Urologic Oncology: Seminars and Original Investigations
Original articleApplying the chicken embryo chorioallantoic membrane assay to study treatment approaches in urothelial carcinoma☆
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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2021, Advanced Drug Delivery ReviewsCitation Excerpt :Subsequently, the tumours are usually fixed, embedded, and sectioned, followed by histological and immunohistochemical analyses (e.g., haematoxylin eosin staining, Ki-67 proliferation antigen staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method counterstained with haematoxylin) [51,66,70,85,89,91,98,100,108,122,123,148–150,154–156,158,172,173,175,176,181–183,185,193,201–204,206,208–212]. The tumour cells can also be examined for their gene expression [85,91,93,108,148,176,203] or protein expression [182,203] profile after the tumour mass is processed. Because of pre-labelling of the grafted tumour cells (e.g., green fluorescent protein [81,96,108,148], firefly luciferase [150,154,203,205], mCherry [148]), it is possible to directly assess tumour growth, tumour size, and the effect of the applied compound by imaging.
Various CAM tumor models
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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.