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Cited by 107Autophagy in Disease and TherapyGlioma Diagnosis and TreatmentElectromagnetic Fields and Biological Effects

The DOI matches its Crossref record; 53 of the 53 references it cites were found in a registry or an index; none of the 27 sources its claims cite is checked for support yet.

AMPK-dependent autophagy upregulation serves as a survival mechanism in response to Tumor Treating Fields (TTFields)

Open access
Shteingauz, Anna, Porat, Yaara, Voloshin, Tali, Schneiderman, Rosa S., Munster, Mijal, Zeevi, Einav, Kaynan, Noa, Gotlib, Karnit, Giladi, Moshe, Kirson, Eilon D., Weinberg, Uri, Kinzel, Adrian, Palti, Yoram·2018·Cell Death & Disease
DOI 10.1038/s41419-018-1085-9PMC PMC6195570
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Abstract

Tumor Treating Fields (TTFields), an approved treatment modality for glioblastoma, are delivered via non-invasive application of low-intensity, intermediate-frequency, alternating electric fields. TTFields application leads to abnormal mitosis, aneuploidy, and increased cell granularity, which are often associated with enhancement of autophagy. In this work, we evaluated whether TTFields effected the regulation of autophagy in glioma cells. We found that autophagy is upregulated in glioma cells treated with TTFields as demonstrated by immunoblot analysis of the lipidated microtubule-associated protein light chain 3 (LC3-II). Fluorescence and transmission electron microscopy demonstrated the presence of LC3 puncta and typical autophagosome-like structures in TTFields-treated cells. Utilizing time-lapse microscopy, we found that the significant increase in the formation of LC3 puncta was specific to cells that divided during TTFields application. Evaluation of selected cell stress parameters revealed an increase in the expression of the endoplasmic reticulum (ER) stress marker GRP78 and decreased intracellular ATP levels, both of which are indicative of increased proteotoxic stress. Pathway analysis demonstrated that TTFields-induced upregulation of autophagy is dependent on AMP-activated protein kinase (AMPK) activation. Depletion of AMPK or autophagy-related protein 7 (ATG7) inhibited the upregulation of autophagy in response to TTFields, as well as sensitized cells to the treatment, suggesting that cancer cells utilize autophagy as a resistance mechanism to TTFields. Combining TTFields with the autophagy inhibitor chloroquine (CQ) resulted in a significant dose-dependent reduction in cell growth compared with either TTFields or CQ alone. These results suggest that dividing cells upregulate autophagy in response to aneuploidy and ER stress induced by TTFields, and that AMPK serves as a key regulator of this process.

Overview

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Written from the paper’s full text.

TL;DR

TTFields upregulate autophagy in glioma cells, especially in daughter cells that divide during treatment, via AMPK activation downstream of aneuploidy/ER stress and low ATP. This autophagy acts as a survival/resistance mechanism, and inhibiting it genetically or with chloroquine sensitizes glioma cells to TTFields.

Key findings

  • TTFields increased cellular granularity across mesothelioma, glioma, lung, and pancreatic cancer cell lines and enlarged the acidic lysosomal pool; in U-87 MG and A172 glioma cells, TTFields increased LC3-II in a treatment-duration-dependent manner, with autophagosome-like and autolysosome structures on EM and increased LC3 staining in F98 rat gliomas in vivo.
  • Autophagy induction was specific to dividing cells: LC3-GFP puncta increased in 51% of TTFields-treated cells vs 17% of untreated cells, and among cells that divided, 60% vs 16% showed increased LC3-GFP; mitotic index was 60% vs 73%, and mitosis duration was 1.5 h vs 1 h (p < 0.05).
  • TTFields increased GRP78 and reduced intracellular ATP by 18% (about 1.2 vs 1.5 nmol per 0.2 × 10^6 cells), activated AMPK/ULK1, and required AMPK for autophagy: siAMPK reduced AMPK protein by 80% and abrogated LC3-II upregulation; siAMPK cells showed 47% vs 32% reduction in cell number versus siVector.
  • Autophagy inhibition enhanced TTFields efficacy: shATG7 reduced cell numbers to 36% vs 64% in U-87 MG and 46% vs 62% in A172; TTFields plus low-dose CQ (3 µM) increased apoptosis to 75%, whereas CQ monotherapy increased apoptosis by 25% in A172 and 8% in U-87 MG.

