@lcolladotor lcolladotor.github.io lcolladotor.github.io/bioc_team_ds Applying Visium Spatial Proteogenomics (Visium-SPG) to study Alzheimer’s Disease Leonardo Collado Torres #Visium_SPG_AD May 17th 2023 #GBD23 Slides available at speakerdeck.com/lcolladotor

twitter.com/sanghokwon17/status/1650589385379962881 from 2023-04-24 Sang Ho Kwon @sanghokwon17 DOI: 10.1101/2023.04.20.537710 #Visium_SPG_AD

DOI: 10.1038/s41593-020-00787-0 twitter.com/lcolladotor/status/1233661576433061888 from 2020-02-29

DOI: 10.1038/s41593-020-00787-0 twitter.com/lcolladotor/status/1233661576433061888 from 2020-02-29 DOI 10.1093/bioinformatics/btac299 Since Feb 2020 spatialLIBD::fetch_data() provides access to SpatialExperiment R/Bioconductor objects

DOI: 10.1038/s41593-020-00787-0 twitter.com/lcolladotor/status/1233661576433061888 from 2020-02-29 twitter.com/CrowellHL/status/1597579271945715717 DOI 10.1093/bioinformatics/btac299 Since Feb 2020 spatialLIBD::fetch_data() provides access to SpatialExperiment R/Bioconductor objects

Visium spatial clustering works for variables with high % variance explained. But what about other ones? DOI: 10.1038/s41593-020-00787-0

Alzheimer’s disease related neuropathology Adopted and modified from B Wang (2018) and the Brain from the Top to Bottom in McGill University 20 um Sang Ho Kwon Amyloid beta pTau

Hypothesis Local tissue microenvironments in close proximity to AD-related neuropathology have distinct cellular and molecular signatures. Sang Ho Kwon

Visium Spatial Proteogenomics (Visium-SPG) Visium-SPG = Visium SRT + immunofluorescence (using identical tissue samples) Sang Ho Kwon

Experimental design & study overview Braak V-VI & CERAD frequent Sang Ho Kwon

AD pathology signal is too small to detect by spatially-resolved gene expression alone research.libd.org/Visium_SPG_AD/

Visium * ~5k spots in honeycomb * gene expression per spot * tissue (H&E staining) Immunofluorescence (IF) * multi-channel (6) images * identifies morphological features of interest * large: might be broken in tiles Channel 1 * triangle feature Channel 2 * cloud feature Channel 6 * xyz feature Tissue (bright field image) Visium spot Channel 1 feature Channel 2 feature + Visium Spatial Proteogenomics (Visium-SPG / IF) raw data: 2 types

Spot ID Gene 1 Gene 2 Gene X spot0001 0 12 39 spot0002 4 0 27 Spot ID Gene 1 Gene 2 Gene X In Tissue # cells spot0001 0 12 39 true 3 spot0002 4 0 27 false 0 * spaceranger * Loupe Browser * VistoSeg on H&E bright field image Visium Analysis @MadhaviTippani @HeenaDivecha cell VistoSeg DOI: 10.1101/2021.08.04.452489

Feature ID X center Y center type intensity feat0001 5 102 triangle 130.4 feat0002 10 30 cloud 99.1 Max (X, Y) Min (X, Y) Area ... SPG / IF Image Analysis * segment each channel * find features Challenges: * morphological features can be quite diverse * images are large * multiple tiles +

Spot ID # Triangle # Cloud % triangle % cloud spot0001 0 12 0 17 spot0002 4 0 27 0 Merge Visium & IF IF Spot ID Gene 1 Gene 2 Gene X In Tissue # cells spot0001 0 12 39 true 3 spot0002 4 0 27 false 0 Visium downstream * QC * analyses spatialLIBD DOI: 10.1186/s12864-022-08601-w VistoSeg DOI: 10.1101/2021.08.04.452489 Check version 2!! @MadhaviTippani

Estimating pathological burden per spot to generate transcriptome-scale maps of AD pathology

Identifying transcriptional signatures of AD-related neuropathology Principal component analysis Sang Ho Kwon

Identifying transcriptional signatures of Abeta-related neuropathology Sang Ho Kwon

Validation study design using RNAscope FISH-IF Sang Ho Kwon

bioconductor.org/packages/spatialLIBD Pardo et al, 2022 DOI 10.1186/s12864-022-08601-w Maynard, Collado-Torres, 2021 DOI 10.1038/s41593-020-00787-0 Brenda Pardo Abby Spangler @PardoBree @abspangler Louise A. Huuki-Myers @lahuuki Check her #GDB2023 poster for more! Session 1: #7

Conclusions • Visium-SPG can identify local transcriptional signatures of late-stage AD pathology • RNAscope FISH-IF can further explore spatial gene expression patterns of a few select features • Proof-of-concept study: our work lays the groundwork. Can now use Visium-SPG for other diseases too • Future studies are needed to produce mechanistic insights • Explore our code & data at research.libd.org/Visium_SPG_AD & download it with spatialLIBD::fetch_data() ○ There’s lots more to explore with this data! Sang Ho Kwon Slide adapted from:

Contributors for this project Investigation Data analysis Mentorship Web resources Sang Ho Kwon @sanghokwon17 #Visium_SPG_AD In collaboration with Slides available at speakerdeck.com /lcolladotor Download data with spatialLIBD::fetch_data() Madhavi Tippani Keri Martinowich Kristen Maynard Sowmya Parthiban Jashandeep Lobana Stephanie Hicks Stephanie Page Heena Divecha Nicholas Eagles

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Identifying transcriptional signatures of Abeta microenvironment VS Neural plasticity* Previously not significant Sang Ho Kwon

6 Abeta-associated DEGs on RNAscope FISH-IF (Kruskal-Wallis test, *p<0.05, &p<0.005, and #p<0.0001) Sang Ho Kwon

Visium + RNAscope to provide additional insights into spatial gene expression gradients Sang Ho Kwon