Abstract 2461: Integrated lifetime and spectral phasor imaging of biomarker expression and metabolism in hormone receptor positive breast cancer models
Authors: Torrado Belen , Vallmitjana Alexander , Olukoya Ayodeji , Bahnassy Shaymaa , Stires Hillary , Rozeboom Aaron , Riggins Rebecca B. , Ranjit Suman 
Published: June 2022

Abstract
In HR+ (hormone receptor positive) breast cancer, endocrine therapies that directly antagonize estrogen receptor or reduce synthesis of its ligand can be highly effective, but endocrine-resistance occurs frequently. Metabolic dysregulation is a mechanistic driver of endocrine-resistant HR+ breast cancer, but most existing methods cannot be multiplexed with assays for biomarker expression. Spectral phasor imaging of multiplex immunofluorescence (mIF) staining for expression of hormone receptors and other biomarkers combined with phasor lifetime analysis of NADH to quantify metabolic state will provide integrated spatial maps of metabolism and biomarker expression in endocrine-resistant HR breast cancer. We used the HCI-013 (013) and HCI-013EI (013EI) patient-derived xenograft (PDX) system, derived from a HR+, invasive lobular metastatic breast cancer which harbors the Y537S ESR1 mutation. Tumors were stained with a multiplex immunofluorescence (mIF) panel for pan-cytokeratin, ER, PR, HER2, and Ki67, detected by spectrally separate OPAL dyes. Hyperspectral phasor fluorescence spectra were obtained using successive single photon excitation at 488 nm, 561 nm and 640 nm and using multiphoton excitation at 1024 nm. Single photon excitation excites specific dyes, and multiphoton excites the entire panel of dyes simultaneously. The emission was collected using a Zeiss 880 laser scanning microscope equipped with a 32 channel spectral detector. Phasor coordinates of each component were resolved into fractional intensities of the five genes. In the same tissues and sections, phasor fluorescence lifetime imaging (FLIM) of NADH was used to quantify metabolic heterogeneity. NADH is present in two forms: free and protein-bound NADH. They have different fluorescence lifetimes and the ratio of these two species are correlated with NAD+/NADH ratio. Free NADH is associated with increased glycolysis. We observed greater metabolic heterogeneity in 013EI PDX samples, with similar levels of free NADH (glycolysis) at the tumor border, but significantly increased bound NADH (oxidative phosphorylation) in the tumor core. Spectral phasor data show differential expression of PR and PanCK (for 640 nm excitation), and HER2 and ERalpha (for 561 nm excitation) in the edge and center of cancer in 013 and 013EI variants. Our results show how a combination of spectral phasor and phasor-FLIM can be used to define metabolic changes in endocrine-resistant, HR+ PDX models and correlate them with biomarker expression. Ongoing work is quantitatively mapping these measurements at single-pixel resolution, and applying this imaging workflow to RNAscope in situ hybridization assays for gene expression. Combining multiphoton spectral (RNAscope) and FLIM (NADH) will allow pixelwise correlation of the gene expression and metabolic changes.
Citation Format: Belen Torrado, Alexander Vallmitjana, Ayodeji Olukoya, Shaymaa Bahnassy, Hillary Stires, Aaron Rozeboom, Rebecca B. Riggins, Suman Ranjit. Integrated lifetime and spectral phasor imaging of biomarker expression and metabolism in hormone receptor positive breast cancer models [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 2461.