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

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.

Keywords: 3Rs; Chorioallantoic membrane; PDX; PET/CT imaging; histology; immunohistochemistry; pleural mesothelioma; preclinical model.

Figure 1.. Preparation of patient tissue for parallel histological assessment and cryopreservation.

A, Workflow diagram of how tissue was processed. Figure created in Adobe Illustrator 2025 (Version 29.4 (64-bit), RRID:SCR_010279). B, Series of photographs showing tissue preparation. Pleural biopsy tissue (Bi) cut into different regions (Bii). A single region (Biii) cut into three equally sized sections (Biv). The middle section (purple box, “fresh-fixed”) was fixed in 10% NBF and the outer sections were cut into 1 mm ³ fragments (Bv) and slow-frozen in either high FBS or low FBS cryopreservation media ( Table 2). One piece from each section (blue box) served as a control for the freezing process.

Figure 2.. Technical considerations for egg experiments.

A, Brinsea egg incubator modifications. Dual humidity and temperature T-Scan probes are fitted inside the egg incubators via the front vent (Ai-ii). 5L plastic containers are used as water reservoirs to increase the water capacity (Aiii). B, Eggs are incubated horizontally and placed to ensure they are touching neighboring eggs and unable to roll.

Figure 3.. Scoring CAM-PDX for vascularization as a surrogate marker of engraftment.

Representative images of radial vessels (black arrowheads) surrounding the CAM-PDX in ovo and feeder vessels (white arrowheads) visible on the underneath of the CAM-PDX post-dissection. Scale bar represents 2.5 mm.

Figure 4.. Technical considerations for histology.

A, Workflow diagram of CAM-PDX processing for histology. CAM-PDX samples are embedded in paraffin so that sectioning the block results in sagittal sections through the tumor nodule and CAM (Ai-iii). Sections (here numbered 1-48 as an example) are placed onto slides in a configuration of 3 sections per slide that provides an overview through the xenograft with minimal slide staining; numbering (black text) refers to sequentially cut sections (Aiv). Sections are collected in up to 10 batches containing 8 slides that will be stained for different markers on contiguous slides. Figure created with Adobe Illustrator 2025 (Version 29.4 (64-bit), RRID:SCR_010279). B, One slide per batch was stained for pan-Cytokeratin to identify areas within the sample with detectable tumor cells (indicated by arrowheads), to identify the most suitable batch for staining for further markers. Scale bars represent 1000 μm.

Figure 5.. Preparation of eggs for preclinical PET/CT imaging.

A, Experimental timeline. PET/CT performed on embryonic day 13 (E13). B, Preparing the eggs for imaging. Radiotracer is injected intravenously, demonstrated here by injecting food dye at the point indicated by the arrow (Bi). Approximately 30 seconds after injection, food dye was completely distributed within the egg (Bii). Immediate compression (a few seconds after injection) should be used to stop any bleeding at the site of injection if required. Two eggs are fixed in position in the imaging bed with tape (Biii) and then loaded into the PET/CT scanner (Biv).

Figure 6.. PET data analysis.

A, User Interface of Invicro Vivoquant software showing different sectional planes (transverse, sagittal and coronal) from a reconstructed CAM-PDX. B, Input controls and operator settings for drawing the region of interest (ROI) manually (painting tool; sphere, size set to 3 pixel). C, CAM-PDX PET signal of example shown in A overlaid with manually drawn ROI. D, Linearity between voxel size and accumulated standardized uptake value (SUVacc) PET signal for different CAM-PDX underlined consistency in manual ROI drawing. Coefficient of determination; R ² = 0.90. Values used are reported in Table 9.

Figure 7.. Characterization of a sarcomatoid mesothelioma CAM-PDX generated from a fresh biopsy tissue sample.

A, Representative brightfield image of freshly dissected CAM-PDX (sarcomatoid; n = 15) at E14, imaged from underneath to show vascularization. Scalebar in overview image represents 2.5 mm and in adjacent zoom image 500 μm. Black arrowheads highlight CAM-PDX feeder vessels. B, Histological and immunohistochemical staining of CAM-PDX generated from fresh sarcomatoid tissue fragments. Images are for example shown in A and are representative of the following CAM-PDXs: H&E n = 6, CalB2 n = 6, BAP1 n = 1, cCasp3 n = 1, Ki67 n = 5, α-SMA n = 5, CD4 n = 5, CD8 n = 5. A minimum of 3 sections per stain per sample were used. Scalebars in overview images represent 500 μm and in adjacent zoom images 50 μm. Black arrowheads highlight Ki67 positive cells.