Methods

  • Glioma and other cancer cell lines were treated with TTFields using the inovitro system at optimal frequencies (150 or 200 kHz) and 1.75 V/cm RMS; assays included flow cytometry, LysoTracker staining, immunoblotting, immunocytochemistry, electron microscopy, ATP measurement, siRNA-mediated AMPK knockdown, shATG7 lentiviral knockdown, time-lapse microscopy of LC3-GFP, and F98 rat intracranial glioma immunohistochemistry.
  • Statistical analysis used means ± SE, two-tailed Student’s t-test, and χ² analysis, with P < 0.05 considered significant; experiments were repeated at least three times with similar results.

Limitations

  • The paper states that it did not examine whether TTFields-induced genotoxic stress pathways such as ATM or PARP1 activate AMPK/autophagy, and says future studies are needed.
  • The paper states that CQ can have autophagy-independent effects, including Golgi and endo-lysosomal disorganization, so some enhanced efficacy of TTFields plus CQ may be unrelated to autophagy inhibition.
  • Evident from the text: the mechanistic autophagy/AMPK and CQ combination experiments are largely in glioma cell lines, while in vivo evidence is limited to LC3 staining in F98 rat tumors without survival or tumor-growth outcomes.

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Integrity signals

Registry

Crossref · 10.1038/s41419-018-1085-9

Cites

53 of 53 references verified0 citation errors · 0 not checkable

Statistics

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Figures

Fig. 1 TTFields application leads to increased cellular granularity. a, b U-87 MG, A172, LN229, MSTO-211H, LLC-1, KLN-205, and AsPC-1 cancer cells were either left untreated or treated with TTFields at the optimal treatment frequency for 72 h. a Side-scatter parameters were measured using flow cytometry. b Median side-scattered values following TTFields application relative to respective control represent the change in cellular granularity (0.01 < *P < 0.05, **P < 0.01 from corresponding control group, Student’s t-test, n = 3). c Fluorescent microscopy images (LysoTracker staining) of U-87 MG, A172, and AsPC-1 cells (upper panels), and light microscopy (phase contrast) (lower panels), following 72 h incubation with or without TTFields application. Original magnification: × 40
Fig. 1 TTFields application leads to increased cellular granularity. a, b U-87 MG, A172, LN229, MSTO-211H, LLC-1, KLN-205, and AsPC-1 cancer cells were either left untreated or treated with TTFields at the optimal treatment frequency for 72 h. a Side-scatter parameters were measured using flow cytometry. b Median side-scattered values following TTFields application relative to respective control represent the change in cellular granularity (0.01 < *P < 0.05, **P < 0.01 from corresponding control group, Student’s t-test, n = 3). c Fluorescent microscopy images (LysoTracker staining) of U-87 MG, A172, and AsPC-1 cells (upper panels), and light microscopy (phase contrast) (lower panels), following 72 h incubation with or without TTFields application. Original magnification: × 40CC BY · PubMed Central
Fig. 2 TTFields induce autophagy in glioma cell lines. a U-87 MG and A172 cells were either left untreated or treated with TTFields at the last 24 h, 48 h, or 72 h of culturing. All cultures were plated on the same time, incubated overnight to allow cell attachment, and collected 72 h afterwards. Cells were collected, lysed, and samples were analyzed using immunoblotting for LC3 and GAPDH. Upper panel: representative blots. Lower panel: densitometric quantification of immunoblot signal, showing an average of at least three independent experiments (0.01 < *P < 0.05, **P < 0.01, Student’s t-test). b Paraffin-embedded sections from sham- or TTFields-treated rats were stained with anti-LC3 Ab (green) and DAPI (blue). Representative images are presented. c Quantification of LC3 intensity, presented as fold increase from corresponding control (*P < 0.05, Student’s t-test)
