Abstract 2337: Label-free single-cell drug response determined by fluorescence lifetime imaging microscopy (FLIM) and RNA sequencing
Authors: Ma Ning , Ren Hui , Ramalingam Naveen , King David , Larijani Banafshé , Jeffrey Stefanie S. 
Published: June 2022

Abstract
Colorectal cancer (CRC) is the second-leading cause of cancer-related death worldwide, and treatment failure in patients with metastatic CRC portends a poor prognosis. Early detection of chemoresistance would help reduce the usage of ineffective and possibly toxic therapies and facilitate alternative treatment strategies. We present a new technique combining phasor-fluorescence lifetime imaging microscopy (phasor-FLIM) and a machine learning algorithm that quantitatively identifies chemoresistant single cells, which may also be potentially applicable for evaluating heterogeneous circulating tumor cells (CTCs) obtained by liquid biopsy. We used parental HCT116 CRC cells sensitive to 5-fluorouracil (5-FU), a common chemotherapeutic agent used in CRC, to generate a cell line resistant to 5-FU; we confirmed resistance by a cell proliferation assay (MTS). We then analyzed phasor-FLIM patterns in sensitive and resistant cells before and after treatment with 5-FU of two endogenously fluorescent biomarkers, NADH and NADPH, which are key co-enzymes involved in energy metabolism. This was performed at 21% and 1% oxygen, which simulates both standard cell culture and tumor hypoxic conditions. Differences in phasor-FLIM signatures were found in resistant cells that suggested upregulation of the electron transport chain under normoxia (21% oxygen) and reprogrammed glucose metabolism under hypoxia (1% oxygen). Such changes were confirmed using NADH, lactate, and pyruvate production colorimetric tests, which are readouts of energy metabolism. To improve the quantification of changes in phasor-FLIM patterns, a new drug resistance indexing algorithm was developed to separate sensitive and resistant cells based on fluorescence lifetime multiparametric analysis of the intrinsic fluorescent biomarkers at the single-cell level. We then used single-cell total RNA sequencing (scRNA-Seq) to explore gene expression differences between sensitive and resistant HCT116 cells. Principal component analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP) clustering showed a clear separation of 5-FU-sensitive and -resistant cells. There was increased expression of proliferation markers in resistant cells and upregulation of genes involved in electron transport from NADPH to ferredoxin, consistent with our single-cell phasor-FLIM findings. We demonstrate proof of concept that the phasor-FLIM method combined with our new drug resistance indexing algorithm provides a marker-free technology for quantitatively assaying drug response and discriminating drug resistance at single-cell resolution.
Citation Format: Ning Ma, Hui Ren, Naveen Ramalingam, David King, Banafshé Larijani, Stefanie S. Jeffrey. Label-free single-cell drug response determined by fluorescence lifetime imaging microscopy (FLIM) and RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2337.