Methods for Monitoring Macroautophagy in Pancreatic Cancer Cells

  • Chiara Vidoni
  • Alessandra Ferraresi
  • Christian Seca
  • Eleonora Secomandi
  • Ciro Isidoro
Part of the Methods in Molecular Biology book series (MIMB, volume 1882)


Macroautophagy is a catabolic process through which redundant, aged, or damaged cellular structures are first enclosed within double-membrane vesicles (called autophagosomes), and thereafter degraded within lysosomes. Macroautophagy provides a primary route for the turnover of macromolecules, membranes and organelles, and as such plays a major role in cell homeostasis. As part of the stress response, autophagy is crucial to determine the cell fate in response to extracellular or intracellular injuries. Autophagy is involved in cancerogenesis and in cancer progression. Here we illustrate the essential methods for monitoring autophagy in pancreatic cancer cells.

Key words

Pancreatic cancer Autophagy LC3 p62 Autophagosome Lysosome 


  1. 1.
    Feng Y, He D, Yao Z, Klionsky DJ (2014) The machinery of macroautophagy. Cell Res 24(1):24–41. Epub 2013 Dec 24. ReviewCrossRefPubMedGoogle Scholar
  2. 2.
    Orsi A, Polson HE, Tooze SA (2010) Membrane trafficking events that partake in autophagy. Curr Opin Cell Biol 22(2):150–156. ReviewCrossRefPubMedGoogle Scholar
  3. 3.
    Metlagel Z, Otomo C, Ohashi K, Takaesu G, Otomo T (2014) Structural insights into E2-E3 interaction for LC3 lipidation. Autophagy 10(3):522–523. ReviewCrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Svenning S, Johansen T (2013) Selective autophagy. Essays Biochem 55:79–92. ReviewCrossRefPubMedGoogle Scholar
  5. 5.
    Lippai M, Lőw P (2014) The role of the selective adaptor p62 and ubiquitin-like proteins in autophagy. Biomed Res Int 2014:832704. ReviewCrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Yin XM, Ding WX (2013) The reciprocal roles of PARK2 and mitofusins in mitophagy and mitochondrial spheroid formation. Autophagy 9(11):1687–1692. ReviewCrossRefPubMedGoogle Scholar
  7. 7.
    Galluzzi L, Pietrocola F, Levine B, Kroemer G (2014) Metabolic control of autophagy. Cell 159(6):1263–1276. ReviewCrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Awan MU, Deng Y (2014) Role of autophagy and its significance in cellular homeostasis. Appl Microbiol Biotechnol 98(12):5319–5328. Epub 2014 Apr 18. ReviewCrossRefPubMedGoogle Scholar
  9. 9.
    Das G, Shravage BV, Baehrecke EH (2012) Regulation and function of autophagy during cell survival and cell death. Cold Spring Harb Perspect Biol 4(6):pii: a008813. ReviewCrossRefGoogle Scholar
  10. 10.
    New M, Van Acker T, Long JS, Sakamaki JI, Ryan KM, Tooze SA (2017) Molecular pathways controlling autophagy in pancreatic cancer. Front Oncol 7:28. eCollection 2017. ReviewCrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Yang S, Wang X, Contino G, Liesa M, Sahin E, Ying H et al (2011) Pancreatic cancers require autophagy for tumor growth. Genes Dev 25:717–729. Scholar
  12. 12.
    Wolpin BM, Rubinson DA, Wang X, Chan JA, Cleary JM, Enzinger PC et al (2014) Phase II and pharmacodynamic study of autophagy inhibition using hydroxychloroquine in patients with metastatic pancreatic adenocarcinoma. Oncologist 19:637. Scholar
  13. 13.
    Rosenfeldt MT, O’Prey J, Morton JP, Nixon C, MacKay G, Mrowinska A et al (2013) p53 status determines the role of autophagy in pancreatic tumour development. Nature 504:296–300. Scholar
  14. 14.
    Eng CH, Wang Z, Tkach D, Toral-Barza L, Ugwonali S, Liu S et al (2016) Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy. Proc Natl Acad Sci U S A 113:182–187. Scholar
  15. 15.
    Thuwajit C, Ferraresi A, Titone R, Thuwajit P, Isidoro C (2017) The metabolic cross-talk between epithelial cancer cells and stromal fibroblasts in ovarian cancer progression: Autophagy plays a role. Med Res Rev. Scholar
  16. 16.
    von Ahrens D, Bhagat TD, Nagrath D, Maitra A, Verma A (2017) The role of stromal cancer-associated fibroblasts in pancreatic cancer. J Hematol Oncol 10(1):76. Scholar
  17. 17.
    Sousa CM, Biancur DE, Wang X, Halbrook CJ, Sherman MH, Zhang L, Kremer D, Hwang RF, Witkiewicz AK, Ying H, Asara JM, Evans RM, Cantley LC, Lyssiotis CA, Kimmelman AC (2016) Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion. Nature 536(7617):479–483CrossRefGoogle Scholar
  18. 18.
    Klionsky DJ et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12(1):1–222. Scholar
  19. 19.
    Trincheri NF, Follo C, Nicotra G, Peracchio C, Castino R, Isidoro C (2008) Resveratrol-induced apoptosis depends on the lipid kinase activity of Vps34 and on the formation of autophagolysosomes. Carcinogenesis 29(2):381–389CrossRefGoogle Scholar
  20. 20.
    Hosokawa N, Hara Y, Mizushima N (2006) Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size. FEBS Lett 580(11):2623–2629CrossRefGoogle Scholar
  21. 21.
    Bjørkøy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn A, Stenmark H, Johansen T (2005) p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol 171(4):603–614CrossRefGoogle Scholar
  22. 22.
    Janda E, Lascala A, Carresi C, Parafati M, Aprigliano S, Russo V, Savoia C, Ziviani E, Musolino V, Morani F, Isidoro C, Mollace V (2015) Parkinsonian toxin-induced oxidative stress inhibits basal autophagy in astrocytes via NQO2/quinone oxidoreductase 2: implications for neuroprotection. Autophagy 11(7):1063–1080. Scholar
  23. 23.
    Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19(21):5720–5728CrossRefGoogle Scholar
  24. 24.
    Kraft LJ, Manral P, Dowler J, Kenworthy AK (2016) Nuclear LC3 associates with slowly diffusing complexes that survey the nucleolus. Traffic 17(4):369–399. Scholar
  25. 25.
    He C, Klionsky DJ (2009) Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 43:67–93. ReviewCrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Nakaso K, Yoshimoto Y, Nakano T, Takeshima T, Fukuhara Y, Yasui K, Araga S, Yanagawa T, Ishii T, Nakashima K (2004) Transcriptional activation of p62/A170/ZIP during the formation of the aggregates: possible mechanisms and the role in Lewy body formation in Parkinson's disease. Brain Res 1012(1–2):42–51CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Chiara Vidoni
    • 1
  • Alessandra Ferraresi
    • 1
  • Christian Seca
    • 1
  • Eleonora Secomandi
    • 1
  • Ciro Isidoro
    • 1
  1. 1.Laboratory of Molecular Pathology and Nanobioimaging, Department of Health SciencesUniversità del Piemonte OrientaleNovaraItaly

Personalised recommendations