Single Cell Analysis of Regions of Interest (SCARI) using a Novel Photoswitchable Tag

Single Cell Analysis of Regions of Interest (SCARI) using a Novel Photoswitchable Tag | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Single Cell Analysis of Regions of Interest (SCARI) using a Novel Photoswitchable Tag Anne van der Leun, Mirjam Hoekstra, Luuk Reinalda, Colinda Scheele, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-71508/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Sep, 2021 Read the published version in Nature Chemical Biology → Version 1 posted You are reading this latest preprint version Abstract The functional activity and differentiation potential of cells is determined by their interaction with surrounding cells. Approaches that allow the unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a trade-off between spatial resolution and cell profiling depth. Here, we developed a photoswitch-based technology that allows the isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran)-cage coupled to nanobodies allowed photoswitching of cells in areas of interest with low-intensity violet light and without detectable phototoxicity. Single cell RNA sequencing of spatially defined CD8 + T cells was used to exemplify the feasibility of identifying location-dependent cell states at the single cell level. Finally, we demonstrate the efficient labeling and photoswitching of cells in live primary human tumor tissue. The technology described here provides a valuable tool for the analysis of spatially defined cells in diverse biological systems, including clinical samples. Immunology Chemical Biology photoswitching unbiased characterization spatial resolution profiling depth Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF. Additional Declarations There is NO Competing Interest. Supplementary Files VanderLeunTableS1.xlsx VanderLeunTableS2.xlsx VanderLeunTableS3.xlsx VanderLeunsupplfig1.pdf VanderLeunsupplfig2.pdf VanderLeunsupplfig3.pdf VanderLeunsupplfig4.pdf VanderLeunsupplfig5.pdf VanderLeunsupplfig6.pdf VanderLeunsupplfig7.pdf Cite Share Download PDF Status: Published Journal Publication published 09 Sep, 2021 Read the published version in Nature Chemical Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-71508","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":2275106,"identity":"62237a00-77ce-42ad-9df1-196e96a00d33","order_by":0,"name":"Anne van der Leun","email":"","orcid":"https://orcid.org/0000-0003-1718-2093","institution":"Netherlands Cancer Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anne","middleName":"van der","lastName":"Leun","suffix":""},{"id":2275107,"identity":"de999c53-f99f-4528-84f8-f70af5900e4b","order_by":1,"name":"Mirjam Hoekstra","email":"","orcid":"","institution":"Netherlands Cancer Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mirjam","middleName":"","lastName":"Hoekstra","suffix":""},{"id":2275108,"identity":"9206fa8b-e93b-41cd-93e3-079f5ee6648c","order_by":2,"name":"Luuk Reinalda","email":"","orcid":"","institution":"Leiden Institute of Chemistry","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Luuk","middleName":"","lastName":"Reinalda","suffix":""},{"id":2275109,"identity":"1f08fb85-66ea-4e41-a45a-381142bb23d0","order_by":3,"name":"Colinda Scheele","email":"","orcid":"https://orcid.org/0000-0001-8999-5451","institution":"VIB-KU Leuven Center for Cancer Biology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Colinda","middleName":"","lastName":"Scheele","suffix":""},{"id":2275110,"identity":"615e0882-ebb2-4b58-885a-af61a81d9ca0","order_by":4,"name":"Mireille Toebes","email":"","orcid":"","institution":"Division of Immunology, Netherlands Cancer Institute, Amsterdam","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mireille","middleName":"","lastName":"Toebes","suffix":""},{"id":2275111,"identity":"7fae1ffe-a85b-4502-b7a7-3f3e92d1b484","order_by":5,"name":"Michel van de Graaff","email":"","orcid":"","institution":"Leiden Institute of Chemistry","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Michel","middleName":"van","lastName":"de Graaff","suffix":""},{"id":2275112,"identity":"a4867813-c3c8-424b-b0ff-ddd427cadfdf","order_by":6,"name":"Hanjie Li","email":"","orcid":"","institution":"Weizmann Institute of Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hanjie","middleName":"","lastName":"Li","suffix":""},{"id":2275113,"identity":"300a9a51-2628-49b7-9958-392f747815d7","order_by":7,"name":"Akhiad