Lipid droplets and ferritin heavy chain: a devilish liaison in human cancer cell radioresistance

  1. Luca Tirinato  Is a corresponding author
  2. Maria Grazia Marafioti
  3. Francesca Pagliari  Is a corresponding author
  4. Jeannette Jansen
  5. Ilenia Aversa
  6. Rachel Hanley
  7. Clelia Nisticò
  8. Daniel Garcia-Calderón
  9. Geraldine Genard
  10. Joana Filipa Guerreiro
  11. Francesco Saverio Costanzo
  12. Joao Seco  Is a corresponding author
  1. Biomedical Physics in Radiation Oncology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld, Germany
  2. Experimental and Clinical Medicine Department, University Magna Graecia of Catanzaro, Italy
  3. Department of Physics and Astronomy, Heidelberg University, Im Neuenheimer Feld, Germany
  4. Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Portugal
5 figures, 1 table and 2 additional files


Figure 1 with 3 supplements
Lipid droplet detection in neuroglioma (H4), lung (H460), breast (MCF7), prostate (PC3), and bladder (T24) 6 Gy X-ray-resistant cancer cells.

Cancer cells have been irradiated with 6 Gy X-ray and left in culture for 72 hrs. Afterwards, surviving and untreated cells have been stained with LD540 and imaged at the fluorescence confocal microscope. Z-projection of the z-stack acquisitions for untreated and 6 Gy treated cells are reported in column (A) (Scale bar, 20 μM). (B) For each cell line, 50 cells have been randomly imaged, and their LD number counted by using FiJi software. (C) qPCR analysis of the PLIN genes in the indicated cell lines. PLIN5 in the H4 6 Gy treated cells is not reported in the graph because it was not expressed. Error bars represent the means ± SD from three independent experiments. *≤0.05; **≤0.01; ***≤0.001, and ****≤0.0001.

Figure 1—figure supplement 1
ROS evaluation in 6 Gy radioresistant cancer cells.
Figure 1—figure supplement 2
ROS and Lipid Droplet Double Staining in Different Cancer Cell Lines.
Figure 1—figure supplement 3
CSC Marker Evaluation in 6 Gy Radioresistant Cells.
Cell survival curves for H4, H460, MCF7, PC3, and T24 cancer cell lines.

All cancer cell lines were stained with LD540 and then sorted in the 10% highest and lowest LD-containing cells (box up-left). For each cell line, the two LD sub-populations were irradiated at 2, 4, and 6 Gy X-ray and their survival fraction calculated. Survival fractions are reported in log-linear scale. Error bar represents the means ± SD from three independent experiments. *≤0.05; **≤0.01; ***≤0.001, and ****≤0.0001.

Figure 3 with 1 supplement
FTH1 silencing downregulates lipid droplets affecting cancer radioresistance.

(A, B) Wester blotting analysis and quantification of FTH1 expression in MCF7, H460, H4, PC3, T24- 0 Gy vs 6 Gy X-ray-treated cells. HSC70 was used as a loading control. (C) H460 and MCF7 were sorted in the 10% Highest (H460 LDHigh and MCF7 LDHigh) and Lowest (H460 LDLow and MCF7 LDLow) LD-containing cells and then FTH1 mRNA expression measured by qRT-PCR in all four sub-populations. Primer sequences are listed in Key resource table. (D) H460 and MCF7 were silenced for FTH1 by lentiviral-driven shRNA strategy. PCR results showed that in H460 shFTH1 and MCF shFTH1 there was a clear FTH1 mRNA reduction compared with their relative controls. (E, F) LD content was measured and quantified in H460 shFTH1 and MCF7 shFTH1 by confocal microscopy. LD540 staining revealed that the FTH1 gene silencing caused a LD decrease in both cell systems. (Scale bars 20 μM). (G) Cellular irradiation response in H460 and MCF7 silenced for FTH1 was investigated by radiobiological clonogenic assay and compared with H460 shRNA and MCF7 shRNA, respectively. Survival fraction (in log-linear scale) is reported in (G). Error bar represents the means ± SD from three independent experiments. *≤0.05; **≤0.01; ***≤0.001, and ****≤0.0001.

