RESUMEN
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.
Asunto(s)
Membrana Corioalantoides , Cromatografía de Gases y Espectrometría de Masas , Neoplasias Pulmonares , Mesotelioma Maligno , Neoplasias Pleurales , Compuestos Orgánicos Volátiles , Compuestos Orgánicos Volátiles/análisis , Animales , Humanos , Mesotelioma Maligno/patología , Neoplasias Pleurales/patología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Biomarcadores de Tumor/análisis , Mesotelioma/patología , Línea Celular Tumoral , Xenoinjertos , Pruebas Respiratorias/métodos , Microextracción en Fase Sólida/métodosRESUMEN
This is the first report of the use of laser ablation-inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS) to analyze human malignant pleural mesothelioma (MPM) samples at the cellular level. MPM is an aggressive, incurable cancer associated with asbestos exposure, with a long latency and poor overall survival. Following careful optimization of the laser fluence, the simultaneous ablation of soft biological tissue and hard mineral fibers was possible, allowing the spatial detection of elements such as Si, Mg, Ca, and Fe, which are also present in the glass substrate. A low-dispersion LA setup was employed, which provided the high spatial resolution necessary to identify the asbestos fibers and fiber fragments in the tissue and to characterize the metallome at the cellular level (a pixel size of 2 µm), with a high speed (at 250 Hz). The multielement LA-ICP-TOFMS imaging approach enabled (i) the detection of asbestos fibers/mineral impurities within the MPM tissue samples of patients, (ii) the visualization of the tissue structure with the endogenous elemental pattern at high spatial resolution, and (iii) obtaining insights into the metallome of MPM patients with different pathologies in a single analysis run. Asbestos and other mineral fibers were detected in the lung and pleura tissue of MPM patients, respectively, based on their multielement pattern (Si, Mg, Ca, Fe, and Sr). Interestingly, strontium was detected in asbestos fibers, suggesting a link between this potential toxic element and MPM pathogenesis. Furthermore, monitoring the metallome around the talc deposit regions (characterized by elevated levels of Al, Mg, and Si) revealed significant tissue damage and inflammation caused by talc pleurodesis. LA-ICP-TOFMS results correlated to Perls' Prussian blue and histological staining of the corresponding serial sections. Ultimately, the ultra-high-speed and high-spatial-resolution capabilities of this novel LA-ICP-TOFMS setup may become an important clinical tool for simultaneous asbestos detection, metallome monitoring, and biomarker identification.
Asunto(s)
Amianto , Terapia por Láser , Mesotelioma Maligno , Amianto/toxicidad , Humanos , Espectrometría de Masas/métodos , Análisis EspectralRESUMEN
RATIONALE: Malignant pleural mesothelioma is an extremely aggressive and incurable malignancy associated with prior exposure to asbestos fibres. Difficulties remain in relation to early diagnosis, notably due to impeded identification of asbestos in lung tissue. This study describes a novel laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) imaging approach to identify asbestos within mesothelioma models with clinical significance. METHODS: Human mesothelioma cells were exposed to different types of asbestos fibres and prepared on plastic slides for LA-ICP-MS analysis. No further sample preparation was required prior to analysis, which was performed using an NWR Image 266 nm laser ablation system coupled to an Element XR sector-field ICP mass spectrometer, with a lateral resolution of 2 µm. Data was processed using LA-ICP-MS ImageTool v1.7 with the final graphic production made using DPlot software. RESULTS: Four different mineral fibres were successfully identified within the mesothelioma samples based on some of the most abundant elements that make up these fibres (Si, Mg and Fe). Using LA-ICP-MS as an imaging tool provided information on the spatial distribution of the fibres at cellular level, which is essential in asbestos detection within tissue samples. Based on the metal counts generated by the different types of asbestos, different fibres can be identified based on shape, size, and elemental composition. Detection of Ca was attempted but requires further optimisation. CONCLUSIONS: Detection of asbestos fibres in lung tissues is very useful, if not necessary, to complete the pathological dt9iagnosis of asbestos-related malignancies in the medicolegal field. For the first time, this study demonstrates the successful application of LA-ICP-MS imaging to identify asbestos fibres and other mineral fibres within mesothelioma samples. Ultimately, high-resolution, fast-speed LA-ICP-MS analysis has the potential to be integrated into clinical workflow to aid earlier detection and stratification of mesothelioma patient samples.
Asunto(s)
Amianto , Neoplasias Pulmonares , Espectrometría de Masas/métodos , Mesotelioma Maligno , Microscopía/métodos , Amianto/análisis , Amianto/química , Línea Celular Tumoral , Humanos , Rayos Láser , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/patología , Mesotelioma Maligno/diagnóstico por imagen , Mesotelioma Maligno/patologíaRESUMEN
BACKGROUND: Mortality rates for leukemia are high despite considerable improvements in treatment. Since polyphenols exert pro-apoptotic effects in solid tumors, our study investigated the effects of polyphenols in haematological malignancies. The effect of eight polyphenols (quercetin, chrysin, apigenin, emodin, aloe-emodin, rhein, cis-stilbene and trans-stilbene) were studied on cell proliferation, cell cycle and apoptosis in four lymphoid and four myeloid leukemic cells lines, together with normal haematopoietic control cells. METHODS: Cellular proliferation was measured by CellTiter-Glo(®) luminescent assay; and cell cycle arrest was assessed using flow cytometry of propidium iodide stained cells. Apoptosis was investigated by caspase-3 activity assay using flow cytometry and apoptotic morphology was confirmed by Hoescht 33342 staining. RESULTS: Emodin, quercetin, and cis-stilbene were the most effective polyphenols at decreasing cell viability (IC50 values of 5-22 µM, 8-33 µM, and 25-85 µM respectively) and inducing apoptosis (AP50 values (the concentration which 50% of cells undergo apoptosis) of 2-27 µM, 19-50 µM, and 8-50 µM respectively). Generally, lymphoid cell lines were more sensitive to polyphenol treatment compared to myeloid cell lines, however the most resistant myeloid (KG-1a and K562) cell lines were still found to respond to emodin and quercetin treatment at low micromolar levels. Non-tumor cells were less sensitive to all polyphenols compared to the leukemia cells. CONCLUSIONS: These findings suggest that polyphenols have anti-tumor activity against leukemia cells with differential effects. Importantly, the differential sensitivity of emodin, quercetin, and cis-stilbene between leukemia and normal cells suggests that polyphenols are potential therapeutic agents for leukemia.