Development of neoplastic disease is a stepwise process involving loss of genetic regulation, changes in cellular physiology, and failure of effective immune surveillance [1–9
]. The progression of neoplastic states to disseminated disease requires interactions between the transformed cells and normal tissues as occurs in invasion, extravasation, migration and neovascularization [10–12
]. Although individual steps in these processes may be modeled in correlative cell culture assays, evaluation of the dynamic and interactive processes of the complete disease requires the context of living animals. However, animal models have typically served as “black boxes” where well-defined signals can be applied, but the ultimate evaluation is performed outside of the animal via ex vivo
assays. Sophistication of these ex vivo
assays has increased dramatically with the development of confocal and two-photon microscopy for the interrogation of thick tissues, reporter proteins with diverse optical signatures, multiparameter flow cytometry and DNA microarrays [13–20
Tremendous sensitivity can be achieved using amplification methods such as polymerase chain reaction (PCR) to detect tumor cell DNA or mRNA levels, but these assays are time-consuming and severely limited by the size of the sample that can be reasonably evaluated. Thus, the fraction of a given tissue that can be analyzed is limited [21,22
]. Studies employing these assays may, therefore, be subject to sampling biases such that results may not represent overall expression levels in the target tissue or organ. In the absence of a signal that can be detected in the intact animal, or even the intact organ, targeting specific tissues for analyses is also difficult, and all tissues cannot be evaluated. Thus, only a small fraction of a few selected tissues is studied when ex vivo
assays are employed. To achieve statistical significance with temporal studies, large numbers of animals are used with groups being sacrificed at each time point. Thus, analyses using ex vivo
assays are both limited and require large numbers of animals.
The limitations of the ex vivo assays indicate that accessible, versatile and sensitive assays that can rapidly reveal cellular and molecular changes as they occur in intact animal models of human neoplasia are necessary to advance our understanding of disease processes and accelerate the development of effective intervention strategies. In particular, refinement of animal models to include markers for imaging the multiple stages of cancer development in vivo would complement current ex vivo assays and would dramatically accelerate and enrich the analyses of animal models of human neoplastic disease by revealing cellular and molecular changes in vivo and directing the ex vivo assays to critical target tissues.
A variety of imaging strategies designed to reveal the physiologic changes associated with neoplasia and response to therapy have been described. The modalities utilized in these approaches include fluorescence imaging, magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), and positron emission tomography (PET) [23–34
]. Contrast agents that enhance MRI are being developed and molecular reporter genes for MRI have enabled cellular and molecular analyses [35,36
]. Similarly, fluorescent dyes that are concentrated at the tumor site or that can be activated by the target cell have been described and utilized to localize tumor cells in animal models [33,37,38
]. PET is, by design, a method of revealing metabolic changes and can be used to localize cells and molecules labeled with radioactive tracers. However, many of these imaging strategies are not well-suited for small animals and can be encumbered by long scan times and expensive instrumentation for detection and support. In addition, some of these methods can be difficult to quantify and lack sensitivity.
Sensitive in vitro
molecular assays have been developed using light-emitting enzymes, luciferases, as reporters [39–42
]. Luciferases comprise a family of photoproteins that can be isolated from a wide variety of species including bacteria and a large number of eukaryotic organisms [43
]. Luciferase from the firefly, Photinus pyralis
, is the most commonly used photoprotein in molecular biology studies and has been used to evaluate gene expression in transformed cell lines in culture [44,45
], and to monitor tumor growth and response of tumors to antineoplastic therapy in animal models of human disease [21,46
]. This reporter gene is not only the most widely used member of the photoprotein family of enzymes; it is also the most-studied and has been modified such that it is well suited for studies of neoplastic disease. These modifications include mutations for optimal mammalian codon usage and increased expression due to removal of a peroxisome targeting site [41,42
]. These features of the firefly enzyme make it an ideal choice for in vivo
monitoring of tumor cell growth. Another luciferase that has been used as a reporter in mammalian cells includes that from the sea pansy (Renilla reniformis
). The wavelength of emission from this enzyme (blue, with a peak at 460 nm) and its use of a substrate other than luciferin have led to its use in dual-reporter assays [47,48
]. Many luciferases from bioluminescent marine organisms, including that of R. reniformis
, use the high-energy compound, coelenterazine, as a substrate. Analyses of these photoproteins as indicators of gene expression have, in the past, required removal of the tissues and assessment of enzymatic activity in cell lysates. This type of assay has many of the same limitations as other ex vivo
assays; however, the fact that light can pass through mammalian tissues and the absence of bioluminescence from mammalian cells, suggested that it may be possible to use these reporter genes for the in vivo
analysis of neoplasia.