Figure 3—figure supplement 1
TfR1 downregulation and FPN upregulation in FTH1-silenced H460 and MCF7 cells.
FTH1 reconstitution as well as DFO treatment restore the LD content re-establishing cancer radioresistance.

(A-B) Western Blotting analysis and quantification of FTH1 expression in MCF7 shFTH1/pcDNA3FTH1 and H460 shFTH1/pcDNA3FTH1. HSC70 was used as loading control. (C) Z-stack representative confocal fluorescence images of LD detection and quantification (D) in MCF7 shFTH1/pcDNA3FTH1 and H460 shFTH1/pcDNA3FTH1 cells and their H460 shFTH1/pcDNA3 and MCF7 shFTH1/pcDNA3 controls. (Scale bars 20 mM). (E) Survival fractions (in log-linear scale) after FTH1 reconstitution in MCF7- and H460- shFTH1 cells. (F) Z-stack representative confocal fluorescence images of LD detection and quantification (G) in MCF7 shFTH1 and H460 shFTH1 treated with DFO (Scale bar 20 mM). (H) Survival curves (in log-linear scale) of FTH1-silenced MCF7 and H460 cells after DFO treatment. F. Error bar represents the means ± SD from three independent experiments. *£ 0.05; **£ 0.01; ***£ 0.001 and ****£ 0.0001.

Figure 5 with 1 supplement
Schematic representation of a putative model at the basis of the cancer radioresistance (Created
Figure 5—figure supplement 1
Keplan-Meier curves showing the Survival Probability related to the PLIN1-5, FTH1, TFR1 and FPN gene expression.


Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Homo sapiens)MCF-7ATCCCat# HTB-22 RRID:CVCL_0031
Cell line (Homo sapiens)H4ATCCCat# HTB-148 RRID:CVCL_1239
Cell line (Homo sapiens)H460ATCCCat# HTB-177 RRID:CVCL_0459
Cell line (Homo sapiens)T24ATCCCat# HTB-4 RRID:CVCL_0554
Cell line (Homo sapiens)PC3ATCCCat# CRL-1435 RRID:CVCL_0035
Antibody(Goat polyclonal) anti-human FTH1Santa Cruz BiotechnologyCat# sc-14416 RRID:AB_2107172WB(1:200)
Antibody(Goat polyclonal) anti-human HSC70Santa Cruz BiotechnologyCat# sc-1059 RRID:AB_2120291WB (1:2000)
Antibody(Goat polyclonal) anti-mouse IgG-HRPSanta Cruz BiotechnologyCat# sc-20550 RRID:AB_631738WB (1:2000)
Recombinant DNA reagentpLKO.1Addgene (gift from F.S. Costanzo) Di Sanzo et al., 2011
Recombinant DNA reagentpcDNA3Addgene (gift from F.S. Costanzo) Aversa et al., 2017
Chemical compound, drugCM-H2DCFDAThermo Fisher ScientificCat# C6827
Chemical compound, drugNile RedThermo Fisher ScientificCat# N1142
Chemical compound, drugDeferoxaminemesylateSigma-AldrichCat# D9533
Chemical compound, drugPuromycin dihydrochlorideAcros organicsCat# BP2965
Chemical compound, drugPower SYBR Green PCR Master mixThermo Fisher ScientificCat# 4367659
Chemical compound, drugLD540Synthetised by Enamine
Commercial assay or kitPierce BCA Protein Assay KitThermo Fisher ScientificCat# 23,225
Commercial assay or kitHigh Pure isolation RNA kitRocheCat# 11828665001
Commercial assay or kitRT2 First strand Kit (50)QiagenCat# 330,404
Commercial assay or kitLipofectamine 3000 Transfection ReagentThermo Fisher ScientificCat# L3000075
Sequence-based reagentCustom-made qPCR primers – GAPDHSigma-AldrichForward – 5'-GCATCCTGGGCTACACTGAG-3' Reverse – 5'-AAAGTGGTCGTTGAGGGCA-3'
Sequence-based reagentCustom-made qPCR primers – FTH1Sigma-AldrichForward – 5′-CATCAACCGCCAGATCAAC-3 Reverse –5′-GATGGCTTTCACCTGCTCAT-3′
Sequence-based reagentCustom-made qPCR primers – TfR1Sigma-AldrichForward – 5'-CTGGTAAACTGGTCCATGCT-3' Reverse – 5'-GTGATTTTCCCTGCTCTGAC-3'
Sequence-based reagentCustom-made qPCR primers – FPNSigma-AldrichForward – 5'-GGTGTCTGTGTTTCTGGT-3' Reverse – 5'-GTCTAGCATTCTTGTCCAC-3'
Sequence-based reagentCustom-made qPCR primers – CD24Sigma-AldrichForward – 5’-CCTGTCAGAGCTGTGTGGAC-3’ Reverse – 5’-GCTGGGTAGAGTGGTGTGT-3’
Sequence-based reagentCustom-made qPCR primers – CD44Sigma-AldrichForward – 5’-GGGTTCATAGAAGGGCACGT-3’ Reverse – 5’-GGGAGGTGTTGGATGTGAGG-3’
Sequence-based reagentCustom-made qPCR primers – CD133Sigma-AldrichForward – 5’-AAGCATTGGCATCTTCTATGG-3’ Reverse – 5‘-AGAGAGTTCGCAAGTCCTTG-3’
Sequence-based reagentCustom-made qPCR primers – CD166Sigma-AldrichForward – 5‘-CGATGAGGCAGACGAGATAAG-3’ Reverse – 5‘-TAGACGACACCAGCAACAAG-3’
Sequence-based reagentCustom-made qPCR primers – ALDH1Sigma-AldrichForward – 5’-AACTGGAATGTGGAGGAGGC-3’ Reverse – 5’-ATGATTTGCTGCACTGGTCC-3’
Sequence-based reagentCustom-made qPCR primers – PLIN1Sigma-AldrichForward – 5'-GACAAGGAAGAGTCAGCCCC-3' Reverse – 5'-GAGAGGGTGTTGGTCAGAGC-3'
Sequence-based reagentCustom-made qPCR primers – PLIN2Sigma-AldrichForward – 5'-ACAGGGGTGATGGACAAGAC-3' Reverse – 5'-ATCATCCGACTCCCCAAGAC-3'
Sequence-based reagentCustom-made qPCR primers – PLIN3Sigma-AldrichForward – 5'-CACCATGTTCCGGGACATTG-3' Reverse – 5'-GCACCTGGTCCTTCACATTG-3'
Sequence-based reagentCustom-made qPCR primers – PLIN4Sigma-AldrichForward – 5’- GTTCCAGGACCACAGACA-3’ Reverse – 5’CCTACACTGAGCACATCC-3’
Sequence-based reagentCustom-made qPCR primers – PLIN5Sigma-AldrichForward – 5'-GATCACTTCCTGCCCATGAC-3' Reverse – 5'-GCTGTCTCCTCTGATCCTCC-3'
Sequence-based reagentCustom-made qPCR primers – SOD1Sigma-AldrichForward – 5'-GCAGATGACTTGGGCAAAGG-3' Reverse – 5'-TGGGCGATCCCAATTACACC-3'
Sequence-based reagentCustom-made qPCR primers – SOD2Sigma-AldrichForward – 5'-CTGGAACCTCACATCAACGC-3' Reverse – 5'-CCTGGTACTTCTCCTCGGTG-3'
Sequence-based reagentCustom-made qPCR primers – GPX1Sigma-AldrichForward – 5’-CCCAAGCTCATCACCTGGTC-3’ Reverse – 5’-TGTCAATGGTCTGGAAGCGG-3’
Sequence-based reagentCustom-made qPCR primers – CatalaseSigma-AldrichForward – 5'-CGTGCTGAATGAGGAACAG-3' Reverse – 5'-GACCGCTTTCTTCTGGATG-3'

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  1. Luca Tirinato
  2. Maria Grazia Marafioti
  3. Francesca Pagliari
  4. Jeannette Jansen
  5. Ilenia Aversa
  6. Rachel Hanley
  7. Clelia Nisticò
  8. Daniel Garcia-Calderón
  9. Geraldine Genard
  10. Joana Filipa Guerreiro
  11. Francesco Saverio Costanzo
  12. Joao Seco
Lipid droplets and ferritin heavy chain: a devilish liaison in human cancer cell radioresistance
eLife 10:e72943.