Single-Objective Multiphoton Light-sheet Microscopy for Lung Cancer Organoid Screening

Cancer organoids are three-dimensional “tumors in dish” which not only can be used to study tumor heterogeneity but can also predict drug response at the individual-patient level. Optimal treatment strategies for individual patients can be formulated based on rapid drug testing among patient-derived organoids (PDO), thus leading to reduced treatment toxicity and prolong patients’ survival. Although PDO provides a physiologically relevant in-vitro 3D model for testing drug response, current organoid imaging methods suffer from shallow penetration depth (single-photon excitation), low speed (single-point scanning), and severe photobleaching and phototoxicity (high peak excitation power for point scanning). Despite the fact that two-photon light-sheet microscopy can overcome many of these limitations and have been applied to image multicellular spheroids, the current methods require the spheroids to be mounted in a manner that precludes high-density microplate imaging. Here we aim to develop a high-speed organoid functional imaging tool that can be used to monitor the dynamic changes of metabolic states of the lung cancer organoids under EGFR inhibitor treatment. Whereas lung cancer patients’ response to EGFR inhibitors can be predicted by genetic tests, resistance to EGFR inhibitors can occur by activation of various molecules (MET or HER2) in different cancer cells, rendering the identification of an effective treatment for resistant tumors complicated. About 14% of patients without EGFR mutation still responded to the EGFR inhibitor treatment, highlighting the limitation of genetic test in drug response prediction of today's lung cancer management. With fluorescence lifetime analysis capability, large imaging depth and compatibility with high-density microplates, our imaging tool will facilitate analysis of mass-produced PDO. The end goal is to identify patients that will respond to a particular drug treatment within a clinically meaningful time frame.