Biopsy-derived oral keratinocytes – A model to potentially test for oral mucosa radiation sensitivity

A.R Thomsen,C Aldrian,B Luka,S Hornhardt,K Huber,M Gomolka,S Moertl,J Hess,H Zitzelsberger,T Heider,N Schlueter,S Rau,B Monroy Ordonez,H Schäfer,G Rücker,M Henke

doi:10.1016/j.ctro.2022.03.007

Abstract

PurposeTo establish stable in vitro growth of keratinocytes from very small biopsy specimens and successfully apply new test systems to determine their radiosensitivity. Materials and MethodsOral mucosa biopsies (diameter: 1.7 mm) from 15 subjects were immobilized with custom-made cups onto culture plates. Outgrowing cells were tested for cytokeratin 5/14 and Ki67, expanded, radiated at different doses, and seeded onto circumscribed areas before being allowed to spread centrifugally. In this newly developed spreading assay, cell-covered areas were measured by image analysis. For statistical analysis, a linear mixed regression model was used; additionally, results were correlated to the radiation dose applied. Colony forming efficiency (CFE) was used to validate the results. DNA damage repair was analysed by gammaH2AX and 53BP1 foci quantification using immunofluorescence microscopy 24 h and 96 h after irradiation. ResultsStable keratinocyte growth continued for up to 7 weeks in 14 biopsies. Cells spread reliably from an initial 16.6 mm2 up to a median of 119.2 mm2 (range: 54.4–290). Radiated cells spread to only 100.7 mm2 (2 Gy; range: 55.3–266.7); 73.2 mm2 (4 Gy; 15–240.4); 47 mm2 (6 Gy; 2–111.9), and 22.7 mm2 (8 Gy; 0–80). Similarly, CFE decreased from 0.223 (0 Gy) to 0.0028 (8 Gy). Using an individual donor as a random factor, cell spread correlated with CFE, where radiation dose was the main driver (decrease by 0.50, adjusted for area). Upon irradiation with 6 Gy, radiation-induced DNA damage was increased after 24 h in all samples, and even after 96 h in 5 out of 7 samples, as detected by a higher number of gammaH2AX/53BP1 foci in irradiated cells (mean 3.7 for 24 h; mean 0.6 for 96 h). ConclusionIn vitro propagation of keratinocytes derived from a small biopsy is feasible. Radiation impairs cellular migration and proliferation, and the newly described spreading assay allows ranking for cellular radioresistance. The keratinocyte model also supports classical functional assays such as clonogenic survival and DNA double strand repair. The clinical relevance awaits upcoming investigations.

Highlights

Using an individual donor as a random factor, cell spread correlated with Colony forming efficiency (CFE), where radiation dose was the main driver
Patients undergoing radiotherapy for head and neck cancer are likely to suffer from inflammation and ulcerations, called radiation mucositis, which causes severe discomfort and can even lead to lifethreatening complications [1–3]
One hundred and forty-four gingiva particles from 16 in dividuals were processed by micro-dissection resulting in 111 primary keratinocyte cultures

Summary

Introduction

While endowed with a remarkable regeneration capacity, the mu cosa of the oral cavity and pharynx is very sensitive to ionizing radia tion. Patients undergoing radiotherapy for head and neck cancer are likely to suffer from inflammation and ulcerations, called radiation mucositis, which causes severe discomfort and can even lead to lifethreatening complications [1–3]. These severe reactions of normal tis sue hamper optimal tumour control by reducing the therapeutically necessary radiation dose. Oral mucosa is a mainly 2-dimensional tissue that can not be assessed by metabolic imaging in a way like other tissues at risk for radiation damage, e.g. salivary glands [4]. Acquisition of oral keratinocytes from individual patients where radiation response and additional cellular properties can be identified could help to better un derstand the pathophysiology of radiation-associated toxicity, and guide personalized treatment

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