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Stratification of chemotherapy-treated stage III colorectal cancer patients using multiplexed imaging and single-cell analysis of T-cell populations

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posted on 02.12.2021, 16:42 authored by Xanthi Stachtea, Maurice B Loughrey, Manuela Salvucci, Andreas Ulrich LindnerAndreas Ulrich Lindner, Sanghee Cho, Elizabeth McDonough, Anup Sood, John Graf, Alberto Santamaria-Pang, Alex Corwin, Pierre Laurent-Puig, Sonali Dasgupta, Jinru Shia, Jonathan R Owens, Samantha Abate, Sandra Van Schaeybroeck, Mark Lawler, Jochen PrehnJochen Prehn, Fiona Ginty, Daniel B Longley
Colorectal cancer (CRC) has one of the highest cancer incidences and mortality rates. In stage III, postoperative chemotherapy benefits <20% of patients, while more than 50% will develop distant metastases. Biomarkers for identification of patients at increased risk of disease recurrence following adjuvant chemotherapy are currently lacking. In this study, we assessed immune signatures in the tumor and tumor microenvironment (TME) using an in situ multiplexed immunofluorescence imaging and single-cell analysis technology (Cell DIVETM) and evaluated their correlations with patient outcomes. Tissue microarrays (TMAs) with up to three 1 mm diameter cores per patient were prepared from 117 stage III CRC patients treated with adjuvant fluoropyrimidine/oxaliplatin (FOLFOX) chemotherapy. Single sections underwent multiplexed immunofluorescence staining for immune cell markers (CD45, CD3, CD4, CD8, FOXP3, PD1) and tumor/cell segmentation markers (DAPI, pan-cytokeratin, AE1, NaKATPase, and S6). We used annotations and a probabilistic classification algorithm to build statistical models of immune cell types. Images were also qualitatively assessed independently by a Pathologist as ‘high’, ‘moderate’ or ‘low’, for stromal and total immune cell content. Excellent agreement was found between manual assessment and total automated scores (p < 0.0001). Moreover, compared to single markers, a multi-marker classification of regulatory T cells (Tregs: CD3+/CD4+FOXP3+/PD1−) was significantly associated with disease-free survival (DFS) and overall survival (OS) (p = 0.049 and 0.032) of FOLFOX-treated patients. Our results also showed that PD1− Tregs rather than PD1+ Tregs were associated with improved survival. These findings were supported by results from an independent FOLFOX-treated cohort of 191 stage III CRC patients, where higher PD1− Tregs were associated with an increase overall survival (p = 0.015) for CD3+/CD4+/FOXP3+/PD1−. Overall, compared to single markers, multi-marker classification provided more accurate quantitation of immune cell types with stronger correlations with outcomes.


National Cancer Institute of the National Institutes of Health under award number R01CA208179

US-Ireland R01 award (NI Partner supported by HSCNI, STL/5715/15)

Science Foundation Ireland and the Health Research Board (16/US/3301)

Health Data Research UK



The original article is available at https://www.nature.com

Published Citation

Stachtea X. et al. Stratification of chemotherapy-treated stage III colorectal cancer patients using multiplexed imaging and single-cell analysis of T-cell populations. Mod Pathol. 2022;35(4):564-576

Publication Date

3 November 2021

PubMed ID



  • Centre for Systems Medicine
  • Physiology and Medical Physics

Research Area

  • Cancer


Springer Nature Limited


  • Published Version (Version of Record)