Bercovich","email":"","orcid":"","institution":"Weizmann Institute of Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akhiad","middleName":"","lastName":"Bercovich","suffix":""},{"id":2275114,"identity":"e0a043e7-a6cb-4fe5-a6fd-7c440f40d51b","order_by":8,"name":"Yaniv Lubling","email":"","orcid":"https://orcid.org/0000-0003-3461-9621","institution":"Weizmann Institute of Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yaniv","middleName":"","lastName":"Lubling","suffix":""},{"id":2275115,"identity":"f9651cd2-bf68-454d-8232-ffa5c4520cc3","order_by":9,"name":"Eyal David","email":"","orcid":"","institution":"Weizmann Institute of Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Eyal","middleName":"","lastName":"David","suffix":""},{"id":2275116,"identity":"cd263d96-1214-4bf3-8b9d-823089b9b72d","order_by":10,"name":"Daniela Thommen","email":"","orcid":"https://orcid.org/0000-0002-7431-2854","institution":"Netherlands Cancer Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"","lastName":"Thommen","suffix":""},{"id":2275117,"identity":"e6f18412-ce9e-4f46-8e88-05f9e6348cd5","order_by":11,"name":"Amos Tanay","email":"","orcid":"","institution":"Weizmann Institute of Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amos","middleName":"","lastName":"Tanay","suffix":""},{"id":2275118,"identity":"a27b8eb0-9e40-454d-b84f-5ac086d6e503","order_by":12,"name":"Jacco van Rheenen","email":"","orcid":"","institution":"Antoni van Leeuwenhoek Hospital/Netherlands Cancer Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jacco","middleName":"van","lastName":"Rheenen","suffix":""},{"id":2275119,"identity":"3fe92807-3e58-430f-8d2c-fd7b41995fa0","order_by":13,"name":"Ido Amit","email":"","orcid":"","institution":"Weizmann Institute of Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ido","middleName":"","lastName":"Amit","suffix":""},{"id":2275120,"identity":"449ccc04-0d6b-48f7-803e-64ee3717ac98","order_by":14,"name":"Sander van Kasteren","email":"","orcid":"https://orcid.org/0000-0003-3733-818X","institution":"Leiden University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sander","middleName":"van","lastName":"Kasteren","suffix":""},{"id":2275121,"identity":"dadc78ce-a308-47dd-83f9-0a77e75fed1f","order_by":15,"name":"Ton Schumacher","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYJACZjApAcQfGBJ4gPwGvMp5kLUwzgBrYSRBCzMPQwIDQS32DOwPPxe2bZPnn9187LPtjjQZBulGQrbwGEvPbLttOOPOseTZuWdyeBhkDhLUwsbM23abcYNEjjFzblsFD4NEIiEt7M9AWuw3SOR/ZrYkTguDGUhLItAWZmbGthwitBwG+oXn3O3kGTfSjBl729J42AhpYW9vf/iZp+y2bf+M5McMP9uS7fklkg/g1QKNFCTAhl/9KBgFo2AUjAJiAACdWDrqkXzWpAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0517-8804","institution":"The Netherlands Cancer Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ton","middleName":"","lastName":"Schumacher","suffix":""}],"badges":[],"createdAt":"2020-09-03 11:25:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-71508/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-71508/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41589-021-00839-x","type":"published","date":"2021-09-09T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":2416989,"identity":"1ed51e4e-8e5d-494f-bf9b-60f3037efe72","added_by":"auto","created_at":"2020-09-15 15:42:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":70367,"visible":true,"origin":"","legend":"Synthesis of the photoswitchable tag (PsT)\na) 1% TFA, DCM, room temperature, 30 minutes (2x); b) 12, DiPEA, DMF, room temperature , 18 hours; c) 20% piperidine, DMF, room temperature, 2 minutes (3x), d) TFA, TIS, H2O 95/2.5/2.5, room temperature, 3 hours; e) CuSO4, sodium ascorbate, THPTA, AF594-N3, H2O, t-BuOH room temperature, 2 hours.