Linking hypoxia, DNA damage and proliferation in multicellular tumor spheroids
Open accessAbstract
BACKGROUND: Multicellular Tumor Spheroids are frequently used to mimic the regionalization of proliferation and the hypoxic environment within avascular tumors. Here we exploit these features to study the activation of DNA damage repair pathways and their correlation to developing hypoxia. METHODS: Activation of DNA damage repair markers, proliferation, cell death, glycogen accumulation and developing hypoxia were investigated using immunofluorescence, immuno-histochemistry, EdU incorporation, Western blots, COMET assays, and pharmacological agents in A673 Ewing sarcoma spheroids and monolayer cultures. RESULTS: DNA damage marker γ-H2AX is observed in the hypoxic, peri-necrotic region of growing spheroids. While most proliferating cells are seen on the spheroid surface, there are also a few Ki-67 positive cells in the hypoxic zone. The hypoxia-induced phosphorylation of H2AX to form γ-H2AX in spheroids is attenuated by the ATM inhibitor KU55933, but not the ATR inhibitor VE-821. CONCLUSION: Tumor spheroids mimic tumor microenvironments such as the anoxic, hypoxic and oxic niches within solid tumors, as well as populations of cells that are viable, proliferating, and undergoing DNA damage repair processes under these different micro-environmental conditions. ATM, but not ATR, is the primary kinase responsible for γ-H2AX formation in the hypoxic core of A673 spheroids. Spheroids could offer unique advantages in testing therapeutics designed to target malignant cells that evade conventional treatment strategies by adapting to the hypoxic tumor microenvironment.
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Fluorescently labeled antibodies targeting caspase-3, Ki-67, and hypoxia-inducible factors, or exogenous hypoxia indicators such as pimonidazole and EF5, allow visualization of apoptotic, proliferating, or hypoxic regions, respectively.
“Fluorescently labeled antibodies targeting markers such as caspase-3, Ki-67, and hypoxia-inducible factors, or exogenous hypoxia indicators such as pimonidazole and EF5, allow visualization of apoptotic, proliferating, or hypoxic regions, respectively [235,236].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [236]
The Methods (page not indicated) describe fluorescent antibody staining for cleaved caspase-3, Ki-67, HIF1-α, and pimonidazole. The Results identify caspase-3 with apoptosis, Ki-67 with proliferation, and pimonidazole with hypoxic regions; the Discussion places HIF1-α staining in a hypoxic region. The paper does not mention EF5, so it does not support that example. (LLM, conf=0.88)
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Cites
NIH Image to ImageJ: 25 years of image analysis
Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012) · Nature Methods · doi:10.1038/nmeth.2089 · Cited by 64,638 · see citing works
Modes of resistance to anti-angiogenic therapy
Bergers, G., & Hanahan, D. (2008) · Nature Reviews Cancer · doi:10.1038/nrc2442 · Cited by 2,953 · cited 1× · see citing works
“While there have been some spectacular clinical successes with anti-angiogenics in cancer treatment, the overall and progression-free survival rates have been disappointing [26].”
Hickson, I., Zhao, Y., Richardson, C. J., Green, S. J., Martin, N. M. B., Orr, A. I., Reaper, P. M., Jackson, S. P., Curtin, N. J., & Smith, G. C. M. (2004) · Cancer Research · doi:10.1158/0008-5472.can-04-2727 · Cited by 1,220 · see citing works
