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

Sep 22, 2026

Uploaded by James

Rectal cancer urgently needs pretreatment biomarkers that are measurable in biopsy specimens. Tumor budding (TB) is linked to epithelial–mesenchymal transition (EMT) and therapy resistance, but EMT-related morphology extends beyond TB, prompting assessment of epithelial marker downregulation and structural features including tumor cluster size and finger-like projections. Using a hyperplex sequential immunofluorescence workflow, researchers built an image analysis pipeline to quantify buds and correlate morphologic features with molecular intensities. CDX2 morpho-molecular transitions relate to cluster size and show that CDX2 downregulation relative to size predicts worse disease-free survival.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages19

Reading Time48 min

AccessFree to download

Background and clinical need

Tumor budding and EMT-related hypotheses

Study design and experimental workflow

Hyperplex sequential immunofluorescence and validation cohort

Image analysis pipeline for morphology quantification

Morphomolecular correlations and biomarker discovery

Epithelial marker downregulation and transition states

CDX2 transitions, tumor cluster size, and outcome association

[www. nature.com/scientificreports](www. nature.com/scientificreports) OPEN CDX2 expression dynamics in tumor clusters: a morphomolecular biomarker in rectal cancer pretreatment biopsies revealed by sequential immunofluorescence Mauro Gwerder1,2,9, Cansaran Saygili Demir1,3,9, 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􀀍 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 epithelialmesenchymal 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 pretreatment 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 atthe 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 ofCDX2 expression in relation to tumor cluster size is associated with worse disease-free survival. Keywords Rectal cancer, Tumor budding, Epithelial–mesenchymal transition, Neoadjuvant chemoradiotherapy, Hyperplex immunofluorescence Colorectal cancer (CRC) is the third most prevalent cancer type in Switzerland and worldwide, with 40% of all CRC cases being of rectal origin1. Although rectal and colon cancers are molecularly comparable, treatment strategies differ strongly due to anatomical differences2. Total neoadjuvant therapy (TNT) is standard of care for locally advanced rectal cancer (LARC)3–5 to facilitate subsequent tumor resection6 and in some cases guarantee organ preservation5. Future treatment strategies could be refined to incorporate two needs: Firstly, more patients responding well to treatment are suited to a watch-and-wait strategy, where no surgery is needed7,8. 1Institute ofTissue Medicine and Pathology, University of Bern, Murtenstrasse 31, 3008 Bern, Switzerland. 2Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland. 3Lunaphore Technologies SA, Tolochenaz, Switzerland. 4Department of Oncology, Geneva University Hospitals, Geneva, Switzerland. 5Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland. 6Center for Scalable Data Analytics and AI (ScaDS.AI), Dresden/Leipzig, Germany. 7Faculty of Computer Science, TU Dresden, Dresden, Germany. 8Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland. 9Mauro Gwerder and Cansaran Saygili Demir contributed equally to this work. 􀀍 email: [inti.zlobec@unibe.ch](inti.zlobec@unibe.ch) [www. nature.com/scientificreports/](www. nature.com/scientificreports/) Secondly, pretreatment assessment of biopsies could guide patient selection for treatment regimens9. To make this paradigm a reality, novel pretreatment biomarkers are needed to improve response prediction. Although histopathological assessment of rectal pretreatment biopsy samples (PBS) is routinely conducted, it acts only asa confirmation of cancer incidence rather than informing treatment decision

FAQs

Why are pretreatment biomarkers needed for rectal cancer biopsies?

Pretreatment assessment from biopsy tissue is needed to improve response prediction. Routine histopathology primarily confirms cancer incidence rather than guiding treatment decisions.

What morphological features besides tumor budding were analyzed?

The study evaluated epithelial marker downregulation and morphological features such as tumor cluster size and finger-like projections.

How did CDX2 morpho-molecular transitions relate to patient outcomes?

Quantification of CDX2-based morpho-molecular transition states in rectal biopsies showed that CDX2 downregulation relative to tumor cluster size is associated with worse disease-free survival.

Sep 22, 2026

Uploaded by James