Analysis of clonogenic growth in vitro

Nikko Brix, Daniel Samaga, Claus Belka, Horst Zitzelsberger, Kirsten Lauber

Published: 2021-10-24 DOI: 10.1038/s41596-021-00615-0

Clonogenic Assay

In Vitro

Cellular Cooperation

Survival Analysis

Protocol Optimization

AI 解读

Supplementary information

Reporting Summary

Supplementary Table 1

MS Excel template file for power regression–based analysis of clonogenic survival

Supplementary Table 2

MS Excel template file for PE-based analysis of clonogenic survival

Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail

Profiling native pulmonary basement membrane stiffness using atomic force microscopy

Genetic circuit design automation with Cello 2.0

Synthesis of siRNA nanoparticles to silence plaque-destabilizing gene in atherosclerotic lesional macrophages

Scalable dual-omics profiling with single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2)

A practical guide to scanning light-field microscopy with digital adaptive optics

Effective high-throughput isolation of fully human antibodies targeting infectious pathogens

Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments

MOF-derived nanoporous carbons with diverse tunable nanoarchitectures

Tutorial: a statistical genetics guide to identifying HLA alleles driving complex disease

查看全部

Sections

Figures

References

Supplementary information

Franken, N. A., Rodermond, H. M., Stap, J., Haveman, J. & van Bree, C. Clonogenic assay of cells in vitro. Nat. Protoc. 1, 2315–2319 (2006).
Puck, T. T. & Marcus, P. I. Action of x-rays on mammalian cells. J. Exp. Med. 103, 653–666 (1956).
Puck, T. T., Marcus, P. I. & Cieciura, S. J. Clonal growth of mammalian cells in vitro; growth characteristics of colonies from single HeLa cells with and without a feeder layer. J. Exp. Med. 103, 273–283 (1956).
Puck, T. T. & Marcus, P. I. A rapid method for viable cell titration and clone production with hela cells in tissue culture: the use of X-irradiated cells to supply conditioning factors. Proc. Natl Acad. Sci. USA 41, 432–437 (1955).
Rheinwald, J. G. & Green, H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331–343 (1975).
Reynolds, B. A. & Weiss, S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707–1710 (1992).
Phillips, T. M., McBride, W. H. & Pajonk, F. The response of CD24-/low/CD44+ breast cancer-initiating cells to radiation. J. Natl Cancer Inst. 98, 1777–1785 (2006).
Wink, D. A. et al. Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species. Proc. Natl Acad. Sci. U. S. A. 90, 9813–9817 (1993).
Liebmann, J. E. et al. Cytotoxic studies of paclitaxel (Taxol) in human tumour cell lines. Br. J. Cancer 68, 1104–1109 (1993).
Wang, Q. et al. UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53. J. Natl Cancer Inst. 88, 956–965 (1996).
Elkind, M. M. & Sutton, H. Radiation response of mammalian cells grown in culture. 1. Repair of X-ray damage in surviving Chinese hamster cells. Radiat. Res. 13, 556–593 (1960).
Huang, P. et al. Folic acid-conjugated silica-modified gold nanorods for X-ray/CT imaging-guided dual-mode radiation and photo-thermal therapy. Biomaterials 32, 9796–9809 (2011).
Elgendy, M., Sheridan, C., Brumatti, G. & Martin, S. J. Oncogenic Ras-induced expression of Noxa and Beclin-1 promotes autophagic cell death and limits clonogenic survival. Mol. Cell 42, 23–35 (2011).
Alimova, I. N. et al. Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro. Cell Cycle 8, 909–915 (2009).
Rochat, A., Kobayashi, K. & Barrandon, Y. of stem cells of human hair follicles by clonal analysis. Cell 76, 1063–1073 (1994).
Sanford, K. K., Earle, W. R. & Likely, G. D. The growth in vitro of single isolated tissue cells. J. Natl Cancer Inst. 9, 229–246 (1948).
Brix, N. et al. The clonogenic assay: robustness of plating efficiency-based analysis is strongly compromised by cellular cooperation. Radiat. Oncol. 15, 248 (2020).
Orth, M., Unger, K., Schoetz, U., Belka, C. & Lauber, K. Taxane-mediated radiosensitization derives from chromosomal missegregation on tripolar mitotic spindles orchestrated by AURKA and TPX2. Oncogene 37, 52–62 (2018).
Hess, J. et al. Genomic amplification of Fanconi anemia complementation group A (FancA) in head and neck squamous cell carcinoma (HNSCC): cellular mechanisms of radioresistance and clinical relevance. Cancer Lett. 386, 87–99 (2017).