Fig. 2 TTFields induce autophagy in glioma cell lines. a U-87 MG and A172 cells were either left untreated or treated with TTFields at the last 24 h, 48 h, or 72 h of culturing. All cultures were plated on the same time, incubated overnight to allow cell attachment, and collected 72 h afterwards. Cells were collected, lysed, and samples were analyzed using immunoblotting for LC3 and GAPDH. Upper panel: representative blots. Lower panel: densitometric quantification of immunoblot signal, showing an average of at least three independent experiments (0.01 < *P < 0.05, **P < 0.01, Student’s t-test). b Paraffin-embedded sections from sham- or TTFields-treated rats were stained with anti-LC3 Ab (green) and DAPI (blue). Representative images are presented. c Quantification of LC3 intensity, presented as fold increase from corresponding control (*P < 0.05, Student’s t-test)CC BY · PubMed Central
Fig. 3 TTFields application leads to increased autophagic flux. a Ultra-structural STEM electron microscopy analysis of U-87 MG (upper panel) and A172 (lower panel) cells treated with TTFields for 48 h. Autophagosomes (blue arrows) and autolysosomes (green arrows) are indicated. b U-87 MG and A172 cells were either left untreated or were treated with TTFields for 24–72 h. CQ (20 µM) was added 4 h before cells were collected. Samples were immunoblotted for LC3 and GAPDH. Upper panel: representative blots. Lower panel: densitometric quantification of immunoblot signal, showing an average of at least three independent experiments (0.01 < *P < 0.05, Student’s t-test). c U-87 MG and A172 cells were either left untreated or were treated with TTFields for 48 h. CQ (20 µM) was added at the last 3 h of treatment and cells were fixed and stained with anti-LC3 Ab (green) and DAPI (blue). Upper panel: representative images. Original magnifications: × 40. Lower panel: quantification of LC3 intensity, presented as average intensity per cell (**P < 0.01, Student’s t-test). d U-87 MG cells were either left untreated or were treated with TTFields (48 h) or with vinblastin 25 nM (0.5 h). CQ (20 µM) was added at the last 3 h of treatment and cells were fixed and stained with anti-LC3 (green), LAMP1 (red), and DAPI (blue) (upper panel). Arrowheads indicate the strongest colocalization staining in each cell. Intensity histograms of LAMP1 and LC3 fluorescent signal calculated from the region of interest indicated by the white bar (lower panel). Representative images are shown
Fig. 3 TTFields application leads to increased autophagic flux. a Ultra-structural STEM electron microscopy analysis of U-87 MG (upper panel) and A172 (lower panel) cells treated with TTFields for 48 h. Autophagosomes (blue arrows) and autolysosomes (green arrows) are indicated. b U-87 MG and A172 cells were either left untreated or were treated with TTFields for 24–72 h. CQ (20 µM) was added 4 h before cells were collected. Samples were immunoblotted for LC3 and GAPDH. Upper panel: representative blots. Lower panel: densitometric quantification of immunoblot signal, showing an average of at least three independent experiments (0.01 < *P < 0.05, Student’s t-test). c U-87 MG and A172 cells were either left untreated or were treated with TTFields for 48 h. CQ (20 µM) was added at the last 3 h of treatment and cells were fixed and stained with anti-LC3 Ab (green) and DAPI (blue). Upper panel: representative images. Original magnifications: × 40. Lower panel: quantification of LC3 intensity, presented as average intensity per cell (**P < 0.01, Student’s t-test). d U-87 MG cells were either left untreated or were treated with TTFields (48 h) or with vinblastin 25 nM (0.5 h). CQ (20 µM) was added at the last 3 h of treatment and cells were fixed and stained with anti-LC3 (green), LAMP1 (red), and DAPI (blue) (upper panel). Arrowheads indicate the strongest colocalization staining in each cell. Intensity histograms of LAMP1 and LC3 fluorescent signal calculated from the region of interest indicated by the white bar (lower panel). Representative images are shownCC BY · PubMed Central