It has previously been demonstrated that internal bioluminescent signals from bacteria and cells of transgenic mice can be externally detected in vivo
, even when located at deep tissue sites [49,50
]. Using this technology, it has been possible to non-invasively monitor infection, gene expression, and passive transfer of mammalian cells [51
]. This approach has recently been applied to the study of tumor progression and response to therapy in living animal models [52,53
]. The reporter genes used in these studies included the modified firefly luciferase for eukaryotic gene expression and a bacterial luciferase from the soil bacterium, Photorhabdus luminescens
, for prokaryotic expression. Tagging biological processes with reporter genes that are propagated along with the labeled cells permit monitoring cell growth, and transcriptional events without the problem of dilution or loss of signal with cell division. The ability to monitor labeled processes from an external vantage point provides a tremendously powerful tool.
The use of external detection of an internal bioluminescent signal differs from other optical imaging strategies and offers some distinct advantages. Optical imaging typically involves the use of external light sources to either interrogate the inherent optical properties of tumor tissue, or to assess the concentration of exogenous dyes that accumulate or are activated at tumor sites [33,37,38,54
]. In contrast, photons originating from photoproteins in labeled cells can serve as internal biological sources of light that transmit through mammalian tissues to reveal spatial and temporal information in the near absence of background bioluminescence [51
]. The weak bioluminescent signal emitted from the labeled cells and transmitted through the animal tissue can be detected and quantified using low light imaging systems such as intensified and ultra-cooled charge-coupled device (CCD) cameras [50
]. Bioluminescent reporters may offer greater versatility than fluorescent or other types of markers in mammalian tissues due to the nearly complete absence of spontaneous emission of light from mammalian cells. The use of outside light sources for fluorescence markers can result in tissue autofluorescence, creating background that is greater than the signal. As reporter genes are integrated into the chromosomes of the tumor cells, they are replicated with cell division, which is a distinct advantage over other labeling techniques using dyes that can be diluted out as the cells divide. Published research has demonstrated that real time, non-invasive analyses of pathogenic events, pharmacological monitoring and assessment of promoter activity can be performed in vivo
]. These studies indicate that reporter genes can provide a window through which biological processes can be viewed in living animals, and thus may be useful in illumination of the temporal and spatial distribution of tumor growth and metastasis in vivo
Detection of bioluminescence from beetle luciferases expressed in mammalian cells either in culture or in vivo
requires exogenous delivery of the substrate for the enzymatic reaction. It has been determined that the substrate for the firefly luciferase, luciferin (d
-(-)-2-(6′-hydroxy-2′-benzothiazolyl)thiazone-4-carboxylic acid), can be added to cell culture medium such that expression in living cells can be monitored [50
]. This contrasts what has been observed for coelenterazine, the substrate used by luciferases from marine eukaryotes (e.g., R. luciferase
from R. reniformis
), which appears to have significant autoluminescence in the presence of serum proteins (Zhang and Contag, unpublished results). The ability of luciferin to enter into cells is likely due to its small size (280.33 g/mol) and its zwitter ionic nature. Typical studies utilizing luciferase genes as transcriptional reporters require cell lysis with enzymatic activity being analyzed in a luminometer with the attendant loss of temporal information. For detection in living animals, the substrate, luciferin, can be supplied via intraperitoneal injection and luciferase activity can be evaluated at many different tissue sites simultaneously [50
], including the central nervous system and fetal tissues in utero (C.H.C., unpublished results). The small size of luciferin also makes it a poor antigen and immune responses to luciferin are unlikely; this will be useful as the models move from xenografts in immunodeficient mice to syngeneic and autologous cell transfers.
Many of the assays for tumor cell growth and regression in animal models of neoplastic disease rely on changes in the volume of large superficial tumors where changes in three orthogonal diameters can be determined using calipers. These tumor models resemble late stages in human disease, and thus, therapies developed using these models are most applicable to late stage disease. In contrast, therapies that target minimal residual disease either after removal of the tumor, via surgery or therapy, or early in the disease course, cannot readily be developed using these conventional assays. Real time accurate assays that employ reporter genes for rapid detection of minimal disease in animal models will change the paradigm of drug development, and therapies that effectively treat small numbers of transformed cells will be developed ().
Figure 1 New developments in cancer therapy. With the advancement of diagnostics and therapy, there is a need to develop new therapies that target small numbers of tumor cells to prevent both initiation of disease and relapse after treatment. Therefore, development (more ...)