The dark side of ID8-Luc2: pitfalls for luciferase tagged murine models for ovarian cancer
© Baert et al. 2015
Received: 16 July 2015
Accepted: 9 November 2015
Published: 15 December 2015
Reliable mouse models are key in the discovery and development of novel anticancer treatments. Non-invasive monitoring techniques such as bioluminescence imaging (BLI) are useful tools to determine tumor engraftment and evaluate tumor growth. However, the development of ascites in ovarian cancer mouse models leads to possible difficulties. Ascites can interfere with the set-up of correct end points and can interfere with the evaluation of tumor volume using BLI. We provide optimized euthanasia criteria and in vivo data underlining the pitfalls of BLI.
KeywordsOvarian cancer ID8 Bioluminescence imaging Mouse model Ascites
With great interest, we read the article of Liao et al. entitled “Preservation of tumor-host immune interactions with luciferase-tagged imaging in a murine model of ovarian cancer”  in the Journal for ImmunoTherapy of Cancer. Adequate mouse models are paramount for translational cancer research. With the development of immunotherapy in the field of anticancer treatment, we should turn to immune competent syngeneic models such as the ID8-Luc2 model described by Liao et al.
Estimating and monitoring tumor load in ovarian cancer is challenging. With innumerable peritoneal implants that are formed, we rely in a clinical setting on CT (computed tomography) or MRI (magnetic resonance imaging) to evaluate tumor growth and disease progression, based on the RECIST 1.1 criteria . The RECIST criteria use target lesions as surrogate measure for tumor load, as total tumor load cannot be quantified in a clinical setting. In animal models, bioluminescence imaging (BLI) is a well-established technique that allows non-invasive quantification of tumor load . Typically, stably integrating retroviral vectors are used to generate stable luciferase expressing reporter lines, that are applied to the respective animal models to monitor tumor growth: cells that express the firefly reporter enzyme generate a photon flux (light) when luciferin (the luciferase substrate) is oxidized in the presence of ATP. As a consequence, only live tumor cells that express the enzyme can be monitored by detecting the emitted photons, rendering BLI an excellent and sensitive tool to examine tumor growth in mouse models, as was demonstrated nicely by the authors.
Figure 2c shows significant increase in BLI signal after drainage of ascites. Therefore, we conclude that the occurrence of ascites affects the BLI photon flux and hence these measurements do not recapitulate the tumor growth. Especially if these mice receive treatments that can affect the development of ascites this is of particular importance. Moreover, in large tumor volumes the photon flux is mostly also an underestimate of tumor load, due to internal necrosis or weak vascularization of the tumor bulk, resulting in diminished/lower luciferin concentrations into the central zone of the tumor .
Improved euthanasia criteria
Criteria of euthanasia
Liao et al.
Loss of 2 g in 48 h
Loss of 3 g in 7 days
Increased respiratory frequency
Increased respiratory frequency
Hunched back with tremor
No spontaneous movement when nudged
In the article of Liao et al. FACS analysis was performed on ascites, spleen and tumor of ID8-WT inoculated mice compared to ID8-Luc2 inoculated mice at 12 to 15 weeks after inoculation to evaluate the tumor microenvironment. These are late stage mice in which immunosuppression has completely taken over from immune control. These results prove that in late stage animals there is no difference in the microenvironment due to the expression of luc2, but do not allow us to draw conclusions concerning early disease, when there is still an equilibrium between immune control and immune escape. Furthermore to determine the change in in vivo tumor growth due to the Luciferase insert, it would be more relevant to flank the firefly Luciferase cDNA with loxP sites, allowing Cre mediated excision of the cassette once the stable cell line is established, providing a perfect control. When comparing the cell line with and without the addition of Cre recombinase, the only difference is the presence firefly Luciferase and not the selection pressure that has been put on the cells during the transfection and selection process.
In conclusion, the use of codon-optimized firefly luciferase Luc2 expressed in ID8 cells as described by Liao et al. is suitable for the evaluation of tumor load in early stage disease. Care should be taken in the interpretation of BLI results once ascites occurs. When using overall survival as an outcome measure in this model we recommend repeated ascites drainages to avoid underestimating survival. The use of orthotopic, immune competent models for ovarian cancer should be encouraged, as they are an adequate representation of the clinical setting of ovarian cancer patients.
Luc2 transfected cell line used by Liao et al.
Luc1 transfected cell line used by our research group
ID8 wild type, before transfection
Photons per second
Magnetic resonance imaging
Response evaluation criteria in solid tumors
We thank Madeline Pe, PhD (Research Group of Quantitative Psychology and Individual Differences KU Leuven, Belgium) for assistance in the statistical analyses.
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