Hypoxic cell labelling using click chemistry

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Chapter 3. Selection of fluorophores for click chemistry based detection of hypoxic cells


Fluorescence is a widely used key technique in life science applications (Drummen, 2012; Fili & Toseland, 2014; Lakowicz, 2007). Each fluorescent compound is characterised by an excitation spectrum (the wavelength and amount of light absorbed) and an emission spectrum (the wavelength and amount of light emitted), usually referred to as compound’s fluorescence signature or fingerprint (Valeur & Berberan-Santos, 2012). The principle that all fluorophores have relatively distinct fluorescence signatures makes fluorometry a highly specific analytical technique (Drummen, 2012; Taraska & Zagotta, 2010).
By selectively generating the wavelength of light required to excite the analyte of interest, selectively transmitting the wavelength of light emitted, and measuring the intensity of the emitted light, fluorescence can be measured based on the principle that the emitted light is proportional to the concentration of the analyte being measured within a certain range of concentration (Fili & Toseland, 2014; Neef & Schultz, 2009; Valeur & Berberan-Santos, 2012).
Due to the ability of labelling biomolecules and staining cells, the use of exogenous synthetic organic fluorophores is still the most-adopted approach to visualise or analyse biological molecules in cells and organisms. Fluorophore-based technologies have made significant contributions to the understanding of dynamic and complicated processes in biological systems since the first attempt to achieve fluorescence staining of cells by von Prowazek exactly a century ago (Lam, Law, Lee, & Wong, 2014; von Prowazek, 1914). Fluorescent probes are versatile research tools, and offer exquisite sensitivity and outstanding selectivity, by which the limits of detecting particular components of complex biomolecular assemblies can be accomplished at the level of single molecules (Valeur & Berberan-Santos, 2012).
Click chemistry is an exciting development in biological chemistry in recent years. As the most widely utilised type of click chemistry, the copper(I)-catalysed reaction between an azide and a terminal alkyne is one of the most successful and versatile bioorthogonal reactions providing novel platforms for development of cell labelling methodologies (Amblard et al., 2009; Best, 2009; Kolb et al., 2001; Meldal & Tornoe, 2008).
Both reaction partners of the copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) are small in size without cross-reactivity with molecules found in living cells (Lim & Lin, 2010; Sletten & Bertozzi, 2009). Before the reaction takes place, both reactants are non-toxic and stable kinetically, thermodynamically, and metabolically. The click chemistry reaction takes place under physiological conditions, i.e. ambient temperature and pressure, neutral pH, and aqueous conditions (Lang & Chin, 2014). During the reaction, the two reactants react selectively with each other and form extremely stable 1,4-disubtituted-1,2,3-triazole covalent linkages between the azide and alkyne in a regiospecific manner without any innocuous byproducts (Agard et al., 2006; Hong et al., 2009; Sletten & Bertozzi, 2009). In the presence of a copper(I) catalyst and a stabilising ligand, the reaction rate of this process is boosted approximately one million times faster than the very sluggish uncatalysed [3+2] cycloaddition (Becer, Hoogenboom, & Schubert, 2009; Lewis et al., 2002; Rodionov et al., 2007).
To establish a methodology capable of selectively labelling target cells using copper(I)-catalysed azide-alkyne cycloaddition, one of the reaction partners has to be modified to carry a specifically designed motif that functions as a biomarker to the feature of interest (Nadler & Schultz, 2013; Uttamapinant et al., 2012).
2-Nitroimidazoles (2-NI) have been demonstrated to undergo hypoxia-selective metabolism. By forming covalent associations (adducts) with cellular macromolecules, their reduction products are able to selectively accumulate in hypoxic cells (Arteel, Thurman, & Raleigh, 1998; Franko & Chapman, 1982). This inherent feature has led to derivatives of nitroimidazole being widely used as hypoxia markers (Arteel et al., 1998; Evans et al., 2006).
A 2-NI compound named SN33267 was synthesised by Dr Moana Tercel for this thesis. Due to the presence of a terminal alkyne moiety, SN33267 is capable of incorporating into cellular macromolecules via covalent associations under a reducing environment. To transfer the information of these covalent adducts into an analytically useful signal, indicators are designed by combining an analyte recognition site with a fluorescent reporter moiety (Boens, Leen, & Dehaen, 2012; Finn & Fokin, 2010; Presolski, Hong, & Finn, 2011; Spiteri & Moses, 2010). In the particular case of this thesis, azide-modified fluorophores were utilised to translate the binding between the analyte (alkyne SN33267) and the recognition site (azide moiety) into a fluorescence output signal by CuAAC.

