Abstract
Macropinocytosis is a mechanism of fluid-phase endocytosis that functions in the nonspecific internalization of extracellular fluid. This uptake pathway has specialized roles in different cell types and organisms, and its importance has recently been established in several diseases, including cancer. In cancer, macropinocytosis is stimulated by oncogenes, such as Ras, and macropinocytic cargo is targeted to lysosomes for degradation, providing a catabolic route for tumor cells to obtain amino acids from the tumor microenvironment. Here, we describe a protocol to employ fluorescently labeled dextran molecules in order to visualize and quantify the extent of macropinocytosis in pancreatic tumors. Multiple samples can be processed in parallel by this method, and the protocol can be easily customized for pancreatic tumor tissue isolated from subcutaneous, orthotopic and genetically engineered mouse models (GEMM), or human patients.
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References
Kerr MC, Teasdale RD (2009) Defining macropinocytosis. Traffic 10(4):364–371. https://doi.org/10.1111/j.1600-0854.2009.00878.x
Donaldson JG, Porat-Shliom N, Cohen LA (2009) Clathrin-independent endocytosis: a unique platform for cell signaling and PM remodeling. Cell Signal 21(1):1–6. https://doi.org/10.1016/j.cellsig.2008.06.020
Lim JP, Gleeson PA (2011) Macropinocytosis: an endocytic pathway for internalising large gulps. Immunol Cell Biol 89(8):836–843. https://doi.org/10.1038/icb.2011.20
Commisso C, Davidson SM, Soydaner-Azeloglu RG, Parker SJ, Kamphorst JJ, Hackett S, Grabocka E, Nofal M, Drebin JA, Thompson CB, Rabinowitz JD, Metallo CM, Vander Heiden MG, Bar-Sagi D (2013) Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells. Nature 497(7451):633–637. https://doi.org/10.1038/nature12138
Recouvreux MV, Commisso C (2017) Macropinocytosis: a metabolic adaptation to nutrient stress in cancer. Front Endocrinol (Lausanne) 8:261. https://doi.org/10.3389/fendo.2017.00261
Marques PE, Grinstein S, Freeman SA (2017) SnapShot:macropinocytosis. Cell 169(4):766–766 e761. https://doi.org/10.1016/j.cell.2017.04.031
Wang JT, Teasdale RD, Liebl D (2014) Macropinosome quantitation assay. MethodsX 1:36–41. https://doi.org/10.1016/j.mex.2014.05.002
Commisso C, Flinn RJ, Bar-Sagi D (2014) Determining the macropinocytic index of cells through a quantitative image-based assay. Nat Protoc 9(1):182–192. https://doi.org/10.1038/nprot.2014.004
Kamphorst JJ, Nofal M, Commisso C, Hackett SR, Lu W, Grabocka E, Vander Heiden MG, Miller G, Drebin JA, Bar-Sagi D, Thompson CB, Rabinowitz JD (2015) Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein. Cancer Res 75(3):544–553. https://doi.org/10.1158/0008-5472.CAN-14-2211
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Lee, SW., Alas, B., Commisso, C. (2019). Detection and Quantification of Macropinosomes in Pancreatic Tumors. In: Su, G. (eds) Pancreatic Cancer. Methods in Molecular Biology, vol 1882. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8879-2_16
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DOI: https://doi.org/10.1007/978-1-4939-8879-2_16
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