Fig. 4 Cells which divided under TTFields application exhibit stress resulting in upregulation of autophagy. Time-lapse microscopy of TTFields-treated (26 h) U-87 MG cells, stably expressing LC3-GFP. a Images of representative location in the plate show increase in LC3-GFP punctate signal (green) following first 15 h of TTFields application. Numbered arrows (1–4) indicate the same cell at two different time points (0 and 15 h). b Quantification of cells demonstrating increased LC3-GFP signal in control and treatment groups is presented as percent of total cells analyzed. Summary of total amount of cells of three independent experiments (***P < 0.001, χ2-test). c Representative fields of view acquired in time laps microscopy during TTFields treatment. Arrows indicate cells entering mitosis and arrowheads denote the resulting daughter cells. d Single-cell analysis of untreated (n = 108) and TTFields-treated (n = 150) cells, summarized observations from three independent experiments. Cells were monitored for mitotic events and increased LC3-GFP punctate signal (**P < 0.01, NS nonsignificant, χ2-test)
Fig. 4 Cells which divided under TTFields application exhibit stress resulting in upregulation of autophagy. Time-lapse microscopy of TTFields-treated (26 h) U-87 MG cells, stably expressing LC3-GFP. a Images of representative location in the plate show increase in LC3-GFP punctate signal (green) following first 15 h of TTFields application. Numbered arrows (1–4) indicate the same cell at two different time points (0 and 15 h). b Quantification of cells demonstrating increased LC3-GFP signal in control and treatment groups is presented as percent of total cells analyzed. Summary of total amount of cells of three independent experiments (***P < 0.001, χ2-test). c Representative fields of view acquired in time laps microscopy during TTFields treatment. Arrows indicate cells entering mitosis and arrowheads denote the resulting daughter cells. d Single-cell analysis of untreated (n = 108) and TTFields-treated (n = 150) cells, summarized observations from three independent experiments. Cells were monitored for mitotic events and increased LC3-GFP punctate signal (**P < 0.01, NS nonsignificant, χ2-test)CC BY · PubMed Central
Fig. 5 Induction of autophagy by TTFields is AMPK dependent. a U-87 MG and A172 cells were either left untreated or were treated with TTFields for indicated time points. Immunoblot analysis of GFP78 protein. Numeric values represent the fold increase in GRP78 signal, normalized to loading control (GAPDH), relative to untreated control. b Quantification of intracellular ATP levels in U-87 MG cells either left untreated or treated with TTFields for 72 h.The results are presented as average ATP concentration (nmol/2 × 106 cells) from three independent experiments (*P < 0.01, Student’s t-test). c U-87 MG and A172 cells were either left untreated or were treated with TTFields for indicated time points. Immunoblot analysis of pAMPK and pULK1proteins. GAPDH was used as loading control. (5D-F) U-87 MG cells were transfected with AMPK-targeting siRNA (siAMPK) or with siRNA sham vector (siVector), and were incubated for 48 h with or without TTFields application. CQ 20 µM was added for the last 4 h of the treatment where indicated. d (left panel) Immunoblot analysis of LC3 and AMPK. Numeric values represent the fold-change in LC3-II signal, normalized to GAPDH signal, relative to respective control. d (right panel) CQ-treated cells were fixed and stained for LC3 (green) and DAPI (blue), original magnifications: × 40. e Cell count of siAMPK- or siVector-expressing cells after the TTFields treatment. (0.01 < *P < 0.05, Student’s t-test, n = 3). f siVector- and siAMPK-transfected U-87 MG cells were either left untreated or were treated with TTFields for 48 h. Cells were then fixed and stained for cleaved caspase-3 (green) and DAPI (blue) (left panel). Images from each treatment were analyzed manually and the fraction of cleaved caspase-3-positive cells was calculated for at least 200 cells from each group (right panel) (**P < 0.01, Student’s t-test, n = 2)
Fig. 5 Induction of autophagy by TTFields is AMPK dependent. a U-87 MG and A172 cells were either left untreated or were treated with TTFields for indicated time points. Immunoblot analysis of GFP78 protein. Numeric values represent the fold increase in GRP78 signal, normalized to loading control (GAPDH), relative to untreated control. b Quantification of intracellular ATP levels in U-87 MG cells either left untreated or treated with TTFields for 72 h.The results are presented as average ATP concentration (nmol/2 × 106 cells) from three independent experiments (*P < 0.01, Student’s t-test). c U-87 MG and A172 cells were either left untreated