Autophagy initiation correlates with the autophagic flux in 3D models of mesothelioma and with patient outcome

Our plain-English summary of Autophagy initiation correlates with the autophagic flux in 3D models of mesothelioma and with patient outcome, by C. Follo, D. Barbone, W.G. Richards, et al (Autophagy, 2016).

The study

• Authors: C. Follo, D. Barbone, W.G. Richards, R. Bueno, V.C. Broaddus
• Journal: Autophagy
• Published: 2016;12(7):1180-94
• Cited by: 20 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Understanding the role of autophagy in cancer has been limited by the inability to measure this dynamic process in formalin-fixed tissue. We considered that 3-dimensional models including ex vivo tumor, such as we have developed for studying mesothelioma, would provide valuable insights. Using these models, in which we could use lysosomal inhibitors to measure the autophagic flux, we sought a marker of autophagy that would be valid in formalin-fixed tumor and be used to assess the role of autophagy in patient outcome. Autophagy was studied in mesothelioma cell lines, as 2-dimensional (2D) monolayers and 3-dimensional (3D) multicellular spheroids (MCS), and in tumor from 25 chemonaive patients, both as ex vivo 3D tumor fragment spheroids (TFS) and as formalin-fixed tissue. Autophagy was evaluated as autophagic flux by detection of the accumulation of LC3 after lysosomal inhibition and as autophagy initiation by detection of ATG13 puncta. We found that autophagic flux in 3D, but not in 2D, correlated with ATG13 positivity. In each TFS, ATG13 positivity was similar to that of the original tumor. When tested in tissue microarrays of 109 chemonaive patients, higher ATG13 positivity correlated with better prognosis and provided information independent of known prognostic factors. Our results show that ATG13 is a static marker of the autophagic flux in 3D models of mesothelioma and may also reflect autophagy levels in formalin-fixed tumor. If confirmed, this marker would represent a novel prognostic factor for mesothelioma, supporting the notion that autophagy plays an important role in this cancer.

In plain English

This laboratory and tissue study sought a way to measure autophagy, cells' breakdown of worn-out parts, in preserved malignant pleural mesothelioma samples. It did not study asbestos exposure. The authors grew six mesothelioma cell lines as flat layers and three-dimensional clusters, plus tumor fragments from 25 patients without prior chemotherapy. Under basal conditions, small clumps (puncta) of the protein ATG13 tracked autophagy, measured directly with blocking drugs, in three-dimensional cultures but not flat layers, where puncta appeared only after strong stimulation. The percentage of mesothelioma cells with ATG13 puncta in the fragments matched that in the original tumors. Among 109 patients operated on without prior chemotherapy, those whose archived tumor had ATG13 puncta in at least 6% of mesothelioma cells had longer median overall survival (17.4 vs 10.6 months) and time to recurrence (13.7 vs 6.6 months). After adjusting for tumor histology and strictly correcting for how the cutpoint was chosen, the link stayed statistically significant for recurrence but not survival. The authors concluded that ATG13 has prognostic value. They said the cutpoint needs validation in other patient groups.

