Volatile organic compound analysis of malignant pleural mesothelioma chorioallantoic membrane xenografts

Abstract

Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with asbestos exposure. MPM is often diagnosed late, at a point where limited treatment options are available, but early intervention could improve the chances of successful treatment for MPM patients. Biomarkers to detect MPM in at-risk individuals are needed to implement early diagnosis technologies. Volatile organic compounds (VOCs) have previously shown diagnostic potential as biomarkers when analysed in MPM patient breath. In this study, chorioallantoic membrane (CAM) xenografts of MPM cell lines were used as models of MPM tumour development for VOC biomarker discovery with the aim of generating targets for investigation in breath, biopsies or other complex matrices. VOC headspace analysis of biphasic or epithelioid MPM CAM xenografts was performed using solid-phase microextraction and gas chromatography-mass spectrometry. We successfully demonstrated the capture, analysis and separation of VOC signatures from CAM xenografts and controls. A panel of VOCs was identified that showed discrimination between MPM xenografts generated from biphasic and epithelioid cells and CAM controls. This is the first application of the CAM xenograft model for the discovery of VOC biomarkers associated with MPM histological subtypes. These findings support the potential utility of non-invasive VOC profiling from breath or headspace analysis of tissues for detection and monitoring of MPM.

Keywords: CAM model; biomarkers; cancer biomarkers; mesothelioma; volatile organic compounds; xenograft.

Figure 1.

Mesothelioma xenografts generated on the CAM. (A) Representative tumour nodules for each mesothelioma cell line evaluated. Bioluminescent signal (top) and corresponding brightfield (BF) image taken post dissection (bottom). Bioluminescence signal is shown as total flux (radiance: p s⁻¹ cm⁻² sr⁻¹). Colour scale: min = 3.79 × 10⁵, max = 1.151 × 10⁷. Scale bar = 500 µm. (B) Representative images of CAM controls acquired prior to dissection.

Figure 2.

PCA (A) and PLS-DA (B) score plots of VOCs from control CAM, mock CAM and CAM xenografts of the mesothelioma cell lines 7 T, 8 T and 12 T. Each point represents the VOC profile from a single sample after normalisation and background filtering.

Figure 3.

Heat-map of peak intensities (log10) for significantly altered volatile organic compounds (VOCs) between control, mock, 7 T, 8 T and 12 T CAM groups. VOCs were first determined to be significantly different from their relative RNAlater controls (Students T-test p < 0.05), these significantly different VOCs were then compared between groups using multivariate (partial least-squares discrimination analysis) and univariate analysis (Students T-test, p < 0.05).

Figure 4.

Multivariate receiver operating characteristic (ROC) curves created by MetaboAnalyst 6.0 from 5 different biomarker models considering different numbers of features (2–19). (A) MPM CAM xenograft groups (7 T, 8 T, 12 T) versus CAM controls (mock, control) and (B) epithelioid MPM (8 T & 12 T) versus biphasic MPM (7 T) CAM xenografts.