Analysis of experimental colorectal liver metastases identifies distinct proliferation-independent transcriptional programs in micro-versus macrometastases

Sep 30, 2026

Background: Distant relapse after surgical removal of primary stage I–III colorectal cancer is driven by outgrowth of micrometastases that are undetectable at first diagnosis, while adjuvant chemotherapy reduces distant metastasis formation by only ~15%, requiring improved prevention strategies. Methods: A p27-based fluorescent reporter was used to mark quiescent tumor cells in vitro and in vivo using patient-derived organoids transplanted into mouse caecum, followed by immunohistochemistry and single-cell RNA sequencing after micro- and macrometastasis isolation. Results: The reporter robustly separates quiescent from cycling tumor cells; micrometastases can be cycling or quiescent. Quiescent macrometastases show TNFα signaling and epithelial-to-mesenchymal transition, whereas micrometastases upregulate metabolic pathways including oxidative phosphorylation. Macrometastases also feature cancer-associated fibroblasts and collagen deposition. Conclusion: A single-cell resolution strategy reveals quiescent micrometastases as a distinct transcriptional tumor entity, suggesting new relapse-prevention approaches targeting quiescent lesions.

LanguageEnglish

CategoryResearch & Report

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Pages17

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Background

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Peters et al. Journal of Translational Medicine (2026) 24:37 [https://doi.org/10.1186/s12967-025-07218-3](https://doi.org/10.1186/s12967-025-07218-3) Journal of Translational Medicine RESEARCH Open Access Analysis of experimental colorectal liver metastases identifies distinct proliferationindependent transcriptional programs in micro-versus macrometastases Niek A. Peters 1†, Maria C. Heinz2,3†, Emre Küçükköse 1, Bastiaan J. Viergever 1, Matthijs Baars2, Mojtaba Amini2, Maarten A. Huismans 1,2,3, Daan Andel 1, AndreVerheem 1, YvonneVercoulen2,4, Hugo Snippert2,3†, Inne H. M. Borel Rinkes 1† and Onno Kranenburg 1*† Abstract Background Distant relapse following surgical removal of primary stage I-III colorectal cancer is caused by the outgrowth of micrometastases that were undetectable at first diagnosis. Adjuvant chemotherapy lowers the risk of distant metastasis formation by only ~15%, indicating that more effective metastasis-prevention strategies are urgently needed. Research aiming to develop such strategies is challenging, due to the low abundance of micrometastases, their small size, and the paucity of model systems. Methods We applied a p27-based fluorescent reporter to identify quiescent tumor cells in vitro and in vivo. Colon cancer patient-derived organoids (PDOs) were transplanted into the mouse caecum, which led to spontaneous formation of micro-and macrometastases. Liver metastases were analyzed by immunohistochemistry. In addition, aliver perfusion and collagenase-based homogenization protocol was developed which allows isolation of micro-and macrometastases. Subsequently, p27-positive and p27-negative tumor cells were isolated and analyzed by bulk and single cell RNA sequencing. Results In vitro and in vivo analyses validated the p27-reporter as a robust tool for separating quiescent from cycling tumor cells. In vivo analyses revealed that micrometastases can be either cycling or quiescent in our metastasis model. In depth analyses of quiescent cells obtained from differently sized metastases showed that macrometastases are characterized by TNFα signaling and epithelial-to-mesenchymal transition. By contrast, micrometastases are | †Niek A. Peters and Maria C. Heinz contributed equally to this article as co-first authors. | | --- | | †Hugo Snippert, Inne H. M. Borel Rinkes and Onno Kranenburg share senior-authorship. | *Correspondence: Onno Kranenburg [o.kranenburg@umcutrecht.nl](o.kranenburg@umcutrecht.nl) Full list of author information is available at the end of the article © The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit [http://creati](http://creati)[vecommons.org/l](vecommons.org/l)icenses/by-nc-nd/4.0/. Peters et al. Journal of Translational Medicine (2026) 24:37 Page 2 of 17 characterized by upregulation of metabolic pathways including oxidative phosphorylation. Macrometastases, but not micrometastases, are characterized by the presence of cancer-associated fibroblasts and collagen deposition. Conclusion We developed a strategy to analyze spontan

FAQs

Why are micrometastases important in colorectal cancer relapse?

Distant relapse after surgery is caused by outgrowth of micrometastases that are not detectable at initial diagnosis. Their growth potential drives recurrence despite treatment.

How were quiescent tumor cells identified in this study?

The study used a p27-based fluorescent reporter to identify quiescent tumor cells in vitro and in vivo, including after organoid transplantation into mouse caecum.

What transcriptional programs distinguish micro- versus macrometastases?

Quiescent macrometastases are characterized by TNFα signaling and epithelial-to-mesenchymal transition, while micrometastases show upregulation of metabolic pathways, including oxidative phosphorylation.

Sep 30, 2026

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