{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T13:32:19Z","timestamp":1783431139873,"version":"3.54.6"},"posted":{"date-parts":[[2026,1,14]]},"group-title":"Genomics","reference-count":42,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,1,14]],"date-time":"2026-01-14T00:00:00Z","timestamp":1768348800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"name":"NIH\/NIAID","award":["DP2AI171161"],"award-info":[{"award-number":["DP2AI171161"]}]},{"name":"Ludwig Institute for Cancer Research"},{"name":"Weill Cancer Hub East"},{"name":"AACR-Bristol-Myers Squibb Immuno-oncology Research Fellowship"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,1,14]]},"abstract":"<jats:title>Summary<\/jats:title>\n                <jats:p>Cell type deconvolution is essential for resolving multicellular composition in spatial transcriptomics data, yet the accuracy and consistency of current methods across biological contexts remain uncertain. We introduce a benchmarking framework using realistic simulations that incorporate spatial and transcriptional complexity. We find that a simple marker gene signature scoring approach performs competitively, often outperforming more complex models, particularly for rare cell types. We further apply this method to a new spatial transcriptomics dataset profiling the early response to a single dose of anti-PD1 checkpoint blockade immunotherapy in a mouse cancer model, revealing pronounced spatially localized immune and microenvironmental changes in both tumors and draining lymph nodes. Our findings have implications for the interpretation of many published spatial transcriptomics studies that rely on cell type deconvolution, and they provide a robust, interpretable strategy for future analyses, even in the absence of matched single-cell references.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Highlights<\/jats:title>\n                  <jats:list list-type=\"simple\">\n                    <jats:list-item>\n                      <jats:p>- Framework for realistic benchmarking of spatial deconvolution methods<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>- Marker gene scoring performs robustly for rare cell types across diverse datasets<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>- Single-dose anti-PD1 cancer immunotherapy triggers profound localized response<\/jats:p>\n                    <\/jats:list-item>\n                  <\/jats:list>\n                <\/jats:sec>","DOI":"10.64898\/2026.01.13.699379","type":"posted-content","created":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T03:45:14Z","timestamp":1768448714000},"source":"Crossref","is-referenced-by-count":3,"title":["Benchmarking cell type deconvolution in spatial transcriptomics and application to cancer immunotherapy"],"prefix":"10.64898","author":[{"given":"Amanda","family":"Sun","sequence":"first","affiliation":[{"name":"Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton NJ"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tamjeed","family":"Azad","sequence":"additional","affiliation":[{"name":"Department of Computer Science, Princeton University, Princeton NJ"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5834-926X","authenticated-orcid":false,"given":"Chrysothemis","family":"Brown","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute and ImmuneOncology Program, Memorial Sloan Kettering Cancer Center, New York NY"},{"name":"Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York NY"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6589-981X","authenticated-orcid":false,"given":"Yuri","family":"Pritykin","sequence":"additional","affiliation":[{"name":"Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton NJ"},{"name":"Department of Computer Science, Princeton University, Princeton NJ"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026020605423310000_2026.01.13.699379v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-022-01409-2"},{"key":"2026020605423310000_2026.01.13.699379v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-022-01480-9"},{"key":"2026020605423310000_2026.01.13.699379v1.3","doi-asserted-by":"crossref","unstructured":"Yan, L. & Sun, X . Benchmarking and integration of methods for deconvoluting spatial transcriptomic data. Bioinformatics 39, btac805 (2023).","DOI":"10.1093\/bioinformatics\/btac805"},{"key":"2026020605423310000_2026.01.13.699379v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-37168-7"},{"key":"2026020605423310000_2026.01.13.699379v1.5","doi-asserted-by":"crossref","unstructured":"Sang-aram, C. , Browaeys, R. , Seurinck, R. & Saeys, Y . Spotless, a reproducible pipeline for benchmarking cell type deconvolution in spatial transcriptomics. eLife 12, RP88431 (2024).","DOI":"10.7554\/eLife.88431"},{"key":"2026020605423310000_2026.01.13.699379v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41576-025-00845-y"},{"key":"2026020605423310000_2026.01.13.699379v1.7","doi-asserted-by":"publisher","DOI":"10.1016\/j.ccell.2014.09.007"},{"key":"2026020605423310000_2026.01.13.699379v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/S41568-020-0285-7"},{"key":"2026020605423310000_2026.01.13.699379v1.9","unstructured":"Human Lymph Node, Spatial Gene Expression Dataset by Space Ranger 1.1.0, 10x Genomics, (2020, June 23)."},{"key":"2026020605423310000_2026.01.13.699379v1.10","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-021-01139-4"},{"key":"2026020605423310000_2026.01.13.699379v1.11","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-021-00830-w"},{"key":"2026020605423310000_2026.01.13.699379v1.12","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-022-01273-7"},{"key":"2026020605423310000_2026.01.13.699379v1.13","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-022-01272-8"},{"key":"2026020605423310000_2026.01.13.699379v1.14","doi-asserted-by":"publisher","DOI":"10.1093\/NAR\/GKAB043"},{"key":"2026020605423310000_2026.01.13.699379v1.15","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-36961-8"},{"key":"2026020605423310000_2026.01.13.699379v1.16","doi-asserted-by":"crossref","unstructured":"Chen, K. H. , Boettiger, A. N. , Moffitt, J. R. , Wang, S. & Zhuang, X . Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015).","DOI":"10.1126\/science.aaa6090"},{"key":"2026020605423310000_2026.01.13.699379v1.17","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1912459116"},{"key":"2026020605423310000_2026.01.13.699379v1.18","unstructured":"Vizgen MERFISH FFPE Human Immuno-oncology Data Set, May 2022."},{"key":"2026020605423310000_2026.01.13.699379v1.19","unstructured":"Xenium FFPE Human Breast with Custom Add-on Panel, In Situ Gene Expression dataset analyzed using Xenium Onboard Analysis 1.3.0, 10x Genomics, (2023, April 18)."},{"key":"2026020605423310000_2026.01.13.699379v1.20","unstructured":"Pancreatic Cancer with Xenium Human Multi-Tissue and Cancer Panel, In Situ Gene Expression dataset analyzed using Xenium Onboard Analysis 1.6.0, (2023, November 15)."},{"key":"2026020605423310000_2026.01.13.699379v1.21","doi-asserted-by":"crossref","unstructured":"Wolf, F. A. , Angerer, P. & Theis, F. J . SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 19, 15 (2018).","DOI":"10.1186\/s13059-017-1382-0"},{"key":"2026020605423310000_2026.01.13.699379v1.22","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac947"},{"key":"2026020605423310000_2026.01.13.699379v1.23","unstructured":"Dom\u00ednguez Conde, C. , et al. Cross-tissue immune cell analysis reveals tissue-specific features in humans. Science 376, eabl5197 (2022)."},{"key":"2026020605423310000_2026.01.13.699379v1.24","doi-asserted-by":"crossref","unstructured":"Franz\u00e9n, O. , Gan, L.-M. & Bj\u00f6rkegren, J. L. M . PanglaoDB: a web server for exploration of mouse and human single-cell RNA sequencing data. Database 2019, (2019).","DOI":"10.1093\/database\/baz046"},{"key":"2026020605423310000_2026.01.13.699379v1.25","doi-asserted-by":"publisher","DOI":"10.1016\/j.ccell.2015.03.001"},{"key":"2026020605423310000_2026.01.13.699379v1.26","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1606774"},{"key":"2026020605423310000_2026.01.13.699379v1.27","doi-asserted-by":"crossref","first-page":"917","DOI":"10.1016\/j.annonc.2021.03.204","article-title":"Delayed immune-related adverse events with anti-PD-1-based immunotherapy in melanoma","volume":"32","year":"2021","journal-title":"Ann. Oncol"},{"key":"2026020605423310000_2026.01.13.699379v1.28","doi-asserted-by":"publisher","DOI":"10.1136\/annrheumdis-2019-216109"},{"key":"2026020605423310000_2026.01.13.699379v1.29","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1016\/j.ejca.2021.03.010","article-title":"Late-onset and long-lasting immune-related adverse events from immune checkpoint-inhibitors: An overlooked aspect in immunotherapy","volume":"149","year":"2021","journal-title":"Eur. J. Cancer"},{"key":"2026020605423310000_2026.01.13.699379v1.30","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20232104"},{"key":"2026020605423310000_2026.01.13.699379v1.31","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2020.11.028"},{"key":"2026020605423310000_2026.01.13.699379v1.32","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2020.03.048"},{"key":"2026020605423310000_2026.01.13.699379v1.33","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0506580102"},{"key":"2026020605423310000_2026.01.13.699379v1.34","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1016\/j.cels.2015.12.004","article-title":"The Molecular Signatures Database Hallmark Gene Set Collection","volume":"1","year":"2015","journal-title":"Cell Syst"},{"key":"2026020605423310000_2026.01.13.699379v1.35","doi-asserted-by":"crossref","unstructured":"Ng, M. S. F. et al. Deterministic reprogramming of neutrophils within tumors. Science 383, eadf6493 (2024).","DOI":"10.1126\/science.adf6493"},{"key":"2026020605423310000_2026.01.13.699379v1.36","doi-asserted-by":"crossref","unstructured":"Chen, Y. , Pal, B. , Lindeman, G. J. , Visvader, J. E. & Smyth, G. K . R code and downstream analysis objects for the scRNA-seq atlas of normal and tumorigenic human breast tissue. Sci. Data 9, 96 (2022).","DOI":"10.1038\/s41597-022-01236-2"},{"key":"2026020605423310000_2026.01.13.699379v1.37","doi-asserted-by":"crossref","unstructured":"Werba, G. et al. Single-cell RNA sequencing reveals the effects of chemotherapy on human pancreatic adenocarcinoma and its tumor microenvironment. Nat. Commun. 14, 797 (2023).","DOI":"10.1038\/s41467-023-36296-4"},{"key":"2026020605423310000_2026.01.13.699379v1.38","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-018-0229-21"},{"key":"2026020605423310000_2026.01.13.699379v1.39","doi-asserted-by":"publisher","DOI":"10.1038\/s41588-021-00911-1"},{"key":"2026020605423310000_2026.01.13.699379v1.40","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-40727-7"},{"key":"2026020605423310000_2026.01.13.699379v1.41","doi-asserted-by":"publisher","DOI":"10.1101\/461954"},{"key":"2026020605423310000_2026.01.13.699379v1.42","doi-asserted-by":"crossref","unstructured":"Choudhary, S. & Satija, R . Comparison and evaluation of statistical error models for scRNA-seq. Genome Biol. 23, 27 (2022).","DOI":"10.1186\/s13059-021-02584-9"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.01.13.699379","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,6]],"date-time":"2026-02-06T13:42:41Z","timestamp":1770385361000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.01.13.699379"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,14]]},"references-count":42,"URL":"https:\/\/doi.org\/10.64898\/2026.01.13.699379","relation":{},"subject":[],"published":{"date-parts":[[2026,1,14]]},"subtype":"preprint"}}