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3R-Project 94-04

Tumor targeted reporter gene expression to improve and refine traditional models of Tumor growth and metastasis

Stefan Vorburger
Dept.Clinical Research; Visceral and Transplantation Surgery, University Hospital Bern, 3010 Bern, Switzerland

Keywords: mice; rat; tumour; tumourigenesis; reduction; refinement; toxicity testing: carcinogenicity

Duration: 2 years Project Completion: 2007

Background and Aim
Background: Until today the use of animal tumor models is still the most informative approach to obtain pre-clinical data of potential anti-neoplastic agents. In most pre-clinical models, assessment of intraabdominal tumor location and size required sacrificing the animal. Furthermore, gene expression patterns between tumor cell implantation and tumor collection remained enigmatic.
- A large number of animals have to be sacrificed to evaluate tumor growth dynamics and kinetics of gene expression:
Over the past several years, methods for in-vivo analyses of tumor growth and gene expression have emerged. The most prominent approach, bioluminescent imaging (BLI) is an imaging method that allows the in-vivo analysis of cells expressing light-emitting enzymes like the luciferase (Luc) through the animal tissues. However, this non-invasive method to visualize tumor cells in-vivo required the cell-lines to be specifically engineered to emit detectable light. Likewise, in the few studies that used soluble reporter peptides like beta-Human Chorionic Gonadotropin (beta HCG) to monitor tumor growth in-vivo through serum level determination, tumor cells had to be stably transfected with the beta-HCG gene. This necessity for stable transfectants not only limits the testing of anti-tumor agents to a few tumor cell-lines, but it has also the disadvantage that the genetic engineering modifies genes of the maternal cell as well, thus altering the phenotype of the tumor cells in question.

- In-vivo transfection of tumor cells would eliminate the necessity for stably transfected cell-lines.
- Expression of reporter genes from a promoter specific to most tumors but not to normal cells would allow the systemic application of transfection vectors:
Re-activation of the human telomerase reverse transcriptase (hTERT) is a general principle of cancer cells, but not in normal somatic cells. We recently showed that tumor-specific transgene expression from the hTERT promoter enables the targeting of pro-apoptotic genes to cancer cells.

Aim: We want to test the possibility of tumor selective reporter gene (luciferase and beta-human chorionic gonadotropin) expression from the human telomerase reverse transcriptase (hTERT) promoter to detect early tumors, follow tumor growth and monitor telomerase activity of tumor cells as a surrogate marker for anti-tumor therapies

Method and Results
the project was terminated at an early stage
Bioluminescence imaging will be used to quantify and locate luciferase (reporter gene) expression after i/p luciferin injection. Serum level determination of beta-HCG will be performed with standard ELISA kits and by real-time PCR. Both reporter genes are expressed by the hTERT promoter, which is basically only activated in tumor cells. Plasmids have been already constructed and showed a satisfactory yield of transgene expression. Preliminary results indicated that the promoter is strong enough to allow detection of the reporter gene by BLI. Further methods will include: in-vitro: MTT-cell proliferation assay, Dual-luciferase assay, Effectene-transfection, Fluoro-Microscopy, Western-blot analysis; in-vivo: Immunohistochemistry, determination of liver transaminases, effect of telomerase-suppressive agents.

Expectations: We expect that both reporter systems will allow to quantify telomerase-active tumor cells after systemic vector application and that the effect of anti-tumor agents and of telomerase-suppressive agents can be monitored in the living animal.

Conclusions and Relevance for 3R
The development of novel anti-cancer strategies requires more sensitive and less invasive methods to detect and monitor minimal tumor formation in a broad variety of cancer models. This is especially true in pre-clinical efficacy testing of molecular anti-tumor agents that display no direct cytotoxicity but rather demonstrate tumerostatic and anti-angiogenic effects. The search for surrogate markers for these agents of tumor-response is becaming increasingly important.
If, as we expect, tumor selective expression of either luciferase or beta HCG reporter genes can indeed be monitored in-vivo, it has the potential to be used in a very broad range of tumor models. This is due to the use of transfection vectors rather than stable transfected cell-lines, which will enable us to follow most established cell-lines as well as primary tumor cells derived directly from patients.

Relevance for 3R:
a) Reduction in animal experimentation
The bioluminescent reporter gene imaging technique (BLI) allows the non-invasive and repetitive image recording of the distribution of luciferase-expressing cells within the body. Hence, tumor-growth can be followed over time.
- It renders the sacrifice of animals in tumor-models that study the dynamics of tumor growth and the kinetics of gene expression patterns at different time-points unnecessary.

It enables the detection of tumors from early on, thus, avoiding treatment of non-tumor bearing animals. Often the implantation of tumor-cells in animals does not induce tumor formation in 100% of the animals. Because, so far, no method to detect tumor formation at an early stage existed, this incomplete tumor formation had to be countered by increasing the number of animals allocated to each treatment arm.
- In-vivo verification of early tumor formation results in a decrease of sample size per treatment group to reach statistical significant results

b) Refining experimental methods
Because telomerase activation is essential for unlimited cellular replication, 85% - 95% of the cancer cells display re-activation of this enzyme complex. The in-vivo monitoring of the hTERT activity in the tumor-cells can be recorded by using the hTERT promoter to enhance luciferase or beta HCG reporter gene expression This is especially useful in the context of novel anti-tumor agents that may act through downregulation of the proliferative capacity in malignant cells rather than through direct cytotoxicity. For these anti-tumor agents hTERT-activity can serve as a surrogate marker to test their efficacy.
- Monitoring of hTERT-activity as surrogate marker will be an increasingly valuable instrument to study efficacy of novel molecular anti-tumor agents.
- Repeated measuring of hTERT-activity will, additionally, allow to determine the kinetics of anti-tumor effects of these agents.

c) Expected achievement towards animal protection
Because both methods to follow tumor growth proposed in this study have been shown to be highly sensitive in previous experiments, the monitoring of tumor growth can start at a very early stage and conclusive results can be obtained at a much earlier stage of the disease.
- Early tumor monitoring allows to minimize tumor manifestation of the animals and to reduce the duration of treatment