**6. References**

132 Bioluminescence – Recent Advances in Oceanic Measurements and Laboratory Applications

significant pH at the lower end. Additionally, it possesses sufficient thermal stability to be applicable to assays that require temperatures lower than 50C or in assays involving short lengths of higher temperature exposure. This mutant could be beneficial for *in vivo* imaging with luciferin, particularly in assays that experience pH fluctuations, and for bioluminescence protease assays or *in vivo* protease imaging, in which the sensitivity of detection may be dependent on the sensitivity of ALH2 detection. The latter assays could

A.

B. FACS histogram showing expression of WT Fluc and thermostable mutants in Raji cells. Cells obtained by fluorescence activated cell sorting were expanded *in vitro* and expression of transgenes was analysed using a Cyan flow cytometer using anti-myc.FITC staining. Non-transduced cells (blue line – filled) were compared to WT Fluc (red line) and x11 Fluc (blue line – dashed) (A*). In vivo* imaging of systemic lymphoma expressing WT Fluc (left) and x11 mutant (right). Mice with lymphomas expressing either

The brightness of x12 Fluc was improved by identifying the mutation at the 295 position as having the major negative impact on the bioluminescent characteristics, reverting it and creating x11 Fluc, which possessed all the desirable properties. x11 Fluc has remarkably high activity and catalytic efficiency with LH2 and ALH2, coupled to a high resistance to thermal inactivation and pH-tolerance. Its highly advantageous properties in terms of stability and brightness in mammalian cells have been demonstrated using systemic lymphoma expressing the mutant in mice as an example, as there was no normalisation for engraftment

WT Fluc or x11 Fluc were imaged in three groups of three mice (B).

Fig. 5. Expression and example of *in vivo* imaging of Flucs in mammalian cells.

and no statistical difference between WT and x11 in this small sample set.

benefit further from increased brightness.


Development of a pH-Tolerant Thermostable

October 26, 1999

5th April 2002

Scientific, Singapore. pp. 189-92

*biology and applications.* pp 243-246

*Nature Letters,* No.400, pp. 372-376

*Biochemistry (Moscow),* No.64, pp. 1141-1150

*Photinus pyralis* Luciferase for Brighter *In Vivo* Imaging 135

Nakatsu, T.; Ichiyama, S., Hiratake, J., Saldanha, A., Kobashi, N., Sakata, K. & Kato, H.

Prebble, S.; Price, R.L., Lingard, B., Tisi, L. & White, P. (2001). Protein Engineering and

Seliger, H. & McElroy, W. (1959). Quantum Yield in the Oxidation of Firefly Luciferin.

Seliger, H. & McElroy, W. (1960). Spectral Emission and Quantum Yield of Firefly Bioluminescence. *Archives of Biochemistry and Biophysics,* No.88, pp. 136-141 Shinde, R.; Perkins, J. & Contag, C. (2006). Luciferin Derivatives for Enhanced in vitro and in

Squirrell, D.; Murphy, M., Price, R., Lowe, C., White, P., Tisi, L. & Murray, J. (1999).

Tisi, L.; Lowe, C. & Murray, J. (2001). Mutagenesis of Solvent-Exposed Hydrophobic

Tisi, L.; White, P., Squirrell, D., Murphy, M., Lowe, C. & Murray, J. (2002). Development of a Thermostable Firefly Luciferase. *Analytica Chimica Acta,* No.457, pp. 115-123 Tisi, L.; Law, E., Gandelman, O., Lowe, C. & Murray, J. (2002b). The Basis of the

Ugarova, N. (1989). Luciferase of Luciola mingrelica Fireflies. Kinetics and Regulation Mechanism. *Journal of Bioluminescence and Chemiluminescence*, No.4, pp. 406-418 Van de Bittner G.; Dubikovskaya, E., Bertozzi, C. & Chang C. (2010). In vivo Imaging of

Viviani, V.; Uchida, A., Suenaga, N., Ryufuku, M. & Ohmiya, Y. (2001). Thr 226 is a Key

*Biochemical and Biophysical Research Communications,* No.280, pp. 1286-1291 Wada, N.; Fujii, H. & Sakai, H. (2007). A Quantum–Chemical Approach to the Amino

