Fluorescence imaging

Over the recent decades, a great number of various fluorescent proteins has been identified, which turned out to be powerful tools for the visualization of dynamic processes in living animals. Predominantly in preclinical oncologic research, the use of such fluorescent proteins has become indispensable for monitoring cancer dynamics, including inter alia, tumor growth, angiogenesis and metastasis [Hoffman, 2009].

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria, a 238 amino acid containing polypeptide, which serves as an energy-transfer acceptor, was the first identified fluorescent protein [Prasher et al., 1992]. In the course of research in this field several gain-of-function mutants of wildtype GFP with enhanced brightness and optimized mammalian expression were generated [Hoffman, 2009]. Cormack et al. [Cormack et al., 1996]

constructed a library of GFP mutants in E. coli strains, showing a 100-fold increase in the fluorescence intensity detected by flow cytometry. The altered fluorescence properties resulted from strongly shifted excitation maxima and improved protein folding of the mutants in prokaryotes compared to wt GFP. Intensified GFP fluorescence was also obtained by DNA-shuffling methods, which yielded, for instance, mutants with 45-fold higher total cell-associated fluorescence intensities than that of the natural protein [Crameri et al., 1996], even without changes in the excitation and emission spectrum. Improved GFP expression in mammalian cells, mainly in cell types from human origin, was accomplished by multiple base substitutions in the coding-sequence leading to efficient codon usage [Zolotukhin et al., 1996].

In 1999 and 2000, the discoveries of red fluorescent proteins in several reef corals were reported [Matz et al., 1999; Fradkov et al., 2000; Gross et al., 2000]. DsRed, a red-fluorescent protein from Discosoma sp., is characterized by massive brightness, pH-stability as well as resistance against denaturants and photobleaching [Campbell et al., 2002]. Furthermore, by mutations an enhanced version of the red fluorescent protein, referred as DsRed2, was generated, which is more soluble and faster in fluorophor maturation than the parent protein.

In search for better red emitting labels, Shaner et al. described a series of new proteins, which were also obtained by mutations of DsRed [Shaner et al., 2004].

Although emission maxima at wavelengths as long as 649 nm are favorable with respect to bioanalytical applications, these proteins are compromised by low quantum yields, resulting in reduced brightness of the fluorescence [Hoffman, 2009].

Quite recently, the isolation of a red fluorescent protein from the sea anemone Entacmaea quadricolor allowed the generation of a new bright protein, named Katushka, emitting far-red light [Shcherbo et al., 2007]. For this purpose, the natural protein was modified with respect to increased velocity of fluorophore maturation, pH-stability and brightness. These modifications yielded the red fluorescent protein TurboRFP [Merzlyak et al., 2007], which served as a starting point for the generation of Katushka by performing multiple cycles of random mutagenesis. Katushka was proven to possess excellent properties for whole body imaging with an excitation peak at 588 nm and an emission maximum at 635 nm.

Furthermore, rapid fluorophor maturation (20 min), an extinction coefficient of 65,000 M^-1 cm^-1 and a quantum yield of 0.34 render Katushka the most promising red fluorescent protein for fluorescence imaging applications up to now. However, the labeling of cellular proteins by Katushka is compromised due to its dimeric structure. For this reason, a monomeric version of TurboRFP (called TagRFP) was generated [Merzlyak et al., 2007]. In addition, modifications of TagRFP yielded the monomeric version of Katushka, termed mKate [Shcherbo et al., 2007].

5.1.2.2 Fluorescent proteins in preclinical oncology

In preclinical oncology, the great number of available fluorescent proteins with different properties enables the monitoring of multiple processes during cancer development. Already in 1997, Chishima et al. [Chishima et al., 1997] described the visualization of cancer invasion and micrometastases in live tissue by using CHO-cells, genetically engineered to stably express the green fluorescent protein [Chishima et al., 1997]. The transfectants were subcutaneously injected into nude mice, and small pieces of s.c. growing tumors were used for the orthotopic implantation into the ovary of nude mice. After 4 weeks, the animals were killed, and micrometastases were microscopically detected in different organs due to GFP fluorescence. As an advancement, noninvasive imaging of GFP-expressing B16F0 mouse melanoma tumors and AC3488 human colon cancer as well as metastases in live mice was performed by Yang et al. [Yang et al., 2000].

