Abstract 6613: Label-free multimodal multiphoton microscopy for predicting DNA damage response in patient derived non-small cell lung cancer organoids

Abstract Background: DNA damage response (DDR) is a fundamental mechanism for evaluating efficacy of cancer therapeutics, many of which target DNA or associated processes (e.g., etoposide: inhibits DNA synthesis, cisplatin: crosslinks DNA). Current techniques to evaluate DDR rely on immunostaining for gamma H2AX foci (γH2AX), which is an indicator of DNA double-strand break formation. While γH2AX immunostaining can provide a snapshot of DDR in fixed cellular and tissue samples, this method is technically cumbersome as: 1) temporal monitoring of DDR requires timepoint replicates; 2) extensive assay development efforts for 3D samples such as organoids, and 3) protocols for γH2AX immunostaining and its evaluation are time-consuming. The goal of this project is to reduce overall burden on assay duration and development in NSCLC organoids by leveraging label-free multiphoton imaging. Method: To predict DDR in 3D organoid samples, label-free multimodal multiphoton techniques such as simultaneous label-free autofluorescence multiharmonic (SLAM) imaging can provide intracellular information based on endogenous contrast mechanisms. SLAM makes use of a single laser to excite autofluorescence in multiple cellular components including FAD and NAD(P)H, enabling the calculation of the optical redox ratio, which estimates the equilibrium of electron donating versus accepting species in cells. Moreover, label-free, multiphoton systems enable imaging of live samples without the need to add labels or generate sacrificial samples and have improved image acquisition in 3D space over conventional confocal microscopy. Using SLAM, we propose to predict DDR in non-small cell lung cancer (NSCLC) organoids. NSCLC organoids recapitulate histological and genetic features of their derived patient tissue, retaining tumor variability. Results: Predictive modeling via gradient boost regressor between handcrafted SLAM image features and γH2AX immunostained images on weakly-paired images confirmed strong correlation between SLAM image features and γH2AX speckles/nuclei. Across multiple patient derived NSCLC organoid lines and multiple DNA targeting chemotherapeutics, the optical redox ratio was demonstrated to robustly predict DDR. Conclusion: Label-free imaging via SLAM can be used to predict DDR in live 3D NSCLC organoids with minimal sample handling and without the need to add labelled reporters, enabling quicker and less technically involved DDR assaying. Citation Format: Terrence T. Roh, Aneesh Alex, Janet E. Sorrells, Prasanna Chandramouleeswaran, Marina Marjanovic, Steve R. Hood, BanuPriya Sridharan, Stephen A. Boppart. Label-free multimodal multiphoton microscopy for predicting DNA damage response in patient derived non-small cell lung cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6613.