Figure 8.. Mesothelioma tumor cells are diffusely dispersed throughout the collected tissue.

Example of pan cytokeratin (panCK) staining of mesothelioma cells for three fresh-fixed regions from two patient biopsy samples (A) and three fragments that were cryopreserved prior to fixation from the same three regions (B). Images are representative for the following samples: R8 n = 7, R11 n = 2. A minimum of 3 sections were used per stain, per sample. Scale bars represent 500 μm.

Figure 9.. Comparison of fresh-fixed and slow-frozen epithelioid mesothelioma biopsy tissue.

Representative histological and immunohistochemical staining of fresh-fixed control tissue and tissue cryopreserved in high or low FBS freezing media. Fresh samples: H&E n = 15, panCK n = 15, BAP1 n = 5, cCasp3 n = 1, Ki67 n = 2, α-SMA n = 1, CD4 n = 1, CD8 n = 1. High FBS: H&E n = 9, panCK n = 10, BAP1 n = 2, cCasp3 n = 1, ki67 n = 2, α-SMA n = 1, CD4 n = 1, CD8 n = 1. Low FBS: H&E n = 7, panCK n = 7, BAP1 n = 2, cCasp3 n = 1, Ki67 n = 2, α-SMA n = 1, CD4 n = 1, CD8 n = 1. A minimum of 3 sections per stain per sample were used. Scalebars in overview images represent 500 μm and in adjacent zoom images 50 μm.

Figure 10.. Epithelioid mesothelioma CAM-PDX generated from cryopreserved biopsy tissue fragments.

A, Representative brightfield images of freshly dissected CAM-PDX at E14, generated from epithelioid mesothelioma tissue cryopreserved in high FBS freezing media ( Table 2, n = 22 CAM-PDX). CAM-PDX imaged from underneath to highlight vascularization. Scalebars in overview images represent 2.5 mm and 500 μm in adjacent zoom images. B, Histological and immunohistochemical staining of engrafted nodule from cryopreserved tissue. Images are representative of the following CAM-PDXs: H&E n = 15, panCK n = 10, BAP1 n = 5, cCasp3 n = 5, ki67 n = 9, α-SMA n = 5, CD4 n = 2, CD8 n = 2. A minimum of 3 sections per stain, per CAM-PDX were used. Scalebars in overview images represent 500 μm and in adjacent zoom images 50 μm.

Figure 11.. Epithelioid mesothelioma CAM-PDX generated from cryopreserved biopsy tissue fragments.

A, Representative brightfield images of freshly dissected CAM-PDX at E14, generated from epithelioid mesothelioma tissue cryopreserved in low FBS freezing medium ( Table 2, n = 20 CAM-PDX). CAM-PDX imaged from underneath to highlight vascularization. Scalebars in overview images represent 2.5 mm and in adjacent zoom images 500 μm. B, Histological and immunohistochemical staining of engrafted nodules from cryopreserved tissue. Images are representative of the following CAM-PDXs: H&E n = 15, panCK n = 10, BAP1 n = 5, cCasp3 n = 5, Ki67 n = 9, α-SMA n = 5, CD4 n = 2, CD8 n = 2. A minimum of 3 sections per stain per sample were used. Scalebars in overview images represent 500 μm and in adjacent zoom images 50 μm (B).

Figure 12.. Passaging of CAM-PDX.

Representative brightfield images of dissected CAM-PDX at embryonic day 14. Images show CAM-PDX generated from epithelioid mesothelioma tissue cryopreserved in high FBS (top, n = 3) or low FBS (bottom, n = 2) freezing media (P0) and the corresponding passaged CAM-PDX (P1; high FBS n = 2, low FBS n = 2). CAM-PDX imaged from underneath to highlight vascularization. Scale bars represent 2.5 mm.

Figure 13.. PET/CT imaging of epithelioid mesothelioma CAM-PDX generated from cryopreserved tissue.

A, Representative reconstruction of [ ¹⁸F]-FDG PET/CT imaging. Color scale represents tracer uptake (Bq/mL, Min = 0, Max = 2.5 × 10 ⁴). Scalebar in overview image represents 1 cm and in inset image 2.5 mm. B, PET quantification of [ ¹⁸F] _-FDG signal from CAM-PDX indicates no significant difference between fragments cryopreserved in high FBS (mean ± SD = 30.52 ± 32.35; n=14) or low FBS (mean ± SD = 34.41 ± 22.09; n = 9) media. Line represents population mean, Two Sample t-test, p=0.76, not significant (ns). Data was acquired from 5 independent experiments. Sample information and SUVacc values provided in Table 9.