, Pleasanton, CA
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Florian Wagner
10x Genomics, Pleasanton, CA
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Morgane Rouault
10x Genomics, Pleasanton, CA
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Ghezal Beliakoff
10x Genomics, Pleasanton, CA
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Michelli Faria de Oliveira
10x Genomics, Pleasanton, CA
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Andrew Kohlway
10x Genomics, Pleasanton, CA
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Jawad Abousoud
10x Genomics, Pleasanton, CA
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Carolyn A. Morrison
10x Genomics, Pleasanton, CA
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Tingsheng Yu Drennon
10x Genomics, Pleasanton, CA
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Seayar H. Mohabbat
10x Genomics, Pleasanton, CA
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Stephen R. Williams
10x Genomics, Pleasanton, CA
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10x Genomics, Pleasanton, CA
Sarah E.B. Taylor
10x Genomics, Pleasanton, CA
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  • For correspondence: correspondence@10xgenomics.com
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Abstract

Single cell and spatial technologies that profile gene expression across a whole tissue are revolutionizing the resolution of molecular states in clinical tissue samples. Commercially available methods that characterize either single cell or spatial gene expression are currently limited by low sample throughput and/or gene plexy, lack of on-instrument analysis, and the destruction of histological features and epitopes during the workflow. Here, we analyzed large, serial formalin-fixed, paraffin-embedded (FFPE) human breast cancer sections using a novel FFPE-compatible single cell gene expression workflow (Chromium Fixed RNA Profiling; scFFPE-seq), spatial transcriptomics (Visium CytAssist), and automated microscopy-based in situ technology using a 313-plex gene panel (Xenium In Situ). Whole transcriptome profiling of the FFPE tissue using scFFPE-seq and Visium facilitated the identification of 17 different cell types. Xenium allowed us to spatially resolve these cell types and their gene expression profiles with single cell resolution. Due to the non-destructive nature of the Xenium workflow, we were able to perform H&E staining and immunofluorescence on the same section post-processing which allowed us to spatially register protein, histological, and RNA data together into a single image. Integration of data from Chromium scFFPE-seq, Visium, and Xenium across serial sections allowed us to do extensive benchmarking of sensitivity and specificity between the technologies. Furthermore, data integration inspired the interrogation of three molecularly distinct tumor subtypes (low-grade and high-grade ductal carcinoma in situ (DCIS), and invasive carcinoma). We used Xenium to characterize cellular composition and differentially expressed genes within these subtypes. This analysis allowed us to draw biological insights about DCIS progression to infiltrating carcinoma, as the myoepithelial layer degrades and tumor cells invade the surrounding stroma. Xenium also allowed us to further predict the hormone receptor status of tumor subtypes, including a small 0.1 mm2 DCIS region that was triple positive for ESR1 (estrogen receptor), PGR (progesterone receptor), and ERBB2 (human epidermal growth factor receptor 2, a.k.a. HER2) RNA. In order to derive whole transcriptome information from these cells, we used Xenium data to interpolate the cell composition of Visium spots, and used Visium whole transcriptome information to discover new biomarkers of breast tumor subtypes. We demonstrate that scFFPE-seq, Visium, and Xenium independently provide information about molecular signatures relevant to understanding cancer heterogeneity. However, it is the integration of these technologies that leads to even deeper insights, ushering in discoveries that will progress oncology research and the development of diagnostics and therapeutics.

Competing Interest Statement

All authors are employees and shareholders of 10x Genomics.

Footnotes

  • ↵* see Supplemental Information

  • Addition of more supplemental information.

  • https://www.10xgenomics.com/products/xenium-in-situ/preview-dataset-human-breast

  • https://www.10xgenomics.com/products/xenium-in-situ/human-breast-dataset-explorer

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted November 03, 2022.
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High resolution mapping of the breast cancer tumor microenvironment using integrated single cell, spatial and in situ analysis of FFPE tissue
Amanda Janesick, Robert Shelansky, Andrew D. Gottscho, Florian Wagner, Morgane Rouault, Ghezal Beliakoff, Michelli Faria de Oliveira, Andrew Kohlway, Jawad Abousoud, Carolyn A. Morrison, Tingsheng Yu Drennon, Seayar H. Mohabbat, Stephen R. Williams, 10x Development Teams, Sarah E.B. Taylor
bioRxiv 2022.10.06.510405; doi: https://doi.org/10.1101/2022.10.06.510405
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High resolution mapping of the breast cancer tumor microenvironment using integrated single cell, spatial and in situ analysis of FFPE tissue
Amanda Janesick, Robert Shelansky, Andrew D. Gottscho, Florian Wagner, Morgane Rouault, Ghezal Beliakoff, Michelli Faria de Oliveira, Andrew Kohlway, Jawad Abousoud, Carolyn A. Morrison, Tingsheng Yu Drennon, Seayar H. Mohabbat, Stephen R. Williams, 10x Development Teams, Sarah E.B. Taylor
bioRxiv 2022.10.06.510405; doi: https://doi.org/10.1101/2022.10.06.510405

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