title: Determining the macropinocytic index of cells through a quantitative image-based assay.
abstract: Macropinocytosis serves as an internalization pathway for extracellular fluid and its contents. Macropinocytosis is upregulated in oncogene-expressing cells and, recently, we have revealed a functional role for macropinocytosis in fueling cancer cell growth through the internalization of extracellular albumin, which is degraded into a usable source of intracellular amino acids. Assessing macropinocytosis has been challenging in the past because of the lack of reliable assays capable of quantitatively measuring this uptake mechanism. Here we describe a protocol for visualizing and quantifying the extent of macropinocytosis in cells both in culture and growing in vivo as tumor xenografts. By using this approach, the 'macropinocytic index' of a particular cell line or subcutaneous tumor can be ascertained within 1-2 d. The protocol can be carried out with multiple samples in parallel and can be easily adapted for a variety of cell types and xenograft or allograft mouse models.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York, USA.
authors.forename: Cosimo
authors.initials: C
authors.lastname: Commisso
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York, USA.
authors.forename: Rory J
authors.initials: RJ
authors.lastname: Flinn
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York, USA.
authors.forename: Dafna
authors.initials: D
authors.lastname: Bar-Sagi
mesh_terms: D000418:Albumins / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D064593:Heterografts / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D008856:Microscopy, Fluorescence / Q000379:methods*
chemical_list: D000418:Albumins