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A group of seven complementary candidate azide-modified fluorophores belonging to three chemical families were synthesised by Dr Moana Tercel. This Chapter evaluated the spectral properties of each fluorophore, in relation to the instrument used. On the basis of their fluorescence properties and capability of labelling hypoxic cells via CuAAC, the selected candidate(s) combined with the clickable 2-NI, SN33267, would be employed to optimise the click chemistry-based hypoxia-dependent cell labelling methodology in the following chapters. The specific objectives of this Chapter were:
To characterise candidate fluorophores by determining absorption- and emission spectra, and ranking the candidates by fluorescence intensity;
To test the performance of each candidate fluorophore in labelling SiHa cells via CuAAC in a hypoxia-dependent manner;
To compare microplate reader with flow cytometer in the assessment of fluorescence in cell suspensions labelled with fluorophores via CuAAC;
To select the best fluorophore(s) in conjunction with SN33267 for hypoxia-selective cell labelling using an initial non-optimised protocol.


One clickable 2-nitroimidazole (SN33267) and 7 azide-modified fluorophores were tested in this chapter (Figure 3.1) (Tercel & Pruijn, 2011). Alexa Fluor 488-azide is commercially available and purchased from Invitrogen New Zealand Limited. The other six azide-modified fluorophores, boron-dipyrromethene 650/665 azide (BODIPY-650/665-azide), boron-dipyrromethene fluorescein-like azide (BODIPY-FL-azide), boron-dipyrromethene Texas Red-like azide (BODIPY- TR-azide), dimethylaminocoumarin acetic acid azide (DMACA-azide), diethylaminocoumarin azide (DEAC-azide), and a mixture of 5- and 6-isomers of tetramethylrhodamine azide (TAMRA-azide), were made in a single step from the corresponding commercially available fluorophore-succinimide esters.
Apart from the commercially available one, all compounds were synthesised by Dr Moana Tercel at the Auckland Cancer Society Research Centre. Each compound was confirmed for >95% purity by HPLC and stored as concentrated stock solution (20 mM for SN33267, 2 mM for all other fluorophores) in DMSO (Merck KGaA, Darmstadt, Germany) at -80 °C.

Chapter 1. Literature review
1.1 Tumour Vasculature and Blood Flow
1.2 Tumour Hypoxia
1.3 Hypoxia-inducible factors
1.4 Hypoxia gene signatures in tumours
1.5 Detection of tumour hypoxia
1.6 Current approaches to isolate hypoxic cell populations from tumour tissues
1.7 Click Chemistry
1.8 Aims and objectives
Chapter 2. Materials and methods
2.1 General materials and compounds
2.2 Cell lines and culture
2.3 Xenograft models
2.4 Hypoxic cell labelling using click chemistry
2.5 Flow cytometric analysis
2.6 Fluorescence-activated cell sorting
2.7 RNA extraction and quality control
2.8 Reverse transcription of RNA to cDNA
2.9 Real-time quantitative PCR using TaqMan® gene expression assays
2.10 Western blotting
2.11 Statistical Analysis
Chapter 3. Selection of fluorophores for click chemistry-based detection of hypoxic cells
3.1 Introduction
3.2 Aims
3.3 Compounds
3.4 Results
3.5 Discussion
Chapter 4. Optimisation of click chemistry-based hypoxia-selective cell labelling method
4.1 Introduction
4.2 Aims
4.3 Results
4.4 Discussion
4.5 Acknowledgements
Chapter 5. Click chemistry-based cell labelling and RNA integrity
5.1 Introduction
5.2 Aims
5.3 Results
5.4 Discussion
Chapter 6. Validation of the click chemistry-based method for labelling of hypoxic cells using a hypoxia gene signature
6.1 Introduction
6.2 Aims
6.3 Statistics
6.4 Results
6.5 Discussion
Chapter 7. The effect of hypoxia on the expression of oxidoreductases
7.1 Introduction
7.2 Aims
7.3 Results
7.4 Discussion
Chapter 8. Future directions and concluding remarks
8.1 Future directions
8.2 Concluding remarks

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