@article{f01fc48d8ea9404cba48eb33102d348d,
title = "Predicting DNA damage response in non-small cell lung cancer organoids via simultaneous label-free autofluorescence multiharmonic microscopy",
abstract = "The DNA damage response (DDR) is a fundamental readout for evaluating efficacy of cancer therapeutics, many of which target DNA associated processes. Current techniques to evaluate DDR rely on immunostaining for phosphorylated histone H2AX (γH2AX), which is an indicator of DNA double-strand breaks. While γH2AX immunostaining can provide a snapshot of DDR in fixed cell and tissue samples, this method is technically cumbersome due to temporal monitoring of DDR requiring timepoint replicates, extensive assay development efforts for 3D cell culture samples such as organoids, and time-consuming protocols for γH2AX immunostaining and its evaluation. The goal of this current study is to reduce overall burden on assay duration and development in non-small cell lung cancer (NSCLC) organoids by leveraging label-free multiphoton imaging. In this study, simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy was used to provide rich intracellular information based on endogenous contrasts. SLAM microscopy enables imaging of live samples eliminating the need to generate sacrificial sample replicates and has improved image acquisition in 3D space over conventional confocal microscopy. Predictive modeling between label-free SLAM microscopy and γH2AX immunostained images confirmed strong correlation between SLAM image features and γH2AX signal. Across multiple DNA targeting chemotherapeutics and multiple patient-derived NSCLC organoid lines, the optical redox ratio and third harmonic generation channels were used to robustly predict DDR. Imaging via SLAM microscopy can be used to more rapidly predict DDR in live 3D NSCLC organoids with minimal sample handling and without labeling.",
keywords = "DNA damage response, Label-free imaging, Organoids",
author = "Roh, \{Terrence T.\} and Aneesh Alex and Chandramouleeswaran, \{Prasanna M.\} and Sorrells, \{Janet E.\} and Alexander Ho and Iyer, \{Rishyashring R.\} and Spillman, \{Darold R.\} and Marina Marjanovic and Ekert, \{Jason E.\} and Sridharan, \{Banu Priya\} and Balabhaskar Prabhakarpandian and Hood, \{Steve R.\} and Boppart, \{Stephen A.\}",
note = "This research was supported by GSK through the Center for Optical Molecular Imaging at the Beckman Institute for Advanced Science and Technology on the campus of the University of Illinois at Urbana-Champaign. To validate SLAM based DDR prediction in organoids, the prediction model was implemented on 3D NSCLC (HUB047) organoids treated with etoposide, bortezomib, and two new DNA damaging compounds, cisplatin and olaparib. SLAM microscopy of 3D organoids embedded in Matrigel leverages optical sectioning at defined z-depths, generating 2D image slices of the 3D structure ( Figs. 1c and 5a). A prediction-based DDR approach with SLAM microscopy enabled collection of longitudinal DDR data from the same samples, whereas \textbackslash{}u03B3H2AX foci labeling required sacrificial samples for each timepoint (Sfig. 4a and b). Implementation of the trained prediction model on SLAM images acquired from 3D organoids show that etoposide and cisplatin induce DDR but not olaparib (Fig. 5d). A separate parallel experiment where \textbackslash{}u03B3H2AX foci labeling and quantification was performed similarly show increased DDR with etoposide and cisplatin but not olaparib in this NSCLC organoid line (Fig. 5b and c). Olaparib, a PARP inhibitor known for synthetic lethal interactions in BRCA-mutated tumors, showed no significant effects, which was supported by the lack of BRCA mutations in this organoid line. The DDR prediction model also correctly identified bortezomib treatment resulting in decreased DDR at days 2 and 5 (Fig. 5c and d). In agreement with previous observations ( Figs. 3b and 4d), increases in the AUC-RR feature tracked closely with the treatment effect from DNA damaging agents (Fig. 5e). The performance of this model in this validation study supports its translation from 2D to 3D organoid formats and across DNA damaging treatments in HUB047 organoids.",
year = "2024",
month = sep,
doi = "10.1016/j.redox.2024.103280",
language = "English (US)",
volume = "75",
journal = "Redox Biology",
issn = "2213-2317",
publisher = "Elsevier B.V.",
}