\n ","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/fig1.png"},{"id":2416990,"identity":"fea5c207-7103-4cf7-a8be-80f4743d5aff","added_by":"auto","created_at":"2020-09-15 15:42:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99514,"visible":true,"origin":"","legend":"Schematic approach for single cell analysis in regions of interest\nA) Schematic representation of the experimental approach for spatial analysis of cells. Nanobodies are labeled with the photoswitchable tag (from here on referred to as PsT) that consists of a FLAG-tag, the NPBF photosensitive cage, and the AF594 fluorochrome. Cells or tissues are stained with PsT-labeled nanobody and PsT stained cells in areas of interest are uncaged using 405nm violet light. After cell harvest or tissue dissociation, single cell suspensions are stained with an αFLAG antibody that only binds to the uncaged FLAG-tag. Cells from the area of interest can thus be identified on the basis of both their low AF594 signal and high FLAG signal. Isolation of cells based on both properties (i.e. AF594low FLAGhigh cells as cells within the area of interest, and AF594high FLAGlow cells as cells outside the area of interest) results in an optimal separation between the two cell populations. \nB) Binding stability of αCD8 nanobodies to CD8+ cells. Top panel: a first population of CD8+ T cells was stained with FITC labeled monomeric nanobody (αCD8M-FITC) and a second population was stained with αCD8M-AF647. Bottom panel: a first population of CD8+ T cells was stained with FITC labeled dimeric nanobody containing recognition domains that are identical to the αCD8M (αCD8D-2-FITC) and a second population was stained with αCD8D-2-AF647. Subsequently, two cell populations labeled with either monomeric or dimeric αCD8 reagents were mixed, incubated at 37oC, and exchange of αCD8 nanobodies was measured by flow cytometry. Results for a second nanobody clone αCD8D-1 are depicted in Fig. S2. \nC) Specific binding of αFLAG antibody clone D6W5B to the uncaged FLAG-tag. Cells stained with CD8D-1 nanobody conjugated to either the FLAG-tag without cage (CD8D-1-T, upper panel) or the FLAG-tag containing the NPBF cage (CD8D-1-PsT, bottom panel) were stained with αFLAG antibody clone D6W5B. Note that appreciable AF488 signal is only observed when the uncaged FLAG tag is used. Representative data from three independent experiments are depicted.\n ","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/fig2.png"},{"id":2416991,"identity":"f5edcc3f-1a27-43bf-bc18-e74e47e1251a","added_by":"auto","created_at":"2020-09-15 15:42:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":273848,"visible":true,"origin":"","legend":"Uncaging of PsT-labeled primary human CD8+ T cells\nA). Left: Confocal image of CD8+ T cells stained with CD8D-1-PsT and CD8D-2-FITC, shown in magenta and green respectively. Overlapping green and magenta signal is shown in white. Area in box with solid line is enlarged in the bottom panel, and shows membrane staining with both αCD8D nanobodies. Scale bars represent 100 μm (top panel) and 10 μm (bottom panel). Right: Flow cytometry analysis of αCD8D-1-PsT labeled CD8+ T cells after αFLAG staining. Representative data from three independent experiments are depicted.\nB) Left: Confocal image of CD8+ T cells stained with αCD8D-1-PsT and αCD8D-2-FITC, shown in magenta and green respectively, as in A. Overlapping green and magenta is shown in white. Box with dotted line indicates area of uncaging by 405 nm light exposure. Area in box with solid line is enlarged in the bottom panel, and shows uncaging in the top half, as detected by loss of AF594 signal (magenta). Scale bars represent 100 μm (top panel) and 10 μm (bottom panel). Right: Flow cytometry analysis of CD8+ T cells following uncaging of cells in the indicated area and subsequent staining of cells with αFLAG antibody. Note the separation between uncaged and caged CD8+ T cells based on both the αCD8D-1-PsT and αFLAG signal. Representative data from three independent experiments are depicted. \nC) Viability of uncaged (AF594low FLAGhigh) and caged (AF594high FLAGlow) CD8+ T cells from partly uncaged samples (as in Fig. 3B), as measured by flow cytometric analysis of cells upon IR-dye live-dead staining. Representative data of three independent experiments are depicted, including one technical replicate.\nD) Correlation between uncaged surface area and the observed fraction uncaged αCD8D-1-PsT labeled CD8+ T cells in peripheral blood mononuclear cells (PBMCs). 