Puck, T. T., Cieciura, S. J. & Fisher, H. W. Clonal growth in vitro of human cells with fibroblastic morphology; comparison of growth and genetic characteristics of single epithelioid and fibroblast-like cells from a variety of human organs. J. Exp. Med. 106, 145–158 (1957).
Fisher, H. W. & Puck, T. T. On the functions of x-irradiated “feeder” cells in supporting growth of single mammalian cells. Proc. Natl Acad. Sci. USA 42, 900–906 (1956).
Terasima, T. & Tolmach, L. J. Changes in x-ray sensitivity of HeLa cells during the division cycle. Nature 190, 1210–1211 (1961).
Son, J. et al. Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway. Nature 496, 101–105 (2013).
Varfolomeev, E. et al. IAP antagonists induce autoubiquitination of c-IAPs, NF-kappaB activation, and TNFalpha-dependent apoptosis. Cell 131, 669–681 (2007).
Till, J. E. & McCulloch, C. E. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14, 213–222 (1961).
Becker, A. J., McCulloch, C. E. & Till, J. E. Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature 197, 452–454 (1963).
Park, C. H., Bergsagel, D. E. & McCulloch, E. A. Mouse myeloma tumor stem cells: a primary cell culture assay. J. Natl Cancer Inst. 46, 411–422 (1971).
Courtenay, V. D. & Mills, J. An in vitro colony assay for human tumours grown in immune-suppressed mice and treated in vivo with cytotoxic agents. Br. J. Cancer 37, 261–268 (1978).
Hamburger, A. W. & Salmon, S. E. Primary bioassay of human tumor stem cells. Science 197, 461–463 (1977).
Whitlock, C. A. & Witte, O. N. Long-term culture of B lymphocytes and their precursors from murine bone marrow. Proc. Natl Acad. Sci. USA 79, 3608–3612 (1982).
Kondo, M., Weissman, I. L. & Akashi, K. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91, 661–672 (1997).
Matsui, W. et al. Characterization of clonogenic multiple myeloma cells. Blood 103, 2332–2336 (2004).
Eliason, J. F., Aapro, M. S., Decrey, D. & Brink-Petersen, M. Non-linearity of colony formation by human tumour cells from biopsy samples. Br. J. Cancer 52, 311–318 (1985).
Pomp, J. et al. Cell density dependent plating efficiency affects outcome and interpretation of colony forming assays. Radiother. Oncol. 40, 121–125 (1996).
Veldwijk, M. R., Zhang, B., Wenz, F. & Herskind, C. The biological effect of large single doses: a possible role for non-targeted effects in cell inactivation. PLoS One 9, e84991 (2014).
Adrian, G., Ceberg, C., Carneiro, A. & Ekblad, L. Rescue effect inherited in colony formation assays affects radiation response. Radiat. Res. 189, 44–52 (2018).
Dakhore, S., Nayer, B. & Hasegawa, K. Human pluripotent stem cell culture: current status, challenges, and advancement. Stem Cells Int. 2018, 7396905 (2018).
Repetto, G., del Peso, A. & Zurita, J. L. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat. Protoc. 3, 1125–1131 (2008).
Lindhagen, E., Nygren, P. & Larsson, R. The fluorometric microculture cytotoxicity assay. Nat. Protoc. 3, 1364–1369 (2008).
Riss, T. L. et al. Cell viability assays. in Assay Guidance Manual (eds. Markossian S. et al.) (Eli Lilly and the National Center for Advancing Translational Sciences, 2004).
Rampersad, S. N. Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors (Basel) 12, 12347–12360 (2012).
Lundin, A., Hasenson, M., Persson, J. & Pousette, A. Estimation of biomass in growing cell lines by adenosine triphosphate assay. Methods Enzymol. 133, 27–42 (1986).
Temple, S. Division and differentiation of isolated CNS blast cells in microculture. Nature 340, 471–473 (1989).
Davis, A. A. & Temple, S. A self-renewing multipotential stem cell in embryonic rat cerebral cortex. Nature 372, 263–266 (1994).
Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).
Ploemacher, R. E., van der Sluijs, J. P., Voerman, J. S. & Brons, N. H. An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse. Blood 74, 2755–2763 (1989).
Sutherland, H. J., Lansdorp, P. M., Henkelman, D. H., Eaves, A. C. & Eaves, C. J. Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc. Natl Acad. Sci. USA 87, 3584–3588 (1990).
Fogg, D. K. et al. A clonogenic bone marrow progenitor specific for macrophages and dendritic cells. Science 311, 83–87 (2006).
