Primary head and neck cancer cell cultures are susceptible to proliferation of Epstein-Barr virus infected lymphocytes

Senyao Shao1?, Lars Uwe Scholtz1?, Sarah Gendreizig1 , Laura Martínez?Ruiz2,3,4, Javier Florido2,3,4, Germaine Escames2,3,4, Matthias Schürmann1 , Carsten Hain5 , Leonie Hose1,6, Almut Mentz6 , Pascal Schmidt5 , Menghang Wang1,7, Peter Goon1 , Michael Wehmeier8 , Frank Brasch6 , J?rn Kalinowski5 , Felix Oppel1*? and Holger Sudhoff1*?

1 Department of Otolaryngology, Head and Neck Surgery, Campus Klinikum Bielefeld Mitte, University Hospital OWL of Bielefeld University, Klinikum Bielefeld, Teutoburger Str. 50, 33604 Bielefeld, Germany. 2 Biomedical Research Center, Health Sciences Technology Park, University of Granada, 18016 Granada, Spain. 3 Department of Physiology, Faculty of Medicine, University of Granada, 18016 Granada, Spain. 4 CIBERFES, Ibs. Granada, San Cecilio University Hospital, 18016 Granada, Spain. 5 Center for Biotechnology (CeBiTec), Universit?t Bielefeld, Bielefeld, Germany. 6 Department of Pathology, Klinikum Bielefeld, Teutoburger Str. 50, 33604 Bielefeld, Germany. 7 Department of Otolaryngology Head and Neck Surgery, Peking University International Hospital, Peking University, Beijing 102206, China. 8 Department of Laboratory Medicine, Klinikum Bielefeld, Teutoburger Str. 50, 33604 Bielefeld, Germany.

Abstract

Background New concepts for a more efective anti-cancer therapy are urgently needed. Experimental faws represent a major counter player of this development and lead to inaccurate and unreproducible data as well as unsuccessful translation of research approaches into clinics. In a previous study we have created epithelial cell cultures from head and neck squamous cell carcinoma (HNSCC) tissue. Methods We characterize primary cell populations isolated from human papillomavirus positive HNSCC tissue for their marker expression by RT-qPCR, fow cytometry, and immunofuorescence staining. Their sensitivity to MDM2-inhibition was measured using cell viability assays. Results Primary HNSCC cell cultures showed the delayed formation of spheroids at higher passages. These spheroids mimicked the morphology and growth characteristics of other established HNSCC spheroid models. However, expres? sion of epithelial and mesenchymal markers could not be detected in these cells despite the presence of the HNSCC stem cell marker aldehyde dehydrogenase 1 family member A1. Instead, strong expression of B- and T-lymphocytes markers was observed. Flow cytometry analysis revealed a heterogeneous mixture of CD3+/CD25+T-lymphocytes and CD19+B-lymphocytes at a ratio of 4:1 at passage 5 and transformed lymphocytes at late passages (≥passage 12) with CD45+CD19+CD20+, of which around 10 to 20% were CD3+CD25+CD56+. Interestingly, the whole population was FOXP3-positive indicative of regulatory B-cells (Bregs). Expression of transcripts specifc for the Epstein-Barr-virus (EBV) was detected to increase in these spheroid cells along late passages, and this population was vulnerable to MDM2 inhibition. HPV+HNSCC cells but not EBV+lymphocytes were detected to engraft into immunodefcient mice.