or were treated with TTFields for indicated time points. Immunoblot analysis of pAMPK and pULK1proteins. GAPDH was used as loading control. (5D-F) U-87 MG cells were transfected with AMPK-targeting siRNA (siAMPK) or with siRNA sham vector (siVector), and were incubated for 48 h with or without TTFields application. CQ 20 µM was added for the last 4 h of the treatment where indicated. d (left panel) Immunoblot analysis of LC3 and AMPK. Numeric values represent the fold-change in LC3-II signal, normalized to GAPDH signal, relative to respective control. d (right panel) CQ-treated cells were fixed and stained for LC3 (green) and DAPI (blue), original magnifications: × 40. e Cell count of siAMPK- or siVector-expressing cells after the TTFields treatment. (0.01 < *P < 0.05, Student’s t-test, n = 3). f siVector- and siAMPK-transfected U-87 MG cells were either left untreated or were treated with TTFields for 48 h. Cells were then fixed and stained for cleaved caspase-3 (green) and DAPI (blue) (left panel). Images from each treatment were analyzed manually and the fraction of cleaved caspase-3-positive cells was calculated for at least 200 cells from each group (right panel) (**P < 0.01, Student’s t-test, n = 2)CC BY · PubMed Central
Fig. 6 Autophagy inhibition triggers apoptosis and results in increased TTFields efficacy in glioma cells. a U-87 MG and A172 were infected with lentiviral particle containing shATG7 or sham vector (shVector). Cells were then either left untreated or were treated with TTFields for 72 h and enumerated by flow cytometry. The data are presented as percent of control. (0.01 < *P < 0.05, Student’s t-test, n = 3). b, c U-87 MG and A172 were treated for 72 h with CQ alone (1–27 µM) and in combination with TTFields. b Dose–response blots for U-87 MG (left) and A172 (right). c Fraction of apoptotic cells as indicated by Annexin V/7-AAD staining in TTFields-treated vs. control cells, with or without CQ (3 μM) (0.01 < *P < 0.05, **P < 0.01, Student’s t-test, n = 3)
Fig. 6 Autophagy inhibition triggers apoptosis and results in increased TTFields efficacy in glioma cells. a U-87 MG and A172 were infected with lentiviral particle containing shATG7 or sham vector (shVector). Cells were then either left untreated or were treated with TTFields for 72 h and enumerated by flow cytometry. The data are presented as percent of control. (0.01 < *P < 0.05, Student’s t-test, n = 3). b, c U-87 MG and A172 were treated for 72 h with CQ alone (1–27 µM) and in combination with TTFields. b Dose–response blots for U-87 MG (left) and A172 (right). c Fraction of apoptotic cells as indicated by Annexin V/7-AAD staining in TTFields-treated vs. control cells, with or without CQ (3 μM) (0.01 < *P < 0.05, **P < 0.01, Student’s t-test, n = 3)CC BY · PubMed Central
Fig. 7 TTFields induce upregulation of autophagy in an AMPK-dependent manner in dividing cells. TTFields application to dividing cancer cells leads to disruption of the mitotic spindle and abnormal chromosome segregation, which mostly trigger different forms of cell death. Aneuploidy, ER stress, and low intracellular ATP levels are characteristics of the surviving daughter cells. These features, and potentially other AMPK-activating cascades, trigger AMPK and sequential ULK1 activation, which results in increased autophagic flux. High autophagy exerts cellular survival under TTFields treatment, thus acting as a resistance mechanism. Addition of CQ increases cell death under treatment and therefore enhances TTFields efficacy
Fig. 7 TTFields induce upregulation of autophagy in an AMPK-dependent manner in dividing cells. TTFields application to dividing cancer cells leads to disruption of the mitotic spindle and abnormal chromosome segregation, which mostly trigger different forms of cell death. Aneuploidy, ER stress, and low intracellular ATP levels are characteristics of the surviving daughter cells. These features, and potentially other AMPK-activating cascades, trigger AMPK and sequential ULK1 activation, which results in increased autophagic flux. High autophagy exerts cellular survival under TTFields treatment, thus acting as a resistance mechanism. Addition of CQ increases cell death under treatment and therefore enhances TTFields efficacyCC BY · PubMed Central

