Quantitative Assessment of Macropinocytosis in mTORC1-Hyperactive Cells using Flow Cytometry

Summary

This protocol provides experimental tools to evaluate macropinocytic uptake of nutrients (carbohydrate and protein) by mTORC1-hyperactive cells. Detailed steps to quantify the uptake of fluorescently labeled dextran and bovine serum albumin (BSA) are described.

Abstract

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. In proliferating cells, macropinocytosis can deliver extracellular nutrients to the lysosome, processed into critical macromolecule building blocks. Recent studies have highlighted the dependence of multiple cancers on macropinocytosis, including breast, colorectal and pancreatic cancer. Ras mutations are thought to be the driver events behind macropinocytosis initiation, leading to the activation of cellular anabolic processes via the mTORC1 signaling pathway. Interestingly, mTORC1 can also be activated by macropinocytosis independently of Ras. Therefore, macropinocytosis represents a metabolic vulnerability that can be leveraged to target macropinocytic tumors by limiting their access to nutrients therapeutically.

In Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM), mTORC1-hyperactivation leads to enhanced macropinocytosis and metabolic reprogramming. Here, we describe a flow cytometry-based protocol to assess macropinocytosis in mammalian cells quantitatively. TSC2-deficient MEFs are employed, which exhibit aberrant activation of mTORC1 and have been shown to have increased macropinocytosis compared to TSC2-expressing cells. Cells treated with pharmacologic inhibitors of macropinocytosis are incubated with fluorescently labeled, lysine-fixable, 70 kDa dextran, or fluorescently labeled bovine serum albumin (BSA) assayed by flow cytometry. To date, robust image-based techniques have been developed to quantitatively assess macropinocytosis in tumor cells in vitro and in vivo. This analysis provides a quantitative assessment of macropinocytosis in multiple experimental conditions and complements existing image-based techniques.

Introduction

Macropinocytosis is an endocytic process dedicated to the bulk uptake of extracellular material followed by the formation of macropinosomes, either recycled to the plasma membrane or fusing with lysosomes to degrade the internalized cargo^1,2. Although cargo uptake is non-selective, macropinocytosis is a multi-step process, tightly regulated by Rab GTPases and membrane phospholipids^3,4. Notably, cancer cells employ macropinocytosis to internalize extracellular nutrients, including proteins, polysaccharides and lipids. Macropinocytosis in cancer cells is....

Protocol

1. Cell treatment

Day 1

1. Seed TSC2-deficient and TSC2-expressing mouse embryonic fibroblasts (MEFs) in triplicate, in each well of a six-well tissue culture plate using DMEM, supplemented with 10% FBS. Cells should be 60-70% confluent by day 3.
   1. Seed additional control wells for each drug condition that will not be stained with FITC-Dextran or TMR-BSA.

Day 2

2. Carefully aspirate media and rinse cells twice with PBS at room temperature.
   1. Treat cells with vehicle (DMSO), 100 µM of phosphatidic acid (PA), 25 µM of....

Results

Ritanserin inhibits macropinocytosis in TSC2-deficient cells
We have previously shown that macropinocytic uptake of nutrients is increased by three-fold in TSC2-deficient cells compared to TSC2-expressing cells¹⁶. In TSC and LAM, macropinocytosis is mediated via diacylglycerol kinase alpha (DGKA). The metabolic product of DGKA is phosphatidic acid (PA), a crucial component of cellular membranes. Therefore, we hypot.......

Discussion

Here, we describe a quantitative approach to assess macropinocytosis using flow cytometry. This method provides an accurate and rapid measurement of the fluorescently labeled macropinocytic cargo dextran and albumin. Prior studies have carefully characterized the macropinocytic index of cancer cells using confocal microscopy approaches^15,20. Although these methods accurately quantify the internalization, size and spacial distribution of macropinosomes, they .......

Materials

Name	Company	Catalog Number	Comments
DMEM	Gibco	11965-092	Growth media
EIPA (amiloride)	Sigma Aldrich	A3085	Macropinocytosis inhibitor
FBS	R&D Systems	S11150	Fetal Bovine Serum
FITC-Dextran	Invitrogen	D1822	Fluorescent polysaccharide (70kDa)
Parafolmadehyde	Pierce	28906	Fixation agent
PBS	Gibco	10010-023	Phosphate Buffer Saline
Penicilin/Streptomycin	Sigma Aldrich	P4458-100ML	Cell culture antibiotics
Phosphatidic Acid	Avanti	840101P	Phospholipid derived from egg
Ritanserin	Tocris	1955	DGKA inhibitor
TMR-BSA	Invitrogen	A23016	Fluorescent albumin
Trypsin	Sigma Aldrich	25300-054	Dissociation agent