Visualization of macropinocytosis in prostate fibroblasts

Abstract

Macropinocytosis has emerged as an important mechanism for non-selective route to internalize extracellular fluids and dissolved molecules in eukaryotic cell. As fundamental cellular behavior, macropinocytosis plays specific and distinct roles in many physiological and pathological processes, such as nutrients uptake, antigen presentation, pathogen capture, and tumorigenesis. It supports tumorigenesis by providing metabolic needs to dividing cells in Ras driven cancer. In recent years, macropinocytosis has gained considerable interest in physiology and various diseases, including cancer, neurodegenerative diseases and atherosclerosis, which in turn has led to the discovery of new endocytic recycling systems. Approaches to assess macropinocytosis will provide insight into its underlying regulatory molecular mechanisms and enable the physiological control of macropinocytosis for controlled drug delivery and targeted cancer therapy. Macropinocytosis is an important phenomenon in Ras-expressing cancer cells and, recently, we have revealed a functional role for macropinocytosis in cancer associated fibroblasts (CAFs) fueling cancer cell growth. Here, we describe a protocol for detection of macropinocytosis in prostatic fibroblasts in vitro by utilizing fluorescently-labeled, lysinefixable, 70 kDa high molecular weight dextran. Macropinosomes are visualized as fluorescent intracellular puncta either by confocal or fluorescent microscopy. To follow, subsequent intracellular events and their underlying mechanisms after macropinosomes formation, we perform co-localization of quenched BSA (DQTM-BSA) along with dextran labeling in cancer associated fibroblasts. Our protocol provides a consistent way to understand macropinocytosis in wild type or genetic manipulated prostatic fibroblast.