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  • The quote only partly states this claim. 1
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  • The paper relies on this without support.

    Senescence is not detected in cells treated with TTFields.

    “As senescence was not detected in cells treated with TTFields, we hypothesized that the origin of the observed granularity may be due to the accumulation of autophagosome vesicles8.”

  • The paper relies on this without support.

    Effective TTFields intensities are present not just in the tumor but also in the surrounding tissues and may potentially affect normal bystander cells.

    “Effective TTFields intensities are present not just in the tumor but also in the surrounding tissues and may potentially affect normal bystander cells.”

  • The paper relies on this without support.

    Both preclinical and clinical evidence did not reveal adverse events in normal tissues.

    “However, both preclinical and clinical evidence did not reveal adverse events in normal tissues.”
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Metadata

Authors
Shteingauz, A., Porat, Y., Voloshin, T., Schneiderman, R. S., Munster, M., Zeevi, E., Kaynan, N., Gotlib, K., Giladi, M., Kirson, E. D., Weinberg, U., Kinzel, A., & Palti, Y.
Corresponding author
Giladi, Moshe
ORCID
Venue
Cell Death & Disease
Publisher
Springer Science and Business Media LLC
Published
2018-10-19
Volume
9
Issue
11
Pages
1074-1074
ISSN
2041-4889 (ISSN-L)
DOI
10.1038/s41419-018-1085-9
PMC
PMC6195570
OpenAlex
W2897336072
Semantic Scholar
0c9028c6bb3ef5d1625b9f88285f8d67f0509e07
Cited by
107
References
53
Citations by year
2026: 8 · 2025: 13 · 2024: 18 · 2023: 16 · 2022: 23 · 2021: 12 · 2020: 9 · 2019: 7 · 2018: 1
FWCI
5.66
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Death and Disease (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
Novocure Ltd
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Health Sciences › Medicine › Oncology › Glioma Diagnosis and Treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Electromagnetic Fields and Biological Effects
Keywords
tumor treating fields, glioblastoma, autophagy, AMPK, ATG7, endoplasmic reticulum stress, GRP78, aneuploidy, chloroquine, tumor cell survival, LC3-II, AC electric field
MeSH terms
Autophagosomes, Autophagy-Related Protein 7, Endoplasmic Reticulum Chaperone BiP, Adenosine Triphosphate, Aneuploidy, Animals, Autophagy, Brain Neoplasms, Cell Survival, Electric Stimulation, Electric Stimulation Therapy, Glioblastoma, Heat-Shock Proteins, Humans, Lysosomes, Microtubule-Associated Proteins, Mitosis, Up-Regulation, Vascular Endothelial Growth Factor A, Cell Line, Tumor, Mice, Rats, AMP-Activated Protein Kinases, Endoplasmic Reticulum Stress

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