White, P.J., Leslie, R.L., Lingard, B., Williams, J.R. and Squirrell, D.J. (2002). Novel in vivo

Scientific Publishing Co., ISBN: 9812381562, Cambridge, U.K., 5th April 2002

Bioluminescent Reporter. *PNAS,* No.14, pp. 21316-21321

Thermostable Photinus pyralis Luciferase Mutant. *U.S. Patent 7906298*, filed

Residues in Firefly Luciferase. In *Proc. 11th Int. Symp. Biolum. Chemilum*. Editors: Case JF, Herring PJ, Robinson BH, Haddock SHD, Kricka LJ and Stanley PE. World

Bathochromic Shift in the Luciferase from Photinus pyralis. *Bioluminescence and Chemiluminescence: Progress and Current Applications.* Editors: Stanley, P. & Kricka, L., pp 57-60. World Scientific Publishing Co., ISBN: 9812381562, Cambridge, U.K.,

Hydrogen Peroxide Production in a Murine Tumor Model with a Chemoselective

Residue for Bioluminescence Spectra Determination in Beetle luciferases.

Analogs of Firefly Luciferin. *Proc. 14th Int. Symp. Biolum. Chemilum.: Chemistry,* 

reporters based on firefly luciferase, *Bioluminescence and Chemiluminescence: Progress and Current Applications.* Editors: Stanley, P. & Kricka, L., pp 509-12. World

*Biochemical and Biophysical Research Communications.* No.1, pp. 21-24

vivo Bioluminescence Assays. *Biochemistry,* No.45, pp. 11103-11112

(2006). Structural Basis for the Spectral Difference in Luciferase Bioluminescence.

Molecular Modelling of Firefly Luciferase. In *Proc. 11th Int. Symp. Biolum. Chemilum*. Editors: Case, J.; Herring, P., Rodinson, B., Haddock, S., Kricka, L. & Stanley, P. pp 181-184. World Scientific Publishing Co, 981024679X, Pacific Grove, CA., U.S.A. Sandalova, T. & Ugarova, N. (1999). Model of the Active Site of Firefly Luciferase.


Frullano, L.; Catana, C., Benner, T., Sherry, A. & Caravan, P. (2010). Bimodal MR–PET Agent

Hall, M.; Gruber, M., Hannah, R., Jennens-Clough, M. & Wood, K. (1999). Stabilisation of

Hanes, C. (1932). Studies on Plant Amylases: The Effect of Starch Concentration upon the

Kutuzova, G.; Hannah, R. & Wood, K. (1997). Bioluminescence Color Variation & Kinetic

Kung A. (2005). Harnessing the Power of Fireflies and Mice for Assessing Cancer Mechanisms. *Drug discovery today: disease mechanisms,* No.2, pp. 153-158 Law, G.; Gandelman, O.; Tisi, L.; Lowe, C. & Murray, J. (2002). Altering the Surface

Law, G.; Gandelman, O., Tisi, L., Lowe, C. & Murray, J. (2006). Mutagenesis of Solvent

Luker K.; Gupta M. & Luker G. (2008). Imaging CXCR4 Signalling with Firefly Luciferase

McCapra, F. & Perring, K. (1985). Luciferin Bioluminescence. In *Chemi-and Bioluminescence.*  Editor: Burr J., pp 359-386. CRC Press, 9780824772772, 5th Aug. 1985 McElroy, W. & Green, A. (1956). Function of Adenosine Triphosphate in Activation of Luciferin. *Archives of Biochemistry and Biophysical,* No.64, pp. 257-271 Mezzanotte L.; Fazzina R., Michelini E., Tonelli R., Pession A., Branchini B. & Roda A.

Michelini E.; Cevenini L., Mezzanotte L., Ablamsky D., Southworth T., Branchini B. & Roda

Monsees, T.; Geiger, R. & Miska, W. (1995). A Novel Bioluminogenic Assay for α-

RetroNectin Culture. *Human Gene Therapy,* No.10, pp. 1743-1752

and pH-Tolerance. *Biochemical Journal.* No.397, pp. 305-312

Complementation. *Analytical Chemistry,* No.80, pp. 5565-5573

2382–2384

I.S.B.C. at Bologna, Italy, Sept. 1998

Woods Hole, MA., U.S.A., 9th Oct. 1996

No.26, pp. 1406–1421

Scientific, Singapore

406-414

pp. 212-217

for Quantitative pH Imaging. *Angewandte Chemie International Edition,* No.49, pp.

