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CDX2 expression dynamics in tumor clusters: a morpho-molecular biomarker in rectal cancer pretreatment biopsies revealed by sequential immunofluorescence
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  • Published: 21 February 2026

CDX2 expression dynamics in tumor clusters: a morpho-molecular biomarker in rectal cancer pretreatment biopsies revealed by sequential immunofluorescence

  • Mauro Gwerder1,2 na1,
  • Cansaran Saygili Demir1,3 na1,
  • Hannah L. Williams1,
  • Alessandro Lugli1,
  • Cristina Graham Martinez1,
  • Joanna Kowal3,
  • Amjad Khan1,
  • Philipp Kirchner1,
  • Thibaud Koessler4,
  • Martin D. Berger5,
  • Martin Weigert6,7,8 &
  • …
  • Inti Zlobec1 

Scientific Reports , Article number:  (2026) Cite this article

We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.

Subjects

  • Biomarkers
  • Cancer
  • Computational biology and bioinformatics
  • Oncology

Abstract

In rectal cancer, there is a need for improved pretreatment biomarkers applicable to biopsies. Tumor budding (TB) is a histological feature used in colon cancer and, due to its link to epithelial-mesenchymal transition (EMT), is hypothesized to be a potential marker for therapy resistance. As EMT-related processes are also seen in other morphological features beyond TB, we investigated epithelial marker downregulation in tumor tissue, as well as morphological features such as tumor cluster size and finger-like projections. We therefore leveraged five colon cancer images to establish a hyperplex immunofluorescence workflow and a validation cohort consisting of rectal cancer pre-treatment biopsies. We built a custom image analysis pipeline to detect and segment tumor buds and other morphological features and correlated them with molecular expression intensities. We found correlations of epithelial marker downregulation and morphological transition states, both at the invasion front and at the tumor center. We furthermore observed a link between morpho-molecular transitions of nuclear CDX2 expression and tumor cluster size, which in turn informs a novel biomarker. Finally, quantification of these CDX2-based morpho-molecular transition states in rectal biopsies showed that downregulation of CDX2 expression in relation to tumor cluster size is associated with worse disease-free survival.

Data availability

Five whole slide images from the colon cancer establishment cohort will be made available upon publication of the paper in the corresponding Zenodo community: https://zenodo.org/communities/morpho-molecularcdx2

Code availability

All code used in this study is accessible on github: https://github.com/digitalpathologybern/MxIF-pipeline.

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Acknowledgements

The authors are thankful to Ana Frei Leni, Elias Baumann, Philipp Zens, Linda Studer, José Francisco Carreño Martinez, Jacob Hanimann, Javier Garcia Baroja and Mohamed Mansour Faye for valuable feedback during this work. The authors are also grateful to Dr. José A. Galvan, Therese Waldburger, Carmen Cardozo, Loredana-Ionela Daminescu and Stefan Reinhard for staining, scanning, data collection and technical assistance. Finally, the authors are also thankful to the UBELIX (http://www.id.unibe.ch/hpc) team, the HPC cluster at the University of Bern, for providing efficient services during this study.

Funding

M.G. was supported by the Swiss cancer league [KFS-5786-02-2023-R].

M.G. and C.S.D. were supported by Innosuisse [51906.1 IP-LS].

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Author notes

  1. Mauro Gwerder and Cansaran Saygili Demir contributed equally to this work.

Authors and Affiliations

  1. Institute of Tissue Medicine and Pathology, University of Bern, Murtenstrasse 31, 3008, Bern, Switzerland

    Mauro Gwerder, Cansaran Saygili Demir, Hannah L. Williams, Alessandro Lugli, Cristina Graham Martinez, Amjad Khan, Philipp Kirchner & Inti Zlobec

  2. Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland

    Mauro Gwerder

  3. Lunaphore Technologies SA, Tolochenaz, Switzerland

    Cansaran Saygili Demir & Joanna Kowal

  4. Department of Oncology, Geneva University Hospitals, Geneva, Switzerland

    Thibaud Koessler

  5. Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland

    Martin D. Berger

  6. Center for Scalable Data Analytics and AI (ScaDS.AI), Dresden/Leipzig, Germany

    Martin Weigert

  7. Faculty of Computer Science, TU Dresden, Dresden, Germany

    Martin Weigert

  8. Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

    Martin Weigert

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Contributions

Mauro Gwerder, Cansaran Saygili Demir and Inti Zlobec performed study conception and design; Cansaran Saygili Demir conducted data acquisition; Mauro Gwerder performed development of methodologies; Mauro Gwerder conducted data analysis and interpretation; Mauro Gwerder, Cansaran Saygili Demir, Cristina Graham Martinez, Martin D. Berger. Hanna L. Williams and Inti Zlobec provided administrative, technical, or material support; Martin Weigert and Inti Zlobec supervised the study. Mauro Gwerder, Cansaran Saygili Demir, Hannah L. Williams, Alessandro Lugli, Cristina Graham Martinez, Joanna Kowal, Amjad Khan, Philipp Kirchner, Thibaud Koessler, Martin D. Berger, Martin Weigert and Inti Zlobec read and approved the final paper.

Corresponding author

Correspondence to Inti Zlobec.

Ethics declarations

Competing interests

JK and CSD are current or former employees of Lunaphore, a Bio-Techne Brand, which is working on commercializing an automated platform to implement seqIF on standard tissue samples. JK is a Bio-Techne shareholder. There are other no conflicts of interest.

Ethics approval and consent to participate

The study was approved by the Ethics committee of the Canton of Bern under project number b2020-00498 and was performed according to the Human Research Act HFG 2014. Informed consent was obtained from all subjects.

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Gwerder, M., Demir, C.S., Williams, H.L. et al. CDX2 expression dynamics in tumor clusters: a morpho-molecular biomarker in rectal cancer pretreatment biopsies revealed by sequential immunofluorescence. Sci Rep (2026). https://doi.org/10.1038/s41598-026-40005-8

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  • Received: 30 September 2025

  • Accepted: 09 February 2026

  • Published: 21 February 2026

  • DOI: https://doi.org/10.1038/s41598-026-40005-8

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Keywords

  • Rectal cancer
  • Tumor budding
  • Epithelial–mesenchymal transition
  • Neoadjuvant chemoradiotherapy
  • Hyperplex immunofluorescence
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