Date Published: July 3, 2017
Publisher: Public Library of Science
Author(s): Florian Guisier, Pierre Bohn, Maxime Patout, Nicolas Piton, Insaf Farah, Pierre Vera, Luc Thiberville, Mathieu Salaün, Aamir Ahmad.
Prediction of treatment outcome of non-small cell lung cancer (NSCLC) with EGFR inhibitors on the basis of the genetic analysis of the tumor can be incorrect in case of rare or complex mutations, bypass molecular activation pathways, or pharmacodynamic variations. The aim of this study was to develop an ex vivo and in vivo real-time quantitative imaging test for EGFR inhibitors sensitivity assessment.
Erlotinib resistant (A549, H460, H1975), insensitive (H1650) and hypersensitive (HCC827) cell lines were injected subcutaneously in Nude mice. Tumor xenografts from mice treated with Erlotinib were imaged ex vivo and in vivo using probe-based confocal laser endomicroscopy (pCLE) and NucView 488 Caspase 3 substrate, a fluorescent probe specific for the activated caspase 3.
Assessment of apoptosis at 24h post treatment, both ex vivo in explanted tumor xenografts and in vivo, showed a significant difference between resistant cell lines (A549, H460 and H1975) and insensitive (H1650) or hypersensitive (HCC827) ones (p<0.05 for ex vivo imaging, p≤0.02 for in vivo imaging). There was also a significant difference between insensitive and hypersensitive cell lines, both ex vivo (p<0.05) and in vivo (p = 0.01). Real-time in vivo and ex vivo assessment of apoptosis using pCLE differentiates resistant from sensitive NSCLC xenografts to Erlotinib.
Over the past decade, identification of oncogenic molecular abnormalities in non-small-cell lung cancer (NSCLC), such as Epidermal Growth Factor Receptor (EGFR) activating mutations, has deeply changed the management of patients with advanced disease . EGFR mutations convey constitutive activation of the EGFR and its downstream signaling pathways. Tumor cells bearing these mutations become highly dependent of the EGFR signal and thus are highly sensitive to EGFR tyrosine kinase inhibitors (EGFR-TKIs).
Our study shows that an in vivo in situ real-time imaging of apoptosis using pCLE enables to differentiate sensitive from resistant tumors to Erlotinib in a mouse model of lung adenocarcinoma, as early as 24 hours after treatment initiation.
This study shows that micro-imaging of apoptosis using pCLE with C3-NucView enables the differentiation of hypersensitive, insensitive and resistant tumors to Erlotinib, both in vivo and ex vivo. Although the technique was applied to Erlotinib sensitivity assessment only, it can be anticipated that it can be used for other drugs that induce apoptosis. If developed for ex-vivo analysis of patients’ samples, giving the growing number of targeted therapies in NSCLC and other tumors -with different resistance mechanisms for several of them-, a unique assessment method could be preferred to multiple genetic analysis, as it limits implementation costs and allows immediate testing for new molecules.