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Determining the macropinocytic index of cells through a quantitative image-based assay

Nature Protocols volume 9, pages 182–192 (2014)Cite this article

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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.

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Figure 1: Representative images typically obtained from an in vitro macropinocytosis assay.
Figure 2: Representative images typically obtained from an in vivo macropinocytosis assay.

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Acknowledgements

We are grateful to members of the Bar-Sagi laboratory for their comments and discussions. This work was supported by US National Institutes of Health (NIH) grant no. R01CA055360 to D.B.-S. C.C. was supported by a Canadian Institutes of Health Research postdoctoral fellowship and an American Association for Cancer Research postdoctoral fellowship provided by the Pancreatic Cancer Action Network. R.J.F. was supported by a NIH NCI NRSA F32 Individual Fellowship F32CA171877. All animal care and procedures were approved by the Institutional Animal Care and Use Committee at New York University (NYU) School of Medicine. The Histopathology Core of NYU School of Medicine is partially supported by the NIH (grant no. 5 P30CA016087-32). Troma I, an antibody that recognizes CK8, was contributed by P. Brulet and R. Kemler and made available by the Developmental Studies Hybridoma Bank under the auspices of the Eunice Kennedy Shriver National Institute of Child Health and Human Development.

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  1. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York, USA

    Cosimo Commisso, Rory J Flinn & Dafna Bar-Sagi

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  1. Cosimo Commisso
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  2. Rory J Flinn
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Contributions

C.C., R.J.F. and D.B.-S. contributed to the experimental design. C.C. performed the experiments and data analysis. C.C., R.J.F. and D.B.-S. wrote the manuscript.

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Correspondence to Cosimo Commisso or Dafna Bar-Sagi.

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The authors declare no competing financial interests.

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Commisso, C., Flinn, R. & Bar-Sagi, D. Determining the macropinocytic index of cells through a quantitative image-based assay. Nat Protoc 9, 182–192 (2014). https://doi.org/10.1038/nprot.2014.004

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