0%, 50%, or 100% of the surface area of wells containing αCD8D-1-PsT labeled cells was uncaged, and samples were analyzed by flow cytometry. \nE) Correlation between size of the uncaged area (microscope) and fractions of uncaged CD8+ T cells as measured with flow cytometry, as shown in Fig. 3D. Blue line represents the fraction of uncaged (AF594low FLAGhigh) cells after local exposure with 405nm light. Green line represents the fraction of AF594low FLAGhigh cells within a second sample that was not exposed to 405nm (i.e. not uncaged) after mixing the sample with cells from the samples in which the indicated percentages were uncaged. Note that αCD8D-1-PsT binding is stable throughout the complete experimental pipeline, as demonstrated by the absence of AF594low FLAGhigh signal on cells from the non-exposed sample. Line graphs show mean of technical triplicates from one experiment. Error bars show lowest and highest values. \n ","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/fig3.png"},{"id":2416992,"identity":"d5694901-c94b-4aa0-82aa-acb6f57bfb35","added_by":"auto","created_at":"2020-09-15 15:42:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":450240,"visible":true,"origin":"","legend":"Uncaging of spatially defined CD8+ T cells in primary human tumor tissue and in vitro tumor-T cell coculture systems\nA) Representative confocal images of CD8+ T cell staining with αCD8D-1-PsT in viable human melanoma (left panels) and non-small cell lung carcinoma (NSCLC) tumor tissue (right panels). Collagen structures are depicted in white, CD8+ T cells are depicted in green. Enlarged areas are indicated with the white boxes. Scale bars represent 100 μm (overview image) and 10 μm (enlarged images).\nB) Uncaging of αCD8D-1-PsT labeled CD8+ T cells in human tumor tissue. Indicated samples were either left unexposed (top panels) or locally exposed to 405nm light (bottom panels). Tumor samples were then processed into single cell suspensions, stained with αFLAG antibody and analyzed by flow cytometry. \nC) Viability of CD8+ T cells from human tumor tissue. CD8+ T cells from locally uncaged regions (AF594low FLAGhigh) are compared to CD8+ T cells from non-exposed areas (AF594highFLAGlow), as measured by flow cytometric analysis of IR-dye live-dead stained cells (n=4 technical replicates per group). Data of one experiment are depicted. \nD) Experimental approach to validate ability to identify local differences in cell state. Islands of GFP+ CDK4R\u003eL antigen positive (Ag+) and Katushka+ antigen negative (Ag-) OVCAR5 cells were generated. αCD8D-1-PsT labeled CDK4R\u003eL-specific CD8+ T cells located in Ag+ or Ag- areas were uncaged, isolated by FACS, and analyzed by single cell RNA sequencing. \nE) Representative confocal images of cocultures of CDK4R\u003eL-specific CD8+ T cells with tumor cell islands containing Ag+ tumor cell areas (green) surrounded by Ag- tumor cell areas (magenta). Bottom panels: control cultures that either only contain Ag+ tumor cells (bottom, left) or Ag- tumor cells (bottom, right). White boxes indicate regions that were uncaged by 405nm light exposure. Scale bars represent 1 mm.\nF) CD69 expression on αCD8D-1-PsT labeled CD8+ T cells that were uncaged in Ag+ or Ag- tumor cell islands. Top panel: Histograms showing CD69 expression on AF594low FLAGhigh CD8+ T cells of cultures in which uncaging was performed on either Ag+ (blue) or Ag- (red) areas. Bottom panel: histogram of CD69 expression on AF594low FLAGhigh CD8+ T cells from control cultures that either only contained Ag+ tumor cells (blue) or Ag- cells (red). \nG) Quantification of the median fluorescence intensity of CD69 on αCD8D-1-PsT labeled CD8+ T cells that were uncaged in Ag+ or Ag- regions, as analyzed by flow cytometry (n=1-3 technical replicates per group). Representative data from three independent experiments are depicted.\n ","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/fig4.png"},{"id":2416993,"identity":"842a6cfc-e494-4a78-8f5f-e1cfc625ad37","added_by":"auto","created_at":"2020-09-15 15:42:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":150438,"visible":true,"origin":"","legend":"Uncaging of spatially defined CD8+ T cells in primary human tumor tissue and in vitro tumor-T cell coculture systems\nA) Representative confocal images of CD8+ T cell staining with αCD8D-1-PsT in viable human melanoma (left panels) and non-small cell lung carcinoma (NSCLC) tumor tissue (right panels). Collagen structures are depicted in white, CD8+ T cells are depicted in green. Enlarged areas are indicated with the white boxes. Scale bars represent 100 μm (overview image) and 10 μm (enlarged images).