Abstract 5282: Transition to and from a cancer stem-like state is marked by rapid metabolic shifts that are observable in real time using fluorescence lifetime imaging microscopy (FLIM)
Authors: Xu Tong , Arnesano Cosimo , Delfarah Alireza , Luo Zhifei , Delijani Kevin , Hodara Emmanuelle , Farnham Peggy , Graham Nicholas A. , Fraser Scott E. , Goldkorn Amir 
Published: August 2019

Abstract
Introduction: Highly tumorigenic, drug resistant cancer stem-like cells play a key role in therapy resistance and disease progression. We and others have previously shown that cancer cells can transition cyclically in and out of this aggressive phenotype in a manner that is rapid and spontaneous and does not require new mutations, drug selection, or ectopic gene expression. Phenotypic plasticity has been linked to altered cellular metabolic states, so we explored whether cancer cells exhibit distinct metabolic shifts as they switch to and from a cancer stem-like phenotype.
Materials and Methods: Bladder cancer cells (J82 cell line) were fractionated by Hoechst staining and FACS into a side population (SP) marked by high tumorigenicity and drug resistance, and a non-side population (NSP) lacking these characteristics. SP and NSP cells were profiled by Liquid Chromatography-Mass Spectrometry (LC-MS) metabolomics after labeling with [U-13C]-glucose or [U-13C]-glutamine, as well as by RNAseq. The results were analyzed by Metabolite Set Enrichment Analysis (MSEA) and Gene Set Enrichment Analysis (GSEA), respectively. Fluorescence Lifetime Imaging Microscopy (FLIM), which measures bound vs. free cellular NADH, was used to image SP and NSP cells separately or in co-culture with or without cisplatin.
Results: SP cells exhibited LC-MS MSEA profiles marked by upregulated TCA cycle activity and increased glutamine usage relative to NSP cells. Consistent with this finding, SP cells also exhibited RNAseq GSEA profiles marked by upregulated oxidative phosphorylation relative to NSP cells. Distinct FLIM metabolic signatures were successfully assigned to SP and NSP cells and used to image these subpopulations over time. When grown separately or in co-culture, SP cells’ FLIM signature gradually shifted to an NSP signature. Conversely, NSP cells’ FLIM signature gradually shifted an SP signature. When treated with cisplatin, SP cells survived and retained an unchanged SP FLIM signature. In contrast, fewer NSP cells survived cisplatin treatment, and NSP cells that did survive shifted towards an SP FLIM signature.
Conclusion: Spontaneous rapid transition to and from a cancer stem-like state involves a metabolic shift to and from oxidative phosphorylation and the TCA cycle. These distinct metabolic profiles can be tracked directly and noninvasively by FLIM, revealing that cancer cells spontaneously switch between the metabolic states over time but preferentially shift toward the cancer stem-like state when exposed to cisplatin. Thus, metabolic plasticity can be characterized, tracked and exploited therapeutically to disrupt cancer resistance and dissemination.
Citation Format: Tong Xu, Cosimo Arnesano, Alireza Delfarah, Zhifei Luo, Kevin Delijani, Emmanuelle Hodara, Peggy Farnham, Nicholas A. Graham, Scott E. Fraser, Amir Goldkorn. Transition to and from a cancer stem-like state is marked by rapid metabolic shifts that are observable in real time using fluorescence lifetime imaging microscopy (FLIM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5282.