Methodology for generating chorioallantoic membrane patient-derived xenograft (CAM-PDX) models of pleural mesothelioma and performing preclinical imaging for the translation of cancer studies and drug screening

Our plain-English summary of Methodology for generating chorioallantoic membrane patient-derived xenograft (CAM-PDX) models of pleural mesothelioma and performing preclinical imaging for the translation of cancer studies and drug screening, by J. Schulze, S. Barnett, L. Shaw, et al (F1000Research, 2025).

The study

• Authors: J. Schulze, S. Barnett, L. Shaw, A. Herrmann, H. Poptani, D.M. Rassl, A. Haragan, M. Shackcloth, J.J. Sacco, J.M. Coulson
• Journal: F1000Research
• Published: 2025;14:481
• Cited by: 2 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Pleural mesothelioma is a cancer of the lung lining associated with asbestos exposure. Platinum/pemetrexed chemotherapy has been used for many years but provides little benefit and, despite recent immunotherapy advances, prognosis remains poor underpinning the need for development of novel therapeutics or drug repurposing. Fertilized hens' eggs provide a rapid and cost-effective alternative to murine models of pleural mesothelioma which are commonly used in preclinical studies, with chorioallantoic membrane (CAM) xenografts being a partial replacement for mouse flank xenografts. Here we describe methods to generate mesothelioma patient-derived xenografts on the CAM (CAM-PDX), and to subsequently assess these PDX nodules by preclinical imaging and histology.

Methods: Fragments of surplus mesothelioma tissue obtained from patient biopsies were implanted onto the CAM on embryonic day 7 (E7), fresh or following cryopreservation, with the established PDX dissected on E14 and fixed for histological/immunohistochemical analysis. The optimal freezing method was determined by comparing tissue integrity and cellular content of cryopreserved tissue fragments with paired fresh samples via histological/immunohistochemical analyses. [ 18F]-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) was used to assess viability of PDXs in ovo.

Results: Methodologies for processing, cryopreservation, re-animation, and engraftment of mesothelioma tissue fragments were established. Cryopreservation of biopsy samples and parallel processing of contiguous sections allows for assessment of mesothelioma cellularity. CAM-PDXs, generated from fresh or slow-frozen tissue, were well vascularized whilst maintaining the architecture and cellular composition of the patient tissue. Furthermore, uptake of [ 18F]-FDG following intravenous injection could be visualized and quantified.

Conclusions: The CAM is a rapid platform for engrafting patient-derived tissue, maintaining elements of the tumor microenvironment and recapitulating heterogeneity observed in mesothelioma. Combining the CAM-PDX model and FDG-PET/CT provides a quantitative in vivo platform for pre-screening of novel treatment strategies and drug combinations, with the potential for development of patient tumor avatars for predicting clinical response.

Copyright: © 2025 Schulze J et al.

In plain English

This methods article did not study asbestos exposure. The authors described how to graft pleural mesothelioma tissue onto the chorioallantoic membrane of fertilized hens' eggs and check grafts by staining and FDG-PET/CT. The tissue was surplus diagnostic biopsy material collected in Liverpool from March 2021 to November 2024. Only half of the samples were later confirmed as mesothelioma, and detailed data came from three patients. Of 15 grafts of fresh sarcomatoid tissue, 93% developed blood vessels by embryonic day 14. Vessels formed in 22 of 27 grafts (82%) from tissue frozen in a high fetal bovine serum (FBS) medium and 20 of 26 from a low-FBS medium. The article prints the low-FBS figure as 74%, which does not match its own count of 20 of 26. The authors reported that grafts kept the patient tissue's structure and cell types. Grafts from the low-FBS medium had fewer immune T-cells. PET tracer uptake did not differ significantly between the two media (p = 0.76). The authors concluded that viable grafts can be made from fresh or frozen tissue, and they recommended the high-FBS medium.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Methods article (laboratory proof-of-principle methods development) using patient tissue grafted onto the chorioallantoic membrane of fertilized hens' eggs, with histology, immunohistochemistry and in ovo FDG-PET/CT imaging
• Who was studied: Surplus biopsy tissue from treatment-naive patients undergoing diagnostic percutaneous biopsies at NHS Liverpool Heart and Chest Hospital, UK. It was collected March 2021 to November 2024 under the Mesobank UK ethics approval. Only 50% of the samples collected were later confirmed as mesothelioma. Detailed data were presented for three patients (one sarcomatoid, two epithelioid), and vascularization was summarized for samples from five patients. The tissue was grafted onto fertilized brown chicken eggs.
• Exposure: Asbestos exposure was not studied; the article mentions asbestos only as background. The experimental conditions compared were fresh tissue versus tissue slow-frozen in a high-FBS medium (90% FBS, 10% DMSO) or a low-FBS medium (80% RPMI, 10% FBS, 10% DMSO). Tissue was grafted on embryonic day 7 and dissected on day 14, with optional intravenous [18F]-FDG for PET/CT on day 13.
• Main results:
  • Fresh sarcomatoid tissue: 93% of grafts (14 of 15) were vascularized by embryonic day 14. Across all fresh grafts, 16 of 17 (94%) were vascularized.
  • Grafts from cryopreserved epithelioid tissue formed well-vascularized nodules after 7 days. This happened in 22 of 27 grafts (82%) from tissue frozen in the high-FBS medium and in 20 of 26 grafts from tissue frozen in the low-FBS medium. The article prints the low-FBS figure as 74%, which does not match its own count of 20 of 26.
  • Grafts from fresh sarcomatoid tissue contained calretinin-positive mesothelioma cells consistent with the patient's diagnosis. Grafts from frozen epithelioid tissue retained mesothelioma cells and BAP1 status, with proliferating Ki67-positive cells and little evidence of apoptosis.
  • The only difference the authors observed between freezing media was a lower frequency of CD4+ and CD8+ T-cells in grafts from tissue frozen in the low-FBS medium.
  • FDG-PET uptake (accumulated standardized uptake value) was similar for grafts from the two freezing media, and the difference was not statistically significant. The high-FBS group had a mean of 30.52 (standard deviation 32.35, n=14) and the low-FBS group a mean of 34.41 (standard deviation 22.09, n=9), with p = 0.76.
  • Only 50% of the surplus biopsy samples collected were later confirmed as mesothelioma by the clinical diagnostic service.
  • Passaged grafts (cut in half and moved to new eggs) became vascularized, but the tissue did not visibly expand over the 7-day period.
• The authors' conclusion: The authors concluded that viable, vascularized mesothelioma grafts can be generated on the chick membrane from both fresh and frozen biopsy fragments, and that these grafts can be assessed for viability with PET/CT. They recommended the high-FBS freezing medium because a higher percentage of nodules became vascularized and immune cells appeared better preserved.
• Limitations the authors state:
  • Detailed data came from only three patients (one sarcomatoid, two epithelioid).
  • The egg model allows only a 7-day window from grafting to the end of the experiment, so animal models may still be needed for longer studies.
  • Egg survival to embryonic day 14 is variable.
  • Patient information such as age and gender was not accessed during method development.
  • The example graft from frozen epithelioid tissue had sparse tumor cell content.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Pleural Mesothelioma, Asbestos Exposure, Pemetrexed (Alimta), Chemotherapy, Immunotherapy, Prognosis, Mesothelioma, Histology.

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• Preclinical Efficacy of an Antibody-Drug Conjugate Targeting Mesothelin Correlates with Quantitative 89Zr-ImmunoPET — A.G. Terwisscha van Scheltinga, A. Ogasawara, et al. (2017)
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