Development and characterization of phasor-based analysis for FLIM to evaluate the metabolic and epigenetic impact of HER2 inhibition on squamous cell carcinoma cultures.

Dan L Pham,Dominick M Myers,Molly S Myers,Christina R Miller,Michael G Nichols,Laura A Hansen

doi:10.1117/1.jbo.26.10.106501

Abstract

.Significance: Deranged metabolism and dysregulated growth factor signaling are closely associated with abnormal levels of proliferation, a recognized hallmark in tumorigenesis. Fluorescence lifetime imaging microscopy (FLIM) of endogenous nicotinamide adenine dinucleotide (NADH), a key metabolic coenzyme, offers a non-invasive, diagnostic indicator of disease progression, and treatment response. The model-independent phasor analysis approach leverages FLIM to rapidly evaluate cancer metabolism in response to targeted therapy.Aim: We combined lifetime and phasor FLIM analysis to evaluate the influence of human epidermal growth factor receptor 2 (HER2) inhibition, a prevalent cancer biomarker, on both nuclear and cytoplasmic NAD(P)H of two squamous cell carcinoma (SCC) cultures. While better established, the standard lifetime analysis approach is relatively slow and potentially subject to intrinsic fitting errors and model assumptions. Phasor FLIM analysis offers a rapid, model-independent alternative, but the sensitivity of the bound NAD(P)H fraction to growth factor signaling must also be firmly established.Approach: Two SCC cultures with low- and high-HER2 expression, were imaged using multiphoton-excited NAD(P)H FLIM, with and without treatment of the HER2 inhibitor AG825. Cells were challenged with mitochondrial inhibition and uncoupling to investigate AG825’s impact on the overall metabolic capacity. Phasor FLIM and lifetime fitting analyses were compared within nuclear and cytoplasmic compartments to investigate epigenetic and metabolic impacts of HER2 inhibition.Results: NAD(P)H fluorescence lifetime and bound fraction consistently decreased following HER2 inhibition in both cell lines. High-HER2 SCC74B cells displayed a more significant response than low-HER2 SCC74A in both techniques. HER2 inhibition induced greater changes in nuclear than cytoplasmic compartments, leading to an increase in NAD(P)H intensity and concentration.Conclusions: The use of both, complementary FLIM analysis techniques together with quantitative fluorescence intensity revealed consistent, quantitative changes in NAD(P)H metabolism associated with inhibition of growth factor signaling in SCC cell lines. HER2 inhibition promoted increased reliance on oxidative phosphorylation in both cell lines.

Highlights

Cellular proliferation is strictly regulated by both the intracellular and extracellular environment
The use of both, complementary Fluorescence lifetime imaging microscopy (FLIM) analysis techniques together with quantitative fluorescence intensity revealed consistent, quantitative changes in NAD(P)H metabolism associated with inhibition of growth factor signaling in squamous cell carcinoma (SCC) cell lines
human epidermal growth factor receptor 2 (HER2) targeting is a well-accepted treatment regimen for HER2 positive breast cancer, and HER1/EGFR targeting is commonly used in head and neck squamous cell carcinoma (HNSCC), where overexpression of EGFR occurs in 80% to 90% of tumors and is associated with poor outcomes.[2]

Summary

Introduction

Cellular proliferation is strictly regulated by both the intracellular and extracellular environment. Cellular metabolism is responsible for energy generation and macromolecule synthesis in cell growth and division, the growth-factor signaling pathway drives the cell cycle and induces epigenetic modifications that initiate and facilitate proliferation. Deranged metabolism and dysregulated growth factor signaling are closely associated with abnormal levels of proliferation, a commonly recognized hallmark in tumorigenesis. Within the growth factor receptor family, human epidermal growth factor receptor 2 (HER2) is overexpressed in breast, skin, gastric, and gastroesophageal cancers, with high-HER2 activity associated with worse prognosis and treatment outcome.[1] HER2 targeting is a well-accepted treatment regimen for HER2 positive breast cancer, and HER1/EGFR targeting is commonly used in head and neck squamous cell carcinoma (HNSCC), where overexpression of EGFR occurs in 80% to 90% of tumors and is associated with poor outcomes.[2] The use of HER2 therapy in cutaneous squamous cell carcinoma (cSCC) has not been thoroughly explored, due to skin cancer’s commonly benign nature, most do not require adjuvant treatment. Skin cancer accounts for more new cases diagnosed than every other cancer combined and SCCs are the second most common subtype.[3,4,5] The majority of skin cancer cases can be attributed to ultraviolet (UV) radiation, and previous studies have found that chronic UV exposure stimulates HER2 dimerization and activation through UV-induced reactive oxygen species (ROS).[6,7,8,9]

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