OpenComet: An automated tool for comet assay image analysis
Gyori, B. M., Venkatachalam, G., Thiagarajan, P. S., Hsu, D., & Clement, M. (2014) · Redox Biology · doi:10.1016/j.redox.2013.12.020 · Cited by 721 · see citing works
Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR
Reaper, P. M., Griffiths, M. R., Long, J. M., Charrier, J.-D., MacCormick, S., Charlton, P. A., Golec, J. M. C., & Pollard, J. R. (2011) · Nature Chemical Biology · doi:10.1038/nchembio.573 · Cited by 633 · see citing works
Tumor Hypoxia in Cancer Therapy
Brown, J. M. (2007) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/s0076-6879(07)35015-5 · Cited by 359 · see citing works
Hypoxia Links ATR and p53 through Replication Arrest
Hammond, E. M., Denko, N. C., Dorie, M. J., Abraham, R. T., & Giaccia, A. J. (2002) · Molecular and Cellular Biology · doi:10.1128/mcb.22.6.1834-1843.2002 · Cited by 321 · see citing works
A method for estimating the oxygen consumption rate in multicellular tumour spheroids
Grimes, D. R., Kelly, M., Bloch, K. M., & Partridge, M. (2014) · Journal of The Royal Society Interface · doi:10.1098/rsif.2013.1124 · Cited by 298 · see citing works
Hammond, E. M., Dorie, M. J., & Giaccia, A. J. (2003) · Journal of Biological Chemistry · doi:10.1074/jbc.m212360200 · Cited by 282 · see citing works
ATR autophosphorylation as a molecular switch for checkpoint activation
Liu, S., Shiotani, B., Lahiri, M., Maréchal, A., Tse, A., Leung, C. C. Y., Glover, J. N. M., Yang, X. H., & Zou, L. (2011) · Molecular Cell · doi:10.1016/j.molcel.2011.06.019 · Cited by 281 · see citing works
mTORC1 Signaling under Hypoxic Conditions Is Controlled by ATM-Dependent Phosphorylation of HIF-1α
Cam, H., Easton, J. B., High, A., & Houghton, P. J. (2010) · Molecular Cell · doi:10.1016/j.molcel.2010.10.030 · Cited by 266 · see citing works
Pescador, N., Villar, D., Cifuentes, D., Garcı́a-Rocha, M., Ortiz-Barahona, A., Vázquez, S., Ordóñez, Á., Cuevas, Y., Saez-Morales, D., García‐Bermejo, L., et al. (2010) · PLoS ONE · doi:10.1371/journal.pone.0009644 · Cited by 265 · see citing works
The multicellular tumor spheroid model for high-throughput cancer drug discovery
LaBarbera, D. V., Reid, B. G., & Yoo, B. H. (2012) · Expert Opinion on Drug Discovery · doi:10.1517/17460441.2012.708334 · Cited by 254 · see citing works
Zois, C. E., & Harris, A. L. (2016) · Journal of Molecular Medicine · doi:10.1007/s00109-015-1377-9 · Cited by 254 · see citing works
Double strand break repair functions of histone H2AX
Scully, R., & Xie, A. (2013) · Mutation research. Fundamental and molecular mechanisms of mutagenesis · doi:10.1016/j.mrfmmm.2013.07.007 · Cited by 251 · see citing works
Prevo, R., Fokas, E., Reaper, P. M., Charlton, P., Pollard, J. R., McKenna, W. G., Muschel, R. J., & Brunner, T. B. (2012) · Cancer Biology & Therapy · doi:10.4161/cbt.21093 · Cited by 242 · see citing works
Gross, M. W., Karbach, U., Groebe, K., Franko, A. J., & Mueller‐Klieser, W. (1995) · International Journal of Cancer · doi:10.1002/ijc.2910610422 · Cited by 206 · see citing works
ATM Activation and Signaling under Hypoxic Conditions
Bencokova, Z., Kaufmann, M. R., Pires, I. M., Lecane, P. S., Giaccia, A. J., & Hammond, E. M. (2008) · Molecular and Cellular Biology · doi:10.1128/mcb.01301-08 · Cited by 205 · see citing works
Pelletier, J., Bellot, G. L., Gounon, P., Lacas‐Gervais, S., Pouysségur, J., & Mazure, N. M. (2012) · Frontiers in Oncology · doi:10.3389/fonc.2012.00018 · Cited by 203 · see citing works
