Behaviour of four different B16 murine melanoma cell sublines: C57BL/6J skinby Corina Danciu, Camelia Oprean, Dorina E. Coricovac, Cioca Andreea, Anca Cimpean, Heinfried Radeke, Codruta Soica, Cristina Dehelean

International Journal of Experimental Pathology

About

Year
2015
DOI
10.1111/iep.12114
Subject
Molecular Biology / Pathology and Forensic Medicine / Cell Biology

Text

ORIG INAL ART ICLE

Behaviour of four different B16 murine melanoma cell sublines:

C57BL/6J skin

Corina Danciu*, Camelia Oprean†, Dorina E. Coricovac‡, Cioca Andreea§, Anca Cimpean¶,

Heinfried Radeke**, Codruta Soica† and Cristina Dehelean‡ *Department of Pharmacognosy, University of Medicine and Pharmacy ‘Victor Babes’, Timisoara, Romania, †Department of

Pharmaceutical Chemistry, University of Medicine and Pharmacy ‘Victor Babes’, Timisoara, Romania, ‡Department of Toxicology,

University of Medicine and Pharmacy ‘Victor Babes’, Timisoara, Romania, §Department of Pathology, ‘Iuliu Hatieganu’ University of Medicine and Pharmacy, Cluj-Napoca, Romania, ¶Department of Microscopic Morphology/Histology, Angiogenesis Research

Center, University of Medicine and Pharmacy ‘Victor Babes’, Timisoara, Romania and **Pharmazentrum Frankfurt/Center for Drug

Research, Development, and Safety, Clinic of J. W. Goethe University, Frankfurt, Germany

INTERNATIONAL

JOURNAL OF

EXPERIMENTAL

PATHOLOGY doi: 10.1111/iep.12114

Received for publication: 17 July 2014

Accepted for publication: 1 December 2014

Correspondence:

Dorina E. Coricovac

Department of Toxicology

University of Medicine and Pharmacy ‘Victor Babes’

Eftimie Murgu Square

No. 2, 300041 Timisoara

Romania

Tel.: +0040744648844

Fax: +0256/490626

E-mail: dorinacoricovac@umft.ro

SUMMARY

Transplantable murine melanomas are well-established models for the study of experimental cancer therapies. The aim of this study was to analyse the behaviour of four different B16 murine melanoma cell sublines after inoculation in C57BL/6J host, more specifically skin-targeted analysis, with respect to two parameters: clinical (tumour volume, melanin amount, erythema) and histological (H & E, S100, VEGF expression). Both non-invasive and invasive determinations showed that B164A5 is the most aggressive melanoma cell line for C57BL/6J’s skin, succeeded by B16F10 and followed in a similar diminished manner of aggressiveness by B16GMCSF and

B16FLT3 cell lines.

Keywords

B164A5, B16F10, B16FLT3, B16GMCSF, C57BL/6J

Skin cancers include basal cell carcinoma, squamous cell carcinoma and malignant melanoma. The first two types of skin cancer are the most frequent malignant neoplasms among fair-skinned population (Andrade et al. 2012). Recent studies showed that the incidence of melanoma was also increasing especially in case of women young adults (Reed et al. 2012).

Ultraviolet (UV) radiation can promote the limited proliferative capacity of melanocytes (Gupta et al. 2013). Although the incidence of melanoma among other types of skin cancer is a parameter with increased variability, the severity of this disease is undisputed. Due to its highly metastatic potential and resistance to chemotherapy, it is responsible for most deaths (Rigel 2005; Svobodova et al. 2006). Over the past years, Food and Drug Administration approved three agents for the treatment of melanoma, namely pegylated interferon alpha-2b, vemurafenib and ipilimumab (Lee et al. 2012).

More research is still required to find a highly effective drug, with low side effects, against this challenging type of cancer.

For this purpose, preclinical studies in animal models provide valuable clues for clinical trials.

Transplantable murine melanomas are well-established models for the study of experimental cancer therapies. Many immunotherapeutic protocols have been tested using the murine B16 melanoma cell line (and its sublines) that originates in the syngeneic C57BL/6 (H-2b) mouse strain. Although

B164A5 is one of the most widely used cell line for the murine melanoma model, as evidenced by the latest papers in the field (Danciu et al. 2013a,b; Lee et al. 2013; Villareal et al. 2013; Ookubo et al. 2014), several subline derivates have been obtained to study different therapeutic strategies. © 2015 The Authors.

International Journal of Experimental Pathology © 2015 International Journal of Experimental Pathology 1

Int. J. Exp. Path. (2015)

The sublines B16F1 and B16F10 were derived from the mother B16 line by selection for their ability to form lung colonies in vivo after intravenous injection and subsequently established in vitro after one (B16F1) or 10 (B16F10) cycles of lung colony formation. B16F10 is a subline that possesses high lung metastasis ability, whereas B16F1 is a subline with low metastatic potential (Fidler 1973; Teicher 2010).

B16GMCSF is a subline derived from B16F10 by transduction, employing an MFG retroviral vector-encoding murine

Granulocyte - Macrophage Colony Stimulating Factor (GMCSF) (Kumar et al. 1999). It has been shown that GM-CSF surface-modified B16F10 melanoma cell vaccine may induce protection against the wild-type tumour challenge (Gao et al. 2006; Danciu et al. 2013a,b). The fourth cell subline analysed in this study is B16FLT3. It was also obtained from the murine B16 cell line, which was transfected with the gene for the Fms-like tyrosine kinase 3 (Flt3)-L cytokine (Vargas et al. 2006; Danciu et al. 2013a,b). Although both GM-CSF and

Flt3 ligand induce marked expansion of dendritic cells, it has been shown that GM-CSF-secreting tumour cells promoted higher levels of protective immunity than vaccination with

FLT3-L-secreting tumour cells (Zarei et al. 2009).

The aim of this study was to analyse the behaviour of this four different B16 murine melanoma cell sublines in the

C57BL/6J host, more specifically skin-targeted analysis, with respect to two parameters: clinical (tumour volume, melanin amount, erythema) and histological features [Haematoxylin and Eosin (H&E), S100, Vascular Endothelial Growth Factor (VEGF) expression].

Materials and methods

Cells

Mouse adherent melanoma cell line B164A5 was purchased from ECACC (European Collection of Cell Cultures,

Salisbury, UK). B16F10, B16GMCSF and B16FLT3 cells were provided by Prof. Radeke, Pharmazentrum Frankfurt/

Center for Drug Research, Development, and Safety, Clinic of JW Goethe University, Frankfurt, Germany. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated foetal calf serum (FCS), 1% non-essential amino acids and 1% penicillin– streptomycin in a humidified atmosphere containing 5%