The development of real-time noninvasive, and minimally invasive, assays for monitoring the progression of neoplastic disease in living systems would offer great benefit for the analyses of host response to disease, as well as in the testing of novel therapeutic strategies [25
]. In vivo
monitoring of optical reporter genes as indicators of expression had previously been limited to cell culture, embryos, plants and small nearly transparent animals such as flies and worms. Scanning techniques such as MRI and CT are modalities ideally suited to reveal structural information, although methods for acquiring functional data using MRI have been developed. MR imaging, however, requires long scan times and expensive instrumentation. PET imaging provides metabolic information and some reporter genes that concentrate radionuclides are available [7,9–11,26,27
]. However, PET is limited by the requirement of radioactive tracers with relatively short half-lives.
Biochemical and molecular assays such as the polymerase chain reaction (PCR) are sensitive and specific; however, these are time consuming and are severely constrained by sampling limitations [28
]. Moreover, a single animal cannot be easily evaluated over time, and animals may need to be sacrificed at serial time points to gain good temporal analyses with statistical endpoints [29,30
]. The large number of animals for these ex vivo
studies can be technically difficult to handle and are prohibitively expensive. A variety of vital dyes have been utilized which have utility in vivo
for tracking cell populations; however, since these agents are not produced by the cell and replicated with the chromosomal DNA they are generally washed out, or diluted out with cell division. Thus, such agents are limited and may not be suitable for imaging biological processes that occur over protracted time periods in adult animals [31
Tagging of biological processes with reporters, which can be monitored and quantified externally provides a powerful tool for evaluating disease progression and response to therapy. In our oncology models we have utilized the optical reporter gene luciferase, which is a modified version of that isolated from the firefly P. pyralis
. This reporter is well suited for these studies since there is essentially no background as other sources of significant bioluminescence are not present in mammals. This contrasts the use of fluorescent tags where tissue autofluorescence and photobleaching can be extremely limiting. Fluorescent reporter genes require the use of excitation light, typically of short wavelengths, and emit in the green region of the spectrum. These wavelengths do not penetrate tissue well and signals from deep tissue sites may not be accessible. The luciferase enzyme produces light in the presence of the substrate luciferin, oxygen and ATP [12
]; the light produced penetrates mammalian tissues, and can be externally detected and quantified using sensitive light imaging systems [21
]. The reporter gene, which is introduced into the chromosomes of the target cells as a stable integration, is replicated with cell division and not lost over time. Images can be produced which provide spatial information and the animals repeatedly imaged which provides temporal information. This ability to produce spatiotemporal data can be used to generate animal models of neoplastic disease that are more predictive of response to therapy and effects of experimental manipulation.
In this report, as few as 1x103 HeLa-luc cells could be visualized following i.p. injection, and tumor progression followed temporally. Growth proceeded in an exponential fashion such that by 28 days a dramatic increase in signal intensity was observed. Despite the signal observed over the 28-day time period these animals remained relatively robust and did not succumb to the tumor for an additional 30 days. Therefore, exponential growth of the tumor could be directly visualized noninvasively, and quantified over the entire body mass of the recipient animal. Similar observations were made following SC injection of the tumor cells where up to four discrete areas of tumor cell growth could be detected and quantified. HeLa-luc cells did not appear to metastasize following i.p. or SC injection and instead grew locally. Using this approach it is possible to study the growth of tumor cells at individual locations, and may also be used to evaluate the potential for metastasis.
There were differences in the sensitivity of detecting labeled cells at each of the three sites tested, and this was likely related to either greater distribution of cells, or metabolic activity at each site. Subcutaneous sites are likely constrained for both oxygen, a requirement for the luciferase enzymatic reaction, and space. It is possible that these constraints limited the signal intensity following SC injection compared to i.p. injection of tumor cells. Hypoxia will likely limit the luciferase reaction, and may alter the ability to quantify total cell numbers as the tumor mass increases. However, use of luciferase as a metabolic indicator may provide some insights into the growth of larger tumors and the propensity of cells at hypoxic sites to metastasize. Combination of MRI and luciferase imaging may be a useful way to obtain both tumor volume and data on metabolic activity for large tumors. When the cells are injected IV they circulate throughout the animal, and are distributed over a larger volume. It is anticipated that greater distribution of the signal in tissues of varied optical properties may account for our inability to detect fewer than 1x106 cells following IV injection.
Among the available imaging modalities for use in laboratory animals, only photoprotein imaging currently has the sensitivity to detect the small numbers of cells present in models of minimal disease states. However, optical reporters are limited by tissue penetration to several centimeters of tissue, and there is a loss of resolution with greater tissue depth. It is reasonable that combined imaging strategies can be used to compensate for limitations of individual imaging modalities. Optical methods, including photoprotein imaging, are well suited to complement other imaging modalities since optical methods tend to be less expensive and provide data not otherwise available. For example, monitoring tumor cells labeled with optical reporter genes may serve as a method to direct MRI investigation by providing a rapid prescreen. The speed and versatility of optical methods suggests that these methods can be used in the development of more versatile MRI reporter genes and imaging strategies. It is likely that combined reporter gene cassettes that provide MRI and optical signatures will be developed for combined imaging strategies.
The strength of PET and MRI in imaging of neoplastic disease is that each of these imaging modalities is currently available for human imaging, and the protocols that are being developed for in vivo cellular and molecular analyses could be applied to clinical investigation. The use of these modalities for monitoring patient response to therapy would offer clinicians great insight for effective management of neoplastic disease.
The model systems and method of detection described in this report will offer tremendous advantages for the study of both gene expression, in tumor and host cells, and response to therapy [19
]. The ability to rapidly evaluate response to a therapeutic intervention in vivo
in a quantitative fashion is an attribute of this approach. Since tumor cell growth in individual animals can be quantitatively assessed, the time and number of animals required can be greatly reduced. The sensitivity of detecting tumor cells labeled with photoprotein reporter genes may allow us to effectively shift the investigation of antineoplastic therapies from those that act on large numbers of cells to minimal disease models. This would permit the development of effective therapies with reduced toxicity to patients.