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Peer-reviewedCC BY 4.0Molecular Oncology

Bridging the gap: a genetically validated avian chorioallantoic membrane platform for investigation of spontaneous circulating tumor cells

  • Dennis Roth1,2
  • Rui P. L. Neves1,2
  • Julia Geheb1,2
  • Christian Ballmeyer1,2
  • Nathalie Gaßmann1,2
  • Kiki C. Andree3
  • Monica Sudarsanam1,2
  • Christiane Driemel1,2
  • Celine Örencik1,2
  • Andrè Franken2,4
  • Eslam Elmaghraby1,2
  • Sven H. Loosen5
6 more authors
  • Roderburg Christoph5
  • Sebastian Schoelch6,7,8
  • Hans Neubauer2,4
  • Wolfram T. Knoefel1,2
  • Nikolas H. Stoecklein1,2
  • Georg Fluegen1,2,9
9 affiliations
  1. 1Department of Surgery (A) Heinrich‐Heine‐University and University Hospital Duesseldorf Germany
  2. 2Liquid Biopsy Center Duesseldorf Center for Integrated Oncology Aachen Bonn, Cologne Germany
  3. 3VyCAP B.V Enschede The Netherlands
  4. 4Department of Obstetrics and Gynecology University Hospital and Medical Faculty of the Heinrich‐Heine‐University Duesseldorf Germany
  5. 5Department of Gastroenterology, Hepatology and Infectious Diseases University Hospital Duesseldorf, Medical Faculty of Heinrich Heine University Duesseldorf Germany
  6. 6JCCU Translational Surgical Oncology (A430) German Cancer Research Center (DKFZ) Heidelberg Germany
  7. 7DKFZ‐Hector Cancer Institute, Medical Faculty Mannheim Heidelberg University Mannheim Germany
  8. 8Department of Surgery Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University Mannheim
  9. 9Department of General, Visceral, Vascular and Transplantation Surgery University Hospital Essen Germany
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Abstract

Circulating tumor cells (CTCs) are clinically relevant markers of metastasis and prognosis in gastrointestinal (GI) cancers. However, their extreme rarity and the limitations of current in vivo models severely restrict mechanistic studies of tumor cell dissemination. Here, we demonstrate for the first time that the avian chorioallantoic membrane (CAM) assay can serve as a biologically relevant and ethically advantageous 3R‐compliant (Replacement, Reduction, and Refinement) in vivo model for investigating spontaneous CTC generation. Six green fluorescent protein (GFP)‐labeled human colorectal, pancreatic, and cholangiocarcinoma cell lines were xenografted onto the CAM. Using a standardized workflow, we achieved reliable detection and isolation of GFP‐positive single CTCs from small‐volume CAM blood samples. Critically, we reproducibly confirmed the presence of spontaneously shed CAM‐derived CTCs independent of GFP expression using three established, clinically relevant CTC detection platforms. Subsequent single‐cell whole‐genome amplification enabled downstream short‐tandem repeat (STR) profiling to authenticate CTC identity, alongside detection of tumor‐specific point mutations and copy number alterations (CNAs). Isolated CTCs exhibited high genetic concordance with their parental cell lines, validating the technical robustness of our workflow and establishing the CAM assay as a versatile, scalable platform for investigating hematogenous dissemination and CTC biology outside of mammalian models. Consequently, the CAM assay opens new avenues for early mechanistic and pharmacological CTC research, including functional studies of patient‐derived material.

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Cite

BibTeXRIS
BibTeX
@article{roth2026bridging,
  title = {{Bridging the gap: a genetically validated avian chorioallantoic membrane platform for investigation of spontaneous circulating tumor cells}},
  author = {Dennis Roth and Rui P. L. Neves and Julia Geheb and Christian Ballmeyer and Nathalie Gaßmann and Kiki C. Andree and Monica Sudarsanam and Christiane Driemel and Celine Örencik and Andrè Franken and Eslam Elmaghraby and Sven H. Loosen and Roderburg Christoph and Sebastian Schoelch and Hans Neubauer and Wolfram T. Knoefel and Nikolas H. Stoecklein and Georg Fluegen},
  journal = {Molecular Oncology},
  year = {2026},
  month = sep,
  doi = {10.1002/1878-0261.70336},
  url = {https://doi.org/10.1002/1878-0261.70336},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Bridging the gap: a genetically validated avian chorioallantoic membrane platform for investigation of spontaneous circulating tumor cells
AU  - Dennis Roth
AU  - Rui P. L. Neves
AU  - Julia Geheb
AU  - Christian Ballmeyer
AU  - Nathalie Gaßmann
AU  - Kiki C. Andree
AU  - Monica Sudarsanam
AU  - Christiane Driemel
AU  - Celine Örencik
AU  - Andrè Franken
AU  - Eslam Elmaghraby
AU  - Sven H. Loosen
AU  - Roderburg Christoph
AU  - Sebastian Schoelch
AU  - Hans Neubauer
AU  - Wolfram T. Knoefel
AU  - Nikolas H. Stoecklein
AU  - Georg Fluegen
PY  - 2026
DA  - 2026/09/18
JO  - Molecular Oncology
DO  - 10.1002/1878-0261.70336
UR  - https://doi.org/10.1002/1878-0261.70336
AB  - Circulating tumor cells (CTCs) are clinically relevant markers of metastasis and prognosis in gastrointestinal (GI) cancers. However, their extreme rarity and the limitations of current in vivo models severely restrict mechanistic studies of tumor cell dissemination. Here, we demonstrate for the first time that the avian chorioallantoic membrane (CAM) assay can serve as a biologically relevant and ethically advantageous 3R‐compliant (Replacement, Reduction, and Refinement) in vivo model for investigating spontaneous CTC generation. Six green fluorescent protein (GFP)‐labeled human colorectal, pancreatic, and cholangiocarcinoma cell lines were xenografted onto the CAM. Using a standardized workflow, we achieved reliable detection and isolation of GFP‐positive single CTCs from small‐volume CAM blood samples. Critically, we reproducibly confirmed the presence of spontaneously shed CAM‐derived CTCs independent of GFP expression using three established, clinically relevant CTC detection platforms. Subsequent single‐cell whole‐genome amplification enabled downstream short‐tandem repeat (STR) profiling to authenticate CTC identity, alongside detection of tumor‐specific point mutations and copy number alterations (CNAs). Isolated CTCs exhibited high genetic concordance with their parental cell lines, validating the technical robustness of our workflow and establishing the CAM assay as a versatile, scalable platform for investigating hematogenous dissemination and CTC biology outside of mammalian models. Consequently, the CAM assay opens new avenues for early mechanistic and pharmacological CTC research, including functional studies of patient‐derived material.
SN  - 1574-7891
SN  - 1878-0261
ER  -