Firefly Luciferase Using Directed Evolution, In *Bioluminescence and Chemiluminescence: Perspectives for the 21st Century.* Editors: Roda, A.; Pazzagli, M. Kricka, L. Stanley, P. pp. 392-395. John Wiley & Sons Inc., ISBN 9780471987338, 10th

Velocity of Hydrolysis by the Amylase of Germinated Barley. *Biochemical Journal,*

Behaviour Relationships Among Beetle Luciferases, In *Bioluminescence and Chemiluminescence: Molecular Reporting with Photons.* Editors: Hastings, J.; Kricka, L. & Stanley P. pp 248-252. John Wiley & Sons Inc., ISBN 9780471975021, 9th I.S.B.C. at

Hydrophobicity of Firefly Luciferase. In *Bioluminescence and Chemiluminescence: Progress and current applications.* Editors: Stanley, P. & Kricka L. pp. 189-92. World

Exposed Amino Acids in Photinus pyralis Luciferase Improves Thermostability

(2010). In vivo Bioluminescence Imaging of Murine Xenograft Cancer Models with a Red-Shifted Thermostable Luciferase. *Molecular Imaging and Biology,* No.12, pp.

A. (2008). Combining Intracellular and Secreted Bioluminescent Reporter Proteins for Multicolor Cell-Based Assays. *Photochemical and Photobiological Sciences,* No.7,

chymotrypsin. *Journal of Chemiluminescence and Bioluminescence,* No.10, pp. 213-218 Murray, L.; Luens, K., Tushinski, R., Jin, L., Burton, M., Chen, J., Forestall, S. & Hill, B.

(1999). Optimization of Retroviral Gene Transduction of Mobilized Primitive Hematopoietic Progenitors by Using Thrombopoietin Flt3, and Kit Ligands and


**7** 

*Canada* 

**Bioluminescence Applications in** 

*5Centre for Drug Research and Development, Vancouver, BC,* 

*4Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC,* 

In vitro studies have offered vast insight into much of cancer biology, however, it is widely accepted that cell based assays are unable to provide a complete picture when attempting to understand the dynamic nature of cancer as it behaves in situ. Critical processes to cancer progression such as angiogenesis, metastasis and response to treatment, rely on complex interactions between tumor cells and their microenvironment. To overcome this challenge, xenografts have been widely used to study cancer biology within the context of a whole organism since as early as the 1950's. These models rely on use of murine (syngeneic) and human (allogeneic) tumor cell lines injected subcutaneously into a rodent host. The subcutaneous animal model has been a valuable tool in the study of cancer and has directly led to the validation of many of the anticancer agents which benefit patients today. Subcutaneous tumor models are easy to implement and monitor due to the accessibility of the tumor tissue. Evaluation of subcutaneous tumors involves calliper measurements of tumor size (width, length and/or height), which are then used to define tumor volume. However, like cell-based assays, subcutaneous tumor models have proven to be poor predictors of therapeutic activity in patients and this is likely due to the reliance on cell lines which when inoculated subcutaneously develop tumors that poorly mimic the biological behaviour of human disease. Cancers arise slowly and evolve into a heterogeneous structure both in terms of cellular composition (host cells and tumor cells) and microenvironment (vascularization and transient regions of hypoxia and nutrient stress). Subcutaneous xenografts are implanted in microenvironments that will be remarkably different from the tissue of origin. This means subcutaneous tumor cells do not receive the same signals from the stroma that influence immunity, angiogenesis and metastasis; all factors that impact tumor progression and response to therapeutic interventions. Although the initiators and drivers of tumors in humans remain poorly understood, it is generally accepted that following initiation, endogenous disease progresses into a primary tumor which in time can invade surrounding tissues. The latter process involves both extravasation and intravasation

**1. Introduction** 

**Preclinical Oncology Research** 

Jessica Kalra1,2 and Marcel B. Bally1,3,4,5 *1Experimental Therapeutics BC Cancer Agency,* 

*3Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC,* 

*2Langara College, Vancouver, BC,* 