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With respect to noninvasive whole-body imaging, the spectral properties of fluorescent proteins are the key. Due to the fact that longer excitation wavelengths cause fewer damages in cellular proteins as well as in DNA, and that the absorbance of emitted light by tissues, containing biomolecules such as hemoglobin, is also less pronounced at longer wavelengths, whole-body imaging can be improved by using red fluorescent proteins [Hoffman, 2009]. The successful application of DsRed2 for in vivo imaging has been demonstrated in several cases [Katz et al., 2004; Bouvet et al., 2005; Jarzyna, 2007]. In contrast, so far, only few reports exist on the application of the promising far-red protein Katushka for visualization of cancer processes in vivo.

A rather new application of fluorescent proteins in cancer research is the so-called color-coded fluorescence imaging, which offers the potential of monitoring tumor-host interactions.

Tumor cells, which express a complementary fluorescent protein are implanted into transgenic nude mice , which expresses either a green or red fluorescent protein in all cells or specific cell types [Hoffman and Yang, 2006]. Moreover, many other applications, for example investigations on tumor angiogenesis [Amoh et al., 2008] or studies on the tumor’s microenvironment [Yang et al., 2009], become feasible by color-coded fluorescence imaging.

Taken together, fluorescent proteins proved to be valuable tools for the visualization of cancer dynamics in vivo, including tumor growth, metastasis or angiogenesis. The recent discovery of bright red-shifted proteins, in particular Katushka, appears promising for whole-body imaging, since limitations of proteins emitting at shorter wavelength, such as the depth of tissue penetration and light absorbance by physiologically present molecules, can be overcome.

5.2 Objective

The objective of this central part of the thesis was to refine an already existing orthotopic human glioblastoma model in nude mice with regard to noninvasive monitoring by optical imaging. For this purpose, the human glioblastoma cell lines U-87 MG and U-373 MG were (co-) transfected with the genes encoding an enhanced version of the firefly luciferase (luc2) and the recently discovered far-red emitting protein Katushka, respectively. A prerequisite for studies in animals was the in vitro characterization of the transfectants with respect to luc2 / Katushka protein expression, growth kinetics and chemosensitivity. The most promising transfectants had to be examined in a subcutaneous tumor model in nude mice concerning tumorigenicity and the stability of luc2 / Katushka expression. To establish a new orthotopic brain tumor model, the transfected malignant human glioblastoma cells U-87 Luc2 / U-87 Katushka were implanted intracerebrally into nude mice to monitor tumor progression. In addition, the advantages and limitations of both techniques, namely BLI and FLI, should be comparatively evaluated by orthotopically implanting U-87 Luc2 / Katushka co-transfectants into the brain of nude mice.

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5.3 Materials and methods

5.3.1 Drugs and chemicals

Water, used for the preparation of drug solutions and culture media, was purified by a Milli-Q system (Millipore, Eschborn, Germany). The selection antibiotics ampicillin sodium (Sigma, Deisenhofen, Germany), puromycin·2HCl (Sigma, Deisenhofen, Germany) and geniticin disulfate (G418, Biochrom AG, Berlin, Germany) were dissolved in water to achieve concentrations of 100 mg/mL (ampicillin sodium), 1 mg/mL (puromycin·2HCl) and 150 mg/mL or 300 mg/mL (G418). The stocks were stored at -20 °C. D-luciferin potassium salt was purchased from Molecular Imaging Products Company, Ann Arbor, MI, USA and SYNCHEM OHG, Felsberg/Altenburg, Germany. For in vivo imaging experiments, 40 mg of D-luciferin potassium salt were dissolved in 1 mL of PBS (8.0 g/L NaCl, 1.0 g/L Na2HPO4 · 2 H2O, 0.20 g/L KCl, 0.20 g/L KH2PO4, 0.15 g/L NaH2PO4· H2O; all chemicals for buffer preparation were purchased from Merck, Darmstadt, Germany). The solution was sterile filtered and aliquoted into autoclaved Eppendorf reaction vessels (Eppendorf, Hamburg, Germany). All preparations of D-luciferin were stored light-protected at -80 °C.