\nB) Uncaging of αCD8D-1-PsT labeled CD8+ T cells in human tumor tissue. Indicated samples were either left unexposed (top panels) or locally exposed to 405nm light (bottom panels). Tumor samples were then processed into single cell suspensions, stained with αFLAG antibody and analyzed by flow cytometry. \nC) Viability of CD8+ T cells from human tumor tissue. CD8+ T cells from locally uncaged regions (AF594low FLAGhigh) are compared to CD8+ T cells from non-exposed areas (AF594highFLAGlow), as measured by flow cytometric analysis of IR-dye live-dead stained cells (n=4 technical replicates per group). Data of one experiment are depicted. \nD) Experimental approach to validate ability to identify local differences in cell state. Islands of GFP+ CDK4R\u003eL antigen positive (Ag+) and Katushka+ antigen negative (Ag-) OVCAR5 cells were generated. αCD8D-1-PsT labeled CDK4R\u003eL-specific CD8+ T cells located in Ag+ or Ag- areas were uncaged, isolated by FACS, and analyzed by single cell RNA sequencing. \nE) Representative confocal images of cocultures of CDK4R\u003eL-specific CD8+ T cells with tumor cell islands containing Ag+ tumor cell areas (green) surrounded by Ag- tumor cell areas (magenta). Bottom panels: control cultures that either only contain Ag+ tumor cells (bottom, left) or Ag- tumor cells (bottom, right). White boxes indicate regions that were uncaged by 405nm light exposure. Scale bars represent 1 mm.\nF) CD69 expression on αCD8D-1-PsT labeled CD8+ T cells that were uncaged in Ag+ or Ag- tumor cell islands. Top panel: Histograms showing CD69 expression on AF594low FLAGhigh CD8+ T cells of cultures in which uncaging was performed on either Ag+ (blue) or Ag- (red) areas. Bottom panel: histogram of CD69 expression on AF594low FLAGhigh CD8+ T cells from control cultures that either only contained Ag+ tumor cells (blue) or Ag- cells (red). \nG) Quantification of the median fluorescence intensity of CD69 on αCD8D-1-PsT labeled CD8+ T cells that were uncaged in Ag+ or Ag- regions, as analyzed by flow cytometry (n=1-3 technical replicates per group). Representative data from three independent experiments are depicted.\n ","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/fig5.png"},{"id":2416994,"identity":"d834c83f-f3a2-4625-ad7d-95e034ae1399","added_by":"auto","created_at":"2020-09-15 15:42:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":206065,"visible":true,"origin":"","legend":"Detection of diverse activation programs in locally activated CD8+ T cells\nA) Barplots showing the expression levels (molecules / 1000 UMIs) of six of the top variable genes throughout the dataset (i.e. all cells from all experiments) across all metacells. For each metacell, the log fold change in expression level relative to the median over all metacells is depicted. Bars are colored by the cell state annotations as used in Fig. 5A.\nB) Heatmap depicting the log fold change in expression relative to the median expression for the top 30 most variable genes in the dataset across all metacells.\nC) Barplots depicting the top 30 genes of which expression correlates most strongly with IFNG expression, together defining the IFNG module.\nD) Expression of the IFNG module per cell in a sample in which αCD8D-1-PsT labeled CD8+ T cells in Ag+ areas were uncaged. Color gradient depicts the fraction of UMIs from the IFNG module of total UMI count per cell divided by the median fraction of IFNG module-related UMIs over all cells. Values were transformed for visualization purposes and are projected on the index plot of the sample in which the Ag+ area was uncaged (same index plot as in Fig. 5B, left panel). Note that expression of the IFNG module is more profound in AF594low FLAGhigh CD8+ T cells relative to AF594high FLAGlow CD8+ T cells from the same sample. Data are representative of two independent experiments.\nE) Expression of the IFNG module in spatially defined CD8+ T cells. Fraction of UMIs from the IFNG module of total UMI count per cell are depicted for uncaged (AF594low FLAGhigh) CD8+ T cells from samples in which uncaging was either restricted to Ag+ areas or Ag- areas. Boxplot shows the median, 25th and 75th percentile. Whiskers depict 1.5x IQR and circles depict outlier cells. Two-tailed Mann-Whitney U test was performed (**** indicates p\u003c0.0001). Data are representative of two independent experiments.\nF) Representative confocal image of tumor island culture in which Ag+ tumor cell areas (green) and Ag- tumor cell areas (magenta) are intermingled. Representative of two independent experiments. Scale bar represents 2mm.