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 study of mesothelioma cell lines and ex vivo tumor fragments, combined with an analysis of archival tumor samples (tissue microarrays) linked to patient outcome data
• Who was studied: The study used six human malignant pleural mesothelioma cell lines (M28, REN, SARC, VAMT, JMN, MSTO-211H) and fresh tumor from 25 chemonaive patients, grown as tumor fragment spheroids. It also used archival tumor from 109 patients with malignant pleural mesothelioma who had surgery at Brigham and Women's Hospital, Boston, without preoperative chemotherapy. Dates are not given.
• Exposure: No asbestos exposure was studied. The experiments used lysosomal inhibitors to measure autophagic flux through LC3 accumulation: ammonium chloride, plus hydroxychloroquine in the cell lines. They measured ATG13 puncta as a marker of autophagy initiation.
• Main results:
  • In the six cell lines, autophagic flux differed between 2D and 3D cultures. It was lower in 3D than in 2D in 3 lines and higher in 3D in the other 3, and flux in 3D did not correlate with flux in 2D.
  • Under basal conditions, ATG13 puncta were almost undetectable in 2D cultures of all cell lines, but in 3D cultures they reflected the autophagic flux. ATG13 protein levels did not reflect the puncta or the flux.
  • After autophagy was stimulated by nutrient deprivation (Earle's balanced salt solution), ATG13 puncta were detectable in all cell lines in both 2D and 3D. The authors concluded that in 2D, ATG13 puncta reflect high autophagic flux only after strong stimulation.
  • In tumor fragment spheroids from the 25 patients, lysosomal inhibition raised the percentage of LC3-positive mesothelioma cells on average from 1.7% to 2.9% in the low-autophagy group. In the high-autophagy group it rose from 2.5% to 20.8%.
  • In the tumor fragment spheroids, the percentage of ATG13-positive cells at baseline correlated with the percentage of LC3-positive cells after lysosomal inhibition (Spearman rank correlation 0.8997, two-tailed P less than 0.0001).
  • The percentage of mesothelioma cells with ATG13 puncta in each tumor fragment spheroid reflected that of the original tumor (Spearman rank correlation 0.9253, two-tailed P less than 0.0001).
  • Among the 109 patients, the median percentage of ATG13-positive mesothelioma cells was 8% (range 0% to 32%; inter-quartile range 2% to 19%).
  • The authors selected a cutpoint of 6% ATG13-positive mesothelioma cells because it minimized hazard ratios for both endpoints. At this cutpoint, 58% of patients were classed as high and 42% as low.
  • Median overall survival was 17.4 months for patients with high ATG13 positivity, versus 10.6 months for those with fewer than 6% ATG13-positive tumor cells (P = 0.0009; Bonferroni corrected P = 0.0162).
  • Time to recurrence was 13.7 months for patients with high ATG13 positivity versus 6.6 months for those with low positivity (P less than 0.0001).
  • Low ATG13 positivity was more common among non-epithelioid tumors (P = 0.01). It was not correlated with age, lymph node status, gender, tumor volume, anemia, resectability, surgical procedure, adjuvant therapy or TNM stage.
  • After adjusting for histology, ATG13 positivity remained significantly prognostic for time to recurrence (P = 0.0017) and overall survival (P = 0.0166).
  • In the histology-adjusted analysis with Bonferroni correction for the repeated testing used to set the cutpoint, ATG13 positivity remained significant for time to recurrence (corrected P = 0.0306). It was not significant for overall survival (corrected P = 0.2988).
• The authors' conclusion: The authors concluded that, using 3D models of mesothelioma, they identified a marker of autophagy (ATG13 puncta) that has prognostic value in a group of patients with mesothelioma. They found that higher autophagy initiation was associated with better outcome.
• Limitations the authors state:
  • The selected cutpoint and effect sizes need validation in additional patient cohorts, given the known pitfalls of dichotomizing continuous measures.
  • The authors cannot say whether the marker would be useful for other tumors.
  • Autophagic flux cannot be measured directly in fixed tumor. Confirming ATG13 as a marker will therefore require comparison with other markers and with matching ex vivo tumor.
  • It is unclear whether autophagy is a cause of the better outcome or only a marker of it; further studies are needed.

Topics

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

Medical terms in this study, explained in our Lexicon: Mesothelioma, Prognosis.

Related studies

• Autophagy Correlates with the Therapeutic Responsiveness of Malignant Pleural Mesothelioma in 3D Models — D. Barbone, C. Follo, et al. (2015)
• Autophagy in 3D In Vitro and Ex Vivo Cancer Models — C. Follo, D. Barbone, et al. (2019)
• Autophagy facilitates the release of immunogenic signals following chemotherapy in 3D models of mesothelioma — C. Follo, Y. Cheng, et al. (2019)
• Inhibition of autophagy initiation potentiates chemosensitivity in mesothelioma — C. Follo, Y. Cheng, et al. (2018)
• MicroRNA-126 induces autophagy by altering cell metabolism in malignant mesothelioma — M. Tomasetti, F. Monaco, et al. (2016)
• AKT/mTOR and c-Jun N-terminal kinase signaling pathways are required for chrysotile asbestos-induced autophagy — Z. Lin, T. Liu, et al. (2014)

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