Effects of Acute versus Chronic Hypoxia on DNA Damage Responses and Genomic Instability
Pires, I. M., Bencokova, Z., Milani, M., Folkes, L. K., Li, J., Stratford, M. R., Harris, A. L., & Hammond, E. M. (2010) · Cancer Research · doi:10.1158/0008-5472.can-09-2715 · Cited by 200 · see citing works
de Feraudy, S. M., Revet, I. M., Bezrookove, V., Feeney, L., & Cleaver, J. E. (2010) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1002175107 · Cited by 195 · see citing works
Regulation of DNA repair in hypoxic cancer cells
Bindra, R. S., Crosby, M. E., & Glazer, P. M. (2007) · Cancer and Metastasis Reviews · doi:10.1007/s10555-007-9061-3 · Cited by 191 · see citing works
Targeting radiation-resistant hypoxic tumour cells through ATR inhibition
Pires, I. M., Olcina, M. M., Anbalagan, S., Pollard, J. R., Reaper, P. M., Charlton, P., McKenna, W. G., & Hammond, E. M. (2012) · British Journal of Cancer · doi:10.1038/bjc.2012.265 · Cited by 171 · see citing works
Multifaceted control of DNA repair pathways by the hypoxic tumor microenvironment
Scanlon, S. E., & Glazer, P. M. (2015) · DNA repair · doi:10.1016/j.dnarep.2015.04.030 · Cited by 147 · see citing works
Histone H2AX is integral to hypoxia-driven neovascularization
Economopoulou, M., Langer, H. F., Celeste, A., Orlova, V. V., Choi, E. Y., Ma, M., Vassilopoulos, A., Callén, E., Deng, C., Bassing, C. H., et al. (2009) · Nature Medicine · doi:10.1038/nm.1947 · Cited by 131 · see citing works
Multicellular tumor spheroid models to explore cell cycle checkpoints in 3D
Laurent, J., Frongia, C., Cazalès, M., Mondésert, O., Ducommun, B., & Lobjois, V. (2013) · BMC Cancer · doi:10.1186/1471-2407-13-73 · Cited by 131 · see citing works
Nam, E. A., Zhao, R., Glick, G. G., Bansbach, C. E., Friedman, D. B., & Cortez, D. (2011) · Journal of Biological Chemistry · doi:10.1074/jbc.m111.248914 · Cited by 126 · see citing works
Replication Stress and Chromatin Context Link ATM Activation to a Role in DNA Replication
Olcina, M. M., Foskolou, I. P., Anbalagan, S., Senra, J. M., Pires, I. M., Jiang, Y., Ryan, A. J., & Hammond, E. M. (2013) · Molecular Cell · doi:10.1016/j.molcel.2013.10.019 · Cited by 110 · see citing works
Targeting Hypoxic Cells through the DNA Damage Response
Olcina, M. M., Lecane, P. S., & Hammond, E. M. (2010) · Clinical Cancer Research · doi:10.1158/1078-0432.ccr-10-0286 · Cited by 109 · see citing works
Langan, L. M., Dodd, N. J. F., Owen, S. F., Purcell, W. M., Jackson, S. K., & Jha, A. N. (2016) · PLOS ONE · doi:10.1371/journal.pone.0149492 · Cited by 101 · see citing works
Shen, G., Zhang, F., Liu, X., & Zhang, J. (2010) · FEBS Letters · doi:10.1016/j.febslet.2010.09.040 · Cited by 99 · see citing works
Comparison of hypoxia-induced replication arrest with hydroxyurea and aphidicolin-induced arrest
Hammond, E. M., Green, S., & Giaccia, A. J. (2003) · Mutation research. Fundamental and molecular mechanisms of mutagenesis · doi:10.1016/j.mrfmmm.2003.08.017 · Cited by 90 · see citing works
Efficacy of ATR inhibitors as single agents in Ewing sarcoma
Nieto-Soler, M., Morgado-Palacin, I., Lafarga, V., Lecona, E., Murga, M., Callén, E., Azorín, D., Alonso, J., Lopez-Contreras, A. J., Nussenzweig, A., et al. (2016) · Oncotarget · doi:10.18632/oncotarget.11643 · Cited by 90 · see citing works
The role of ATM and ATR in the cellular response to hypoxia and re-oxygenation
Hammond, E. M., & Giaccia, A. J. (2004) · DNA repair · doi:10.1016/j.dnarep.2004.03.035 · Cited by 84 · see citing works
Growth of mammalian multicellular tumor spheroids.
