Luminol-based bioluminescence imaging of mouse mammary tumors
Graphical abstract
Introduction
Cancer is a major cause of disease and death in the United States and other countries around the world. For example, the American Cancer Society estimates that 580,350 people will die of cancer in the US in 2013 [1]. Detecting tumors at earlier stages could significantly improve therapeutic outcomes [2], [3]. In pursuing this goal, understanding the tumor microenvironment may facilitate tumor diagnosis at the earliest stages. It has been known that tumor microenvironment differs from that of normal tissue in that it is often characterized by low pH, hypoxia, over-expressed proteases [4], and infiltration of defensive and immune cells [5].
These features can potentially be exploited for detection and treatment. Several imaging techniques have been used to examine the tumor microenvironment, such as optical imaging, positron emission tomography (PET) and magnetic resonance imaging (MRI) [4], [6], [7], [8], [9], [10], [11], [12]. Bioluminescence imaging (BLI) is a non-invasive imaging technique used in pre-clinical oncology research to image tumors by generating visible light, that is usually generated by luciferase-expressing cells [2], [13]. The luciferase-based BLI mechanism involves the oxidation of luciferin in the presence of adenosine tri-phosphate (ATP), magnesium (Mg2+) and molecular oxygen (O2) to create an electronically excited oxy-luciferin, which emits visible radiation in the yellow-green to yellow-orange spectrum, with an emission maximum of 560 nm [2], [13]. Firefly luciferase transfected mouse mammary gland tumor cells 4T1-luc2 show stable light emission in the presence of luciferin and permit the detection of early tumors [2]. In contrast to luciferase-based BLI mechanism, Liu and Mason showed beta galactosidase based chemiluminescent imaging for in vivo detection of tumors expressing a beta galactosidase transgene [14]. However, naturally occurring cancers do not express luciferase. Therefore, luciferase-based BLI is not available when treating cancers in humans and/or not transfected tumors in mammals. Note that in the research reported here, luciferase-based BLI is only used to define the region of interest.
Luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) is a known chemiluminescent molecule that emits light at maximum of 425 nm when oxidized [15], [16]. Luminol has been used in various fields, such as biochemistry, analytical chemistry, and clinical diagnostics for detecting reactive intermediates [17]. For instance, luminol has been used as an analytical tool to examine the role of granulocyte-derived reactive oxygen species in heart muscle damage, to screen polymorphonuclear leukocyte function in patients with diabetes mellitus, and to detect leukocyte activity in patients with peritonitis [15], [18]. Luminol detects phagocytic oxidative bursts and subsequent myeloperoxidase (MPO) activity [17]. Gross et al. have demonstrated that luminol-based imaging can be used for quantitative longitudinal monitoring of MPO activity in animal models of acute dermatitis, mixed allergic contact hypersensitivity, focal arthritis and spontaneous large granular lymphocytic tumors [17]. The work presented here consists of tailoring their detection method for revealing early breast tumors (4T1) in mice. Chronically inflamed tissues like cancer are populated by defensive cells, such as neutrophils, monocytes, and macrophages, which can produce reactive oxygen species (ROS) by respiratory bursts and subsequent chemical reactions. Many of these ROS react with luminol and produce light by means of luminol chemiluminescence [19]. Since this chemiluminescence occurs from a chemical and oxidizers that are produced by enzymes in living cells, we will use the term luminol-based bioluminescence imaging (BLI). Here, we present first evidence that administration of luminol to mice that have been transplanted with 4T1 mammary tumor cells permits early stage imaging of tumors by bioluminescence-based detection of MPO activity.
Section snippets
Theory of luminol-based bioluminescence
When PMNs are activated in inflamed tissues such as tumors, they start releasing myeloperoxidase (MPO) [20], [21]. MPO is an abundant protein produced in azurophilic granules of neutrophils, where it can constitute more than 5% of the granule’s protein [22]. Respiratory burst is initiated by phagocytic NADPH oxidase (Phox). Phox reduces molecular oxygen (O2) to give superoxide anions (O2.-). In the presence of protons (H+), the superoxide anion reacts to the hydroperoxy free radical (HO2.).
Materials and cell culture
Mouse mammary gland adenocarcinoma tumor cell line 4T1luc2 was purchased from Caliper Life Sciences, Hopkinton, MA. This cell line has been engineered to stably express the firefly luciferase gene luc2 [24]. Cells were grown in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS; Sigma–Aldrich, St. Louis, MO) and 1% penicillin/ streptomycin (Invitrogen, Grand Island, NY). Cells were incubated at 37 °C at 95% humidity in 5% CO2. Luminol sodium salt was purchased from Gold Biotechnology
D-luciferin imaging
4T1luc2 cell-transplanted mice showed a bioluminescence signal at mammary fat pad number seven as early as day 1 with D-luciferin imaging (Fig. 2A), suggesting that with D-luciferin imaging we can detect 4T1luc2 cells before they form palpable tumors. This finding is in agreement with the literature [2]. Note that luciferin bioimaging is used as positive control for luminol imaging. Mice injected with PBS showed no bioluminescence signal with D-luciferin imaging (Fig. 2A). D-luciferin imaging
Discussion
The work described above demonstrates that orthotopically transplanted 4T1luc2 mammary adenocarcinoma cells can be successfully imaged by means of luminol-based biolumincescence imaging even before tumors are palpable. Although control mice injected with saline did show a small signal, the control bioluminescence signal disappeared after two days, in contrast to the transplanted tumor signal. Also, mice showed luminescence at other areas apart from the tumor cell injection site, primarily in
Abbreviations
PMNs Polymorphonuclear neutrophils BLI bioluminescence imaging ROS reactive oxygen species MPO myeloperoxidase PBS phosphate buffered saline ROI region of interest
Acknowledgements
Hamad S. Alshetaiwi acknowledges financial support from College of Medicine, University of Hail, Saudi Arabia. The authors gratefully acknowledge financial support from National Science Foundation (NSF/CBET, Award # 0933701 and NSF/DMR, Award # 1242765) and from the Johnson Cancer Research Center at Kansas State University.
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