\nG) Quantification of T cell states in uncaged αCD8D-1-PsT labeled CD8+ T cells from Ag+ and Ag- areas in a mixed tumor culture as shown in fig. 6G. Barplots show the quantification of T cell states as defined in Fig. 5A of uncaged CD8+ T cell populations from samples in which either Ag+ or Ag- areas were uncaged (252 and 236 cells, respectively). Tumor cells are excluded from the analysis. Two-tailed Chi-square test was performed (**** indicates p\u003c0.0001). Data shown were obtained in a single experiment.\n\n \n ","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/fig6.png"},{"id":15779729,"identity":"83cdab54-9dcb-4816-aa55-92734e912219","added_by":"auto","created_at":"2021-11-22 15:38:40","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1560335,"visible":true,"origin":"","legend":"","description":"","filename":"VanderLeunManuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1_covered.pdf"},{"id":13530633,"identity":"fc4834cc-722f-44b3-b69e-b4b7c3861243","added_by":"auto","created_at":"2021-09-17 01:09:35","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1555369,"visible":true,"origin":"","legend":"","description":"","filename":"VanderLeunManuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1_covered.pdf"},{"id":2417006,"identity":"2e0193b6-091c-4715-8b58-2139322d69a0","added_by":"auto","created_at":"2020-09-15 15:42:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1663164,"visible":true,"origin":"","legend":"","description":"","filename":"VanderLeunManuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1_stamped.pdf"},{"id":2416995,"identity":"d7e672a4-46db-4e40-a164-f2bbf6354754","added_by":"auto","created_at":"2020-09-15 15:42:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1609804,"visible":true,"origin":"","legend":"","description":"","filename":"VanderLeunManuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/VanderLeunManuscript.pdf"},{"id":2416996,"identity":"d68819cb-c5c6-4a0a-9cef-d7ae55207505","added_by":"auto","created_at":"2020-09-15 15:42:37","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12652,"visible":true,"origin":"","legend":"","description":"","filename":"VanderLeunTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/VanderLeunTableS1.xlsx"},{"id":2416997,"identity":"c7a35702-d56a-4cfd-8792-99f4e3e3f39c","added_by":"auto","created_at":"2020-09-15 15:42:38","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":44995,"visible":true,"origin":"","legend":"","description":"","filename":"VanderLeunTableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/VanderLeunTableS2.xlsx"},{"id":2416998,"identity":"b1a360dc-cb77-45ec-8c1a-efa1465c4f48","added_by":"auto","created_at":"2020-09-15 15:42:38","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13248,"visible":true,"origin":"","legend":"","description":"","filename":"VanderLeunTableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/VanderLeunTableS3.xlsx"},{"id":2416999,"identity":"1ed2909c-1f20-4ebc-8002-61f584ccb6a8","added_by":"auto","created_at":"2020-09-15 15:42:38","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":126924,"visible":true,"origin":"","legend":"","description":"","filename":"VanderLeunsupplfig1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/VanderLeunsupplfig1.pdf"},{"id":2417000,"identity":"e0a46b74-6711-4c59-a0c9-0209e63b9592","added_by":"auto","created_at":"2020-09-15 15:42:38","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":253739,"visible":true,"origin":"","legend":"","description":"","filename":"VanderLeunsupplfig2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/VanderLeunsupplfig2.pdf"},{"id":2417001,"identity":"dd910cf9-4435-4bf2-80d9-835638aba0b3","added_by":"auto","created_at":"2020-09-15 15:42:38","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":220309,"visible":true,"origin":"","legend":"","description":"","filename":"VanderLeunsupplfig3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/VanderLeunsupplfig3.pdf"},{"id":2417002,"identity":"3538d5c7-9924-4d9d-9d93-098e0f5fc116","added_by":"auto","created_at":"2020-09-15 15:42:39","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":166681,"visible":true,"origin":"","legend":"","description":"","filename":"VanderLeunsupplfig4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/VanderLeunsupplfig4.pdf"},{"id":2417003,"identity":"a6b96127-089f-4a8f-9402-1bd7db9ee99b","added_by":"auto","created_at":"2020-09-15 