Fluorescent canine prostate cancer cell lines for in vivo studies

Tumour-derived cell lines provide a valuable resource for cancer researches and offer several possibilities for the basic drug evaluation. But studies in vitro remain limited to evaluate complex interactions. Whole animal models are key to understand the underlying mechanisms of tumorigenesis and metastasis of cancer as well as essential to develop novel therapeutic approaches. In addition to the in vitro

charac-terization of the generated sphere cells, in vivo evaluating the tumorigenic potential of the generated sphere cells is necessary to verify the potential CSC character. There-fore, an in vivo model is needed allowingdetecting and analysingof the enriched cell subpopulations in order to evaluate if these populations are able to initiate new tu-mour. Such a model will also be a valuable tool to further characterize the role of MYC and ITGA6 in canine cancer cells. To achieve this, first of all, cell lines that can be detected also in early phases in animal models need to be established and well characterized. Fluorescence reporters provide the opportunity to study early tumour progression before palpation as well as drug intervention longitudinal in living animals.

In this thesis, three canine prostate cancer cell lines stably expressing fluorescent proteins were established. These cell lines could be used to generate spheres as done before in this thesis and be analysed in vivo taking advantage of their fluores-cent features. The cell line CT1258 is established from a highly malignant canine ad-enocarcinoma with mesentery metastasis. This cell line showed highly tumourigenic behaviour in vivo (Fork et al. 2008, Sterenczak et al. 2012). Its generated fluorescent cell lines CT1258-Fusionred, CT1258-mKate2C and CT1258-TurboFP650 express FusionRed protein (red), mKate2 (far-red) and TurboFP650 protein (near infar-red) respectively. CT1258-Fusionred and CT1258-TurboFP650 are polyclonal cell lines without monoclonal selecting after selection with geneticin (G418). They maintained the heterogeneous character of the initial cell line CT1258. CT1258-mKate2C is a monoclonal subline generated by limiting dilution assay. In CT1258-mKate2C, mKate2 positive cell can achieve 97% and able to maintain a considerable level after long-term cultivation without antibiotic selection. However, the monoclonal cell line may have higher risk of morphology, proliferation or gene expression changes.

One of the technical challenges for in vivo inmaging is the photon attenuation in living tissue (Rao et al. 2007). Therefore, fluorescent proteins with long emission at the red, far-red, and near infra-red (NIR) region were chosed in current study. All

General discussion

90

Random integration of the herein used plasmid-DNA into the genome can easily result in alteration of basic cell line characteristics, such as cell morphology, proliferation, and genomic stabilty. Compared with their parental cell line CT1258, the biological characteristics of the generated cell lines, such as cell proliferation and metabolic activity, did not display significant differences. Furthermore, the expression of stem cell marker genes also remained substantially stable within the lines.

Different from the other two cell lines, CT1258-mKate2C was found to be characterised by an increased metabolic activity. During cultivation, an interesting phenomenon is observed in mKate2C. The cell clusters formed by CT1258-mKate2C were observed similar to the spheres generated by serum-free CT1258 culture in a previous study (Liu et al. 2015). The increased metabolic activity of CT1258-mKate2C mentioned above was also consistent to the prior characterising of the CT1258 generated spheres (Liu et al. 2015). These result led to an interesting hypothesis that the CT1258-mKate2C cell line might be the result of a clone selected from a sphere-formation subpopulation. To further validate this hypothesis, expres-sion of ITGA6 gene and the surface marker CD49f (aka ITGA6) was analysed in CT1258-mKate2C. Increased ITGA6 gene expression was detected in CT1258 gen-erated spheres by real-time PCR in previous study (Liu et al. 2015). However, in-creased expression of ITGA6 and CD49f was not revealed in CT1258-mKate2C cells by the real-time PCR and flow cytometric analyses. Nevertheless, CT1258-mKate2C cells exhibited higher sphere-forming capacity compared to CT1258 cells in serum-free medium. The ability to form non-adherent spheres in serum-serum-free condition is an indicator to evaluate self-renewal ability of the cells, thus it was considered to be one of the important phenotypic characteristics of cancer stem-like cells (Visvader and Lindeman 2012). Taken together, these results indicate that the CT1258-mKate2C cell line may have an enriched potential cancer stem-like cell population. Further val-idation still need to be performed.

Furthermore, to characterize the genomic stability after transfection, copy number variations (CNVs) were analyzed by whole genome sequencing. In general, genomic profiling of the three generated fluorescent cell lines showed no significant changes of copy numbers. However, different from CT1258, Fusionred and CT1258-TurboFP650, CT1258-mKate2C is the only cell line which does not show a distal CFA16 deletion. The preserved CFA16 part contains 270 encoding genes. DAVID

pathway analyses (https://david.ncifcrf.gov/summary.jsp) revealed that the lysoso-mal, insulin and MAPK signalling pathways are modulated by twelve of the genes;

however, the p-value did not show significance.

To conclude, key characteristics of the generated cell lines remained comparable to the parental line CT1258. The generated cell lines that stably expressing strong fluorescent protein provided a valuable option for deep tissue in vivo imaging and a stable model for further studies characterizing the previously described potential CSC subpopulations.