Conger, A. D., & Ziskin, M. C. (1983) · PubMed · Cited by 81 · see citing works
Hoogsteen, I. J., Marres, H. A. M., Wijffels, K. I. E. M., et al. (2005) · Clinical Cancer Research · doi:10.1158/1078-0432.97.11.1 · Cited by 74 · see citing works
Characterization and Drug Resistance Patterns of Ewing's Sarcoma Family Tumor Cell Lines
May, W. A., Grigoryan, R. S., Keshelava, N., Cabral, D. J., Christensen, L. L., Jenabi, J. M., Ji, L., Triche, T. J., Lawlor, E. R., & Reynolds, C. P. (2013) · PLoS ONE · doi:10.1371/journal.pone.0080060 · Cited by 60 · see citing works
Gomes, A., Guillaume, L., Grimes, D. R., Fehrenbach, J., Lobjois, V., & Ducommun, B. (2016) · PLoS ONE · doi:10.1371/journal.pone.0161239 · Cited by 55 · see citing works
Evans, S. M., Jenkins, K. W., Chen, H. I., Jenkins, W. T., Judy, K. D., Hwang, W., Lustig, R. A., Judkins, A. R., Gradyt, M. S., Hahn, S. M., et al. (2010) · Translational Oncology · doi:10.1593/tlo.09265 · Cited by 47 · see citing works
ATM activation in hypoxia - causes and consequences
Olcina, M. M., Grand, R. J. A., & Hammond, E. M. (2014) · Molecular & Cellular Oncology · doi:10.4161/mco.29903 · Cited by 35 · see citing works
The Eyes Absent Proteins in Developmental and Pathological Angiogenesis
Wang, Y., Tadjuidje, E., Pandey, R. N., et al. (2016) · American Journal Of Pathology · doi:10.1016/j.ajpath.2015.10.031 · Cited by 18 · see citing works
Self-citationMartin, L., Rainey, M. D., Santocanale, C., & Gardner, L. B. (2011) · Oncogene · doi:10.1038/onc.2011.585 · Cited by 15 · see citing works
Bringing H2AX into the Angiogenesis Family
Rankin, E. B., Giaccia, A. J., & Hammond, E. M. (2009) · Cancer Cell · doi:10.1016/j.ccr.2009.05.004 · Cited by 14 · see citing works
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Tuttle, C. S. L., Waaijer, M. E. C., Slee‐Valentijn, M. S., Stijnen, T., Westendorp, R., & Maier, A. B. (2020) · Aging Cell · doi:10.1111/acel.13083 · Cited by 211 · see citing works
Ng, S. S., Saeb-Parsy, K., Blackford, S. J. I., Segal, J. M., Serra, M. P., Horcas-Lopez, M., No, D. Y., Mastoridis, S., Jassem, W., Frank, C. W., et al. (2018) · Biomaterials · doi:10.1016/j.biomaterials.2018.07.043 · Cited by 133 · see citing works
Shah, S., & D’Souza, G. G. M. (2025) · Cells · doi:10.3390/cells14100732 · Cited by 66 · see citing works
“Fluorescently labeled antibodies targeting markers such as caspase-3, Ki-67, and hypoxia-inducible factors, or exogenous hypoxia indicators such as pimonidazole and EF5, allow visualization of apoptotic, proliferating, or hypoxic regions, respectively [235,236].”
Quílez, C., Jeon, E. Y., Pappalardo, A., Pathak, P., & Abaci, H. E. (2024) · Advanced Healthcare Materials · doi:10.1002/adhm.202400405 · Cited by 22 · see citing works
Boussommier-Calleja, A. (2020) · Bioengineering Innovative Solutions for Cancer · doi:10.1016/b978-0-12-813886-1.00013-9 · Cited by 10 · see citing works
- Authors
- Riffle, S., Pandey, R. N., Albert, M., & Hegde, R. S.
- Affiliations
- Cincinnati Children's Hospital Medical Center (US)
- Corresponding author
- Hegde, Rashmi S.
- Venue
- BMC Cancer
- Published
- 2017-05-18
- Volume
- 17
- Issue
- 1
- Pages
- 338-338
- ISSN
- 1471-2407 (ISSN-L)
- PMC
- PMC5437385
- OpenAlex
- W2615171435
- Cited by
- 142
- References
- 43
- Citations by year
- 2026: 9 · 2025: 19 · 2024: 22 · 2023: 10 · 2022: 15 · 2021: 24 · 2020: 18 · 2019: 13 · 2018: 10 · 2017: 2
- FWCI
- 4.81
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- BMC Cancer (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Cancer Free Kids
- National Eye Institute (EY014648)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
- Keywords
- tumor spheroids, tumor hypoxia, DNA repair, γH2AX, ATM kinase, ATR kinase, KU55933, VE-821, Ewing sarcoma, A673 cells, cell proliferation, tumor microenvironment, Ki-67, cell death, glycogen accumulation
- MeSH terms
- Animals, DNA Damage, Histones, Humans, Cell Hypoxia, Apoptosis, Carcinoma, Lewis Lung, Spheroids, Cellular, Cell Line, Tumor, Cell Proliferation, Mice
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