Riether, C. et al. Targeting CD70 with cusatuzumab eliminates acute myeloid leukemia stem cells in patients treated with hypomethylating agents. Nat. Med. 26, 1459–1467 (2020).
Hu, Y. & Smyth, G. K. ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J. Immunol. Methods 347, 70–78 (2009).
Fiebig, H. H., Maier, A. & Burger, A. M. Clonogenic assay with established human tumour xenografts: correlation of in vitro to in vivo activity as a basis for anticancer drug discovery. Eur. J. Cancer 40, 802–820 (2004).
Borowicz, S. et al. The soft agar colony formation assay. J. Vis. Exp. 92, e51998 (2014).
Leivas, A. et al. Natural killer cells efficiently target multiple myeloma clonogenic tumor cells. Cancer Immunol. Immunother. (in the press).
Kukreja, A. et al. Enhancement of clonogenicity of human multiple myeloma by dendritic cells. J. Exp. Med. 203, 1859–1865 (2006).
Eke, I., Hehlgans, S., Sandfort, V. & Cordes, N. 3D matrix-based cell cultures: automated analysis of tumor cell survival and proliferation. Int. J. Oncol 48, 313–321 (2016).
Nielson, L., Smyth, G. & Greenfield, P. Hemacytometer cell count distributions: implications of non-Poisson behavior. Biotechnol. Prog. 7, 560–563 (1991).
Harnicek, D. et al. Hyperthermia adds to trabectedin effectiveness and thermal enhancement is associated with BRCA2 degradation and impairment of DNA homologous recombination repair. Int. J. Cancer 139, 467–479 (2016).
Weiss, E. M. et al. High hydrostatic pressure treatment generates inactivated mammalian tumor cells with immunogeneic features. J. Immunotoxicol. 7, 194–204 (2010).
Preciado, S. et al. The incorporation of extracellular vesicles from mesenchymal stromal cells into CD34+ cells increases their clonogenic capacity and bone marrow lodging ability. Stem Cells 37, 1357–1368 (2019).
Yao, T. & Asayama, Y. Animal-cell culture media: history, characteristics, and current issues. Reprod. Med. Biol. 16, 99–117 (2017).
Nuryadi, E., Mayang Permata, T. B., Komatsu, S., Oike, T. & Nakano, T. Inter-assay precision of clonogenic assays for radiosensitivity in cancer cell line A549. Oncotarget 9, 13706–13712 (2018).
Lacerda, L. et al. Simvastatin radiosensitizes differentiated and stem-like breast cancer cell lines and is associated with improved local control in inflammatory breast cancer patients treated with postmastectomy radiation. Stem Cells Transl. Med. 3, 849–856 (2014).
Dahle, J., Kakar, M., Steen, H. B. & Kaalhus, O. Automated counting of mammalian cell colonies by means of a flat bed scanner and image processing. Cytometry A 60, 182–188 (2004).
Choudhry, P. High-throughput method for automated colony and cell counting by digital image analysis based on edge detection. PLoS One 11, e0148469 (2016).
Bewes, J. M., Suchowerska, N. & McKenzie, D. R. Automated cell colony counting and analysis using the circular Hough image transform algorithm (CHiTA). Phys. Med. Biol. 53, 5991–6008 (2008).
Meijering, E., Carpenter, A. E., Peng, H., Hamprecht, F. A. & Olivo-Marin, J. C. Imagining the future of bioimage analysis. Nat. Biotechnol. 34, 1250–1255 (2016).
Moen, E. et al. Deep learning for cellular image analysis. Nat. Methods 16, 1233–1246 (2019).
Segebarth, D. et al. On the objectivity, reliability, and validity of deep learning enabled bioimage analyses. eLife 9, e59780 (2020).
Parker, W. S. & Risbey, J. S. False precision, surprise and improved uncertainty assessment. Philos. Trans. A Math. Phys. Eng. Sci. 373, 20140453 (2015).
Kellerer, A. M. & Rossi, H. H. A generalized formulation of dual radiation action. Radiat. Res. 75, 471–488 (1978).
Unkel, S., Belka, C. & Lauber, K. On the analysis of clonogenic survival data: statistical alternatives to the linear-quadratic model. Radiat. Oncol. 11, 11 (2016).
Vembadi, A., Menachery, A. & Qasaimeh, M. A. Cell cytometry: review and perspective on biotechnological advances. Front. Bioeng. Biotechnol. 7, 147 (2019).
Gerweck, L. E., Dullea, R., Zaidi, S. T., Budach, W. & Hartford, A. Influence of experimental factors on intrinsic radiosensitivity assays at low doses of radiation: cell multiplicity. Radiat. Res. 138, 361–366 (1994).
Rockwell, S. Effects of clumps and clusters on survival measurements with clonogenic assays. Cancer Res. 45, 1601–1607 (1985).

Analysis of clonogenic growth in vitro

Supplementary information

Reporting Summary

Supplementary Table 1

Supplementary Table 2

推荐阅读