Distribution of Cholesterol and HER2 in Patient Breast Cancer Cells using Quantitative Single Molecule Localization Microscopy

Abstract

The nano-scale organization of cholesterol in the plasma membrane is important for cell signaling and function. Cholesterol content of the outer membrane has been assessed previously with domain 4 of the Perfringolysin O toxin (PFO-D4). Here, we used PFO-D4 (C459A)-Alexa Fluor 647 to image cholesterol with quantitative single molecule localization microscopy (qSMLM). We first assessed cholesterol in cultured breast cancer cells. SK-BR-3 cells exhibited cholesterol-enriched domains with an average domain size of 29 nm. We then assessed tumors from breast cancer patients using a combination of qSMLM and tissue touch preparation (touch prep-qSMLM). Patient tumor samples exhibited cholesterol-enriched domains; their sizes ranged from ∼20 to 110 nm. Additionally, the tumor samples were assessed for human epidermal growth factor receptor 2 (HER2) using fluorescently labeled trastuzumab. Importantly, HER2 copy numbers from traditional testing (e.g. fluorescence in situ hybridization) showed a significant positive correlation with detected densities from touch prep-qSMLM. In the future, these single molecule studies on HER2 and cholesterol may help clarify mechanisms of oncogenic signaling and, ultimately, resistance to therapy.