Original article
Applying the chicken embryo chorioallantoic membrane assay to study treatment approaches in urothelial carcinoma

https://doi.org/10.1016/j.urolonc.2017.05.003Get rights and content

Highlights

  • CAM model suitable for xenografting of various urothelial carcinoma cell lines.

  • 2D cell culture, CAM model, and recultured CAM tumors are molecularly similar.

  • Cisplatin is as efficient in CAM tumors as in 2D-cultured cells.

  • HDACi are less efficacious in CAM tumors compared to 2D-cultured cells.

  • HDACi pretreatment increases cisplatin efficiency in 2D cultures and CAM tumors.

Abstract

Background

Rapid development of novel treatment options demands valid preclinical screening models for urothelial carcinoma (UC). The translational value of high-throughput drug testing using 2-dimensional (2D) cultures is limited while for xenograft models handling efforts and costs often become prohibitive for larger-scale drug testing. Therefore, we investigated to which extent the chicken chorioallantoic membrane (CAM) assay might provide an alternative model to study antineoplastic treatment approaches for UC.

Methods

The ability of 8 human UC cell lines (UCCs) to form tumors after implantation on CAMs was investigated. Epithelial-like RT-112 and mesenchymal-like T-24 UCCs in cell culture or as CAM tumors were treated with cisplatin alone or combined with histone deacetylase inhibitors (HDACi) romidepsin and suberanilohydroxamic acid. Tumor weight, size, and bioluminescence activity were monitored; tumor specimens were analyzed by histology and immunohistochemistry. Western blotting and quantitative real time polymerase chain reaction were used to measure protein and mRNA expression.

Results

UCCs were reliably implantable on the CAM, but tumor development varied among cell lines. Expression of differentiation markers (E-cadherin, vimentin, CK5, CK18, and CK20) was similar in CAM tumors and 2D cultures. Cellular phenotypes also remained stable after recultivation of CAM tumors in 2D cultures. Bioluminescence images correlated with tumor weight.

Cisplatin and HDACi decreased weight and growth of CAM tumors in a dose-dependent manner, but HDACi treatment acted less efficiently as in 2D cultures, especially on its typically associated molecular markers. Synergistic effects of HDACi and subsequent cisplatin treatment on UCCs were neither detected in 2D cultures nor detected in CAM tumors.

Conclusion

Our results demonstrate that the CAM assay is a useful tool for studying tumor growth and response to conventional anticancer drugs under 3D conditions, especially cytotoxic drugs as cisplatin. With some limitations, it might serve as a cost- and time-effective preclinical screening assay for novel therapeutic approaches before further assessment in expensive and cumbersome animal models.

Section snippets

Background

Novel antineoplastic drugs are usually first assessed in 2-dimensional (2D) cell cultures. For urothelial carcinoma (UC), a broad range of established cell culture models are available reflecting the different subtypes and heterogeneity of UC [1], [2], [3]. Cell culture models are usually simple, easy to handle, yielding results quickly, and at low cost. However, cell culture models differ substantially from the original tumor, lacking the natural tumor environment including stromal cells and

Cell culture and transfection

UCCs RT-112, VM-CUB-1, 5637, HT-1376, UM-UC-3, T-24, 639 V, and J82 were grown in DMEM GlutaMAX-I (Gibco) containing 10% FCS (Merck Millipore) as described [13]. Immortalized human urothelial HBLAK cells were cultured in CnT-Prime Epithelial Culture Medium [14]. Cell lines were authenticated by standard DNA fingerprint analysis [12]. RT-112 cells were stably transduced with a lentivirus expressing luciferase (RT-112 Luc).

CAM assay

The assay was performed as described and detailed in supplemented methods

Most UCCs form CAM tumors

UCCs HT-1376, RT-112, VM-CUB-1, and 5637 cells (epithelial phenotype) as well as J82, UM-UC-3, T-24, and 639 V, (mesenchymal phenotype) and benign HBLAK were tested for their ability to form CAM tumors [14], [18]. Except for 5637 cells, implantation was successful for all UCCs and for HBLAK. Notably, tumor weights and sizes differed for each cell line and did not always correlate (Fig. 1, Table S6).

Macroscopic growth patterns of RT-112 and T-24 cells were further compared. Take rate (RT-112:

Discussion

In vivo tumor models are essential for understanding cancer progression mechanisms, identifying therapeutic targets, and evaluating cytotoxic drugs. As an intermediate between 2D cultures and experiments using rodents, the CAM model represents a cost-effective, quick, reproducible, and naturally immunodeficient model, which moreover raises few ethical or legal concerns [20], [21]. Various cancer cell lines of different tumor entities have been seeded onto the CAM (Table S1), but only few

Authors’ contributions

M.A.S., G.N., M.J.H., W.A.S., and R.N. conceived and designed the study. M.A.S. performed most of the experiments, and additional experiments were conducted by A.S. and A.R. A.S. and R.N. helped developing the CAM assay. G.N. and M.J.H. supervised the project with the support of W.A.S. M.A.S. and G.N. prepared the initial manuscript. W.A.S., M.J.H., R.N., G.A.K., A.R., and P.A. contributed to the article. All authors reviewed and approved the final article.

Acknowledgments

The authors thank Christine Walter and Christoph Fleisgarten from the Institute of Neuropathology, University Hospital Duesseldorf for embedding the tumor samples in paraffin wax. The authors gratefully acknowledge Joep G. H. van Roermund, Department of Urology, Maastricht University Medical Centre, The Netherlands, for supporting the project by helpful discussions. We thank Gerhard Fritz, Institute of Toxicology, Duesseldorf, Germany, for critically reading the article.

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    This study was supported by a fellowship of the Duesseldorf School of Oncology (funded by the Comprehensive Cancer Centre Dusseldorf/Deutsche Krebshilfe and the Medical Faculty of the Heinrich-Heine University Duesseldorf) to M.A.S. and by the Strategischer Forschungsförderungs-Fond of the Heinrich-Heine University.

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