15:42:39","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":200027,"visible":true,"origin":"","legend":"","description":"","filename":"VanderLeunsupplfig5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/VanderLeunsupplfig5.pdf"},{"id":2417004,"identity":"85f5e49b-3c0a-4325-a551-954e189d12c9","added_by":"auto","created_at":"2020-09-15 15:42:39","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":5132369,"visible":true,"origin":"","legend":"","description":"","filename":"VanderLeunsupplfig6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/VanderLeunsupplfig6.pdf"},{"id":2417005,"identity":"c7db8b09-3154-43dc-a698-a7c7ef3a2553","added_by":"auto","created_at":"2020-09-15 15:42:39","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":6786941,"visible":true,"origin":"","legend":"","description":"","filename":"VanderLeunsupplfig7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-71508/v1/VanderLeunsupplfig7.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003eSingle Cell Analysis of Regions of Interest (SCARI) using a Novel Photoswitchable Tag\u003c/p\u003e","fulltext":[{"header":"Full Text","content":"\u003cp\u003eDue to technical limitations, full-text HTML conversion of this \nmanuscript could not be completed. However, \nthe manuscript can be downloaded and accessed as a PDF.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"photoswitching, unbiased characterization, spatial resolution, profiling depth","lastPublishedDoi":"10.21203/rs.3.rs-71508/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-71508/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe functional activity and differentiation potential of cells is determined by their interaction with surrounding cells. Approaches that allow the unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a trade-off between spatial resolution and cell profiling depth. Here, we developed a photoswitch-based technology that allows the isolation and in-depth analysis of live cells from regions of interest in complex \u003cem\u003eex vivo\u003c/em\u003e systems, including human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran)-cage coupled to nanobodies allowed photoswitching of cells in areas of interest with low-intensity violet light and without detectable phototoxicity. Single cell RNA sequencing of spatially defined CD8\u003csup\u003e+\u003c/sup\u003e\u003cstrong\u003e \u003c/strong\u003eT cells was used to exemplify the feasibility of identifying location-dependent cell states at the single cell level. Finally, we demonstrate the efficient labeling and photoswitching of cells in live primary human tumor tissue. The technology described here provides a valuable tool for the analysis of spatially defined cells in diverse biological systems, including clinical samples.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Single Cell Analysis of Regions of Interest (SCARI) using a Novel Photoswitchable Tag","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-09-15 15:42:35","doi":"10.21203/rs.3.rs-71508/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-chemical-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nchembio","sideBox":"Learn more about [Nature Chemical Biology](http://www.nature.com/nchembio/)","snPcode":"","submissionUrl":"","title":"Nature Chemical Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"39dcccab-a013-45c8-845c-d0188bc43fd7","owner":[],"postedDate":"September 15th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":521807,"name":"Immunology"},{"id":521808,"name":"Chemical Biology"}],"tags":[],"updatedAt":"2021-11-22T15:35:31+00:00","versionOfRecord":{"articleIdentity":"rs-71508","link":"https://doi.org/10.1038/s41589-021-00839-x","journal":{"identity":"nature-chemical-biology","isVorOnly":false,"title":"Nature Chemical Biology"},"publishedOn":"2021-09-09 04:00:00","publishedOnDateReadable":"September 9th, 2021"},"versionCreatedAt":"2020-09-15 15:42:35","video":"","vorDoi":"10.1038/s41589-021-00839-x","vorDoiUrl":"https://doi.org/10.1038/s41589-021-00839-x","workflowStages":[]},"version":"v1","identity":"rs-71508","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-71508","identity":"rs-71508","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}