In this work, we report successful generation of a new transgenic mouse strain expressing an enhanced form of firefly luciferase (Luc2) in a mammary tissue-specific manner. We also report successful application of this new strain for visualisation of primary tumour development and longitudinal monitoring of local tumour progression in oncogene-bearing transgenic animals.
Improvement of currently existing transgenic animal models for breast cancer is a pressing issue and has been addressed by different approaches in the past. Conceptually, MMTV-Luc2 mouse model stands in line with previously generated models of luciferase expression driven by other tissue-specific promoters, which are currently available from Taconic Farms, Inc. (Hudson, NY). These models include such strains as FVB/N-Tg(Vegfr2-luc)-Xen (vascular-specific expression ) or FVB/N-EL1-Luc/EL1-Tag (pancreatic expression).
Theoretically, another strain available from Taconic, the LucRep (FVB) mice , can be engineered for mammary tissue-specific expression using a Cre-activated system . It has been previously shown that Cre-activated luminescence permits, for instance, a temporally and spatially controlled expression of responder genes in embryonic and multiple adult tissues . Therefore, from a mechanistic perspective, using a Cre-activated system would provide decreased variability of the model system as compared to MMTV-driven expression. It must be considered, however, that Cre-activated luminescence brings the need for using yet another mouse strain (e.g. WAP-Cre ) within the crossbreeding schedule. Conversely, one of the strengths of the MMTV-Luc2 mice lies in the increased feasibility of this system over Cre-activated models; indeed, the MMTV-Luc2 model could be found desirable for some transgenic projects, especially ones utilising non-Cre-activated oncogenic strains, such as MMTV-PyVT. It is also important to mention that using the Cre-activated system can also lead to mosaic expression profile in some instances . In summary, while certain advantages of Cre-activated systems are obvious, MMTV-Luc2 mice can provide a practical alternative to these models. ODD-luciferase mice [24, 25] are another universal strain with potential use for imaging of mammary tumours in transgenic animals. In this model, expression of luciferase is induced by hypoxic conditions within tumours. However, in vivo imaging in ODD-luciferase strain has shown significant generalised background luminescence of the body , as hypoxia is intrinsic in some tissues under normal conditions, which can hamper exact localising of early overgrowth of mammary tissue via
in vivo imaging. Notably, this task can be successfully achieved by the use of MMTV-Luc2 model (Figure 3C and Additional file 1 Figure S6B)
Our model shows also close conceptual similarities with previously generated MMTV- or WAP- GFP mice, which are mouse strains expressing green fluorescent protein (GFP) under various mammary tissue-specific promoters . GFP-based imaging has been also successfully used for longitudinal studies of mammogenesis . However, although successfully generated, the applicability of GFP-based systems is of very limited value for whole animal in vivo imaging, mainly because of autofluorescence-related problems and low tissue penetration of the light emitted by GFP. Conversely, detection of bioluminescence from cells expressing firefly luciferase is considered the most sensitive technique for optical imaging in vivo, as light produced after the reaction catalysed by luciferase penetrates the tissues with high efficacy. The issue of autofluorescence is also alleviated, as bioluminescence does not require the excitation light. Hence, our model holds substantial advantages over GFP-based systems.
It is important to point out that the MMTV-Luc2 model by design is not expected to be capable of detecting metastases (e.g. into the lungs) directly in living transgenic animals, primarily because of the robust localisation of mammary tissue in female mice. In result, the background signal from superficially located mammary tumours and also healthy mammary tissue would set up the imaging threshold too high for potential detection of metastases in whole body imaging in vivo. Therefore, we wish to recommend the usage of the MMTV-Luc2 mice mainly for monitoring the growth of primary tumours, at least with the currently available bioluminescent imaging systems.
Another issue related to the applicability of the MMTV-Luc2 model system is the evidence from previous studies that the MMTV-driven expression of the transgene is primarily active in luminal epithelial cells rather than basal cells in the gland (reviewed in ). Therefore, while MMTV-Luc2 mice appear relevant for imaging of other MMTV-driven oncogenic model systems, as exemplified by the MMTVPyVT model in this work, the applicability of MMTV-Luc2 imaging in basal-type breast cancer models (such as BRCA1-knockout mice ) is yet to be studied.
As mentioned above, the MMTV-Luc2 model has been primarily designed for rendering the currently available MMTV-oncogene-based strains applicable for advanced luminescent imaging. Such enhancement greatly increases the capability of monitoring local tumour development and progression, but potentially also response to therapies in such models. We present an example of such an application by intercrossing the MMTV-Luc2 mice with the MMTV-PyVT strain. However, what must be mentioned is that luciferase-based quantification of the signal is not primarily designed to not be used for direct quantification of the tumour burden in living animals, but rather as an indicative marker. One of the reasons for this fact is the well established phenomenon of mosaic expression of the MMTV-driven transgene, especially in heterozygous animals. Indeed, in the MMTV-Luc2 model, we do see some differences in production of the luminescent signal among the tumours taken out from the MMTV-Luc2PyVT females (Figure 3E and Additional file 1 Figure S7A). Secondly, MMTV promoter activation is known to be modulated by steroid and peptide hormones that, in turn, can alter a linear correlation between cell number and the potency of luciferase signal. Therefore, we would like to recommend the MMTV-Luc2 imaging as supportive tool to manual methods of tumour monitoring. At the same time, we would like to strongly point out that homozygous MMTV-Luc2 females express a much more unified pattern of luminescent signal considering the promoter being used, which indicates they can be feasibly applicable for tumour visualisation purposes in transgenic models for breast cancer.
For future applications, as the MMTV-Luc2 homozygous mice are viable and fertile, this strain can be also used instead of wild-type FVB mice as a primary strain for generating new models incorporating mammary tissue-specific expression of a molecule of choice. Ideally, bicistronic vectors encoding the molecule of interest (most frequently a putative oncogene) and fluorescent markers (to highlight oncogenic transgene expression) would be introduced into MMTV-Luc2 mice and then visualised in a dual-modality manner for co-localisation of fluorescence and luminescence. This would provide information on both levels of transgene expression (by fluorescence) and potential local changes in mammary tissue ‘content’ (by luminescence) in these new models. Importantly, the use of the homozygous MMTV-Luc2 mice in such systems will decrease the intra-strain variability of tissue-specific Luc2 expression in comparison to imaging of heterozygous MMTV-Luc2 mice. This, in turn, would correspond to the current ethical considerations regarding studies involving animals, i.e. our model will facilitate reduction in the number of transgenic animals per group necessary for obtaining statistically significant results.