Autophagy in 3D In Vitro and Ex Vivo Cancer Models

Our plain-English summary of Autophagy in 3D In Vitro and Ex Vivo Cancer Models, by C. Follo, D. Barbone, W.G. Richards, et al (Methods in Molecular Biology, 2019).

The study

• Authors: C. Follo, D. Barbone, W.G. Richards, R. Bueno, V. Courtney Broaddus
• Journal: Methods in Molecular Biology
• Published: 2019;1880:491-510
• Cited by: 3 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Three-dimensional (3D) models are acquiring importance in cancer research due to their ability to mimic multiple features of the tumor microenvironment more accurately than standard monolayer two-dimensional (2D) cultures. Several groups, including our laboratory, are now accumulating evidence that autophagy in solid tumors is also better represented in 3D than in 2D. Here we detail how we generate 3D models, both in vitro multicellular spheroids generated from cell lines and ex vivo tumor fragment spheroids generated from tumor samples, and how autophagy can be measured in 3D cultures.

In plain English

This book chapter is a laboratory methods protocol. It is not a study of asbestos exposure or of patients' health. The authors described how they grow mesothelioma cells in three dimensions (3D), either as multicellular spheroids made from cell lines or as tumor fragment spheroids made from fresh mesothelioma surgical samples. They also described how they measure autophagy, a process by which cells break down and recycle their own parts. Example figures used two mesothelioma cell lines (JMN and M28) and tumor fragment spheroids from 25 chemonaive mesothelioma patients. In these examples, autophagic flux differed between cells grown flat (2D) and cells grown as spheroids (3D), and some differences in LC3 puncta ratios were marked as statistically significant (P less than 0.05). ATG13, a marker of the start of autophagy, reflected autophagic flux in 3D but not in 2D. The authors used a 6% ATG13 cutoff taken from their own earlier study. They cautioned that long exposure to lysosomal inhibitors can distort measurements and that extra caution is needed when studying autophagy in 3D. The authors stated that their work supports using 3D models to study autophagy.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory methods chapter (protocol) describing how to build 3D mesothelioma models and measure autophagy, with illustrative experimental figures
• Who was studied: Mesothelioma cell lines (JMN and M28) grown as monolayers or multicellular spheroids, and tumor fragment spheroids made from fresh mesothelioma resection samples from Brigham and Women's Hospital; one example figure used tumor biopsies from 25 chemonaive mesothelioma patients. No dates given.
• Exposure: No asbestos or fiber exposure was studied. The experimental treatments were the lysosomal inhibitors ammonium chloride (10 mM) or hydroxychloroquine (20 μM), used to measure autophagy.
• Main results:
  • In JMN and M28 mesothelioma cells, autophagic flux measured by LC3B-II immunoblotting differed between 2D monolayers and 3D spheroids (representative of three independent experiments)
  • LC3 puncta ratios in these cell lines were marked as significantly different between 2D and 3D at P less than 0.05
  • In the cell lines, ATG13 puncta (a marker of the start of autophagy) reflected autophagic flux in 3D spheroids but not in 2D monolayers
  • In tumor fragment spheroids, LC3 immunofluorescence separated tumors with low autophagy from tumors with high autophagy, and the level of autophagy initiation by ATG13 matched the level of autophagy by LC3
  • The authors used a 6% ATG13-positivity cutoff (below 6% = low autophagy initiation) for tumor fragment spheroids, chosen from their previous results linking ATG13 positivity to better patient outcome
  • No significant difference was found in LC3 puncta counts between cytospun cells and cells grown on cover slips
• The authors' conclusion: The authors stated that their work supports using 3D models to study autophagy, while advising extra caution when studying autophagy in 3D.
• Limitations the authors state:
  • Prolonged exposure to lysosomal inhibitors may trigger a secondary autophagic response and give an incorrect measurement
  • In tumor fragment spheroids, LC3 immunoblotting cannot distinguish mesothelioma LC3 from that of other cell types
  • In tissue sections, puncta overlap and cannot be counted individually, so the percentage of positive cells was used instead
  • Other autophagy markers (p62, Beclin 1) did not correlate with autophagy levels in 3D mesothelioma models, so standard markers may not apply in 3D and extra caution is needed
  • Spheroids cultured more than 48 hours may develop a dark, possibly necrotic, core

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Tumor Microenvironment.

Related studies

• Autophagy facilitates the release of immunogenic signals following chemotherapy in 3D models of mesothelioma — C. Follo, Y. Cheng, et al. (2019)
• Berberine induces mitochondrial‑mediated apoptosis and protective autophagy in human malignant pleural mesothelioma NCI‑H2452 cells — Z. Yao, Y. Wan, et al. (2018)
• Inhibition of autophagy initiation potentiates chemosensitivity in mesothelioma — C. Follo, Y. Cheng, et al. (2018)
• Changes in global gene expression associated with 3D structure of tumors: an ex vivo matrix-free mesothelioma spheroid model — H. Kim, Y. Phung, et al. (2012)
• Curcumin induces autophagy in ACC-MESO-1 cells — Y. Yamauchi, Y. Izumi, et al. (2012)
• Pro-oxidant activity of sulforaphane and cisplatin potentiates apoptosis and simultaneously promotes autophagy in malignant mesothelioma cells — Y.J. Lee, S.H. Lee (2017)

Browse more research in Methods in Molecular Biology, or return to medical research.