Thomsen A R, Aldrian C, Luka B, Hornhardt S, Gomolka M, Moertl S, Hess J, Zitzelsberger H, Heider T, Schlueter N, Rau S, Monroy Ordonez B, Schäfer H, Rücker G, Henke M
Department of Radiation Oncology, University Medical Center, University of Freiburg, Freiburg/Breisgau, Germany.
German Cancer Consortium (DKTK) Partner Site Freiburg, German Cancer Research Center (dkfz), Heidelberg, Germany.
Clin Transl Radiat Oncol. 2022 Mar 16;34:51-56. doi: 10.1016/j.ctro.2022.03.007. eCollection 2022 May.
To establish stable growth of keratinocytes from very small biopsy specimens and successfully apply new test systems to determine their radiosensitivity.
Oral 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.
Stable keratinocyte growth continued for up to 7 weeks in 14 biopsies. Cells spread reliably from an initial 16.6 mm up to a median of 119.2 mm (range: 54.4-290). Radiated cells spread to only 100.7 mm (2 Gy; range: 55.3-266.7); 73.2 mm (4 Gy; 15-240.4); 47 mm (6 Gy; 2-111.9), and 22.7 mm (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).
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.
从小活检标本中建立角质形成细胞的稳定生长，并成功应用新的测试系统来确定其放射敏感性。
将15名受试者的口腔黏膜活检标本（直径：1.7毫米）用定制杯固定在培养板上。对长出的细胞进行细胞角蛋白5/14和Ki67检测，扩增后，给予不同剂量的辐射，然后接种到限定区域，使其离心扩散。在这个新开发的扩散试验中，通过图像分析测量细胞覆盖区域。进行统计分析时，使用线性混合回归模型；此外，将结果与所施加的辐射剂量相关联。采用集落形成效率（CFE）来验证结果。在照射后24小时和96小时，通过免疫荧光显微镜对γH2AX和53BP1焦点进行定量分析，以分析DNA损伤修复情况。
14份活检标本中的角质形成细胞稳定生长持续长达7周。细胞从最初的16.6毫米可靠地扩散至中位数为119.2毫米（范围：54.4 - 290）。接受辐射的细胞扩散至仅100.7毫米（2 Gy；范围：55.3 - 266.7）；73.2毫米（4 Gy；15 - 240.4）；47毫米（6 Gy；2 - 111.9），以及22.7毫米（8 Gy；0 - 80）。同样，CFE从0.223（0 Gy）降至0.0028（8 Gy）。将个体供体作为随机因素，细胞扩散与CFE相关，其中辐射剂量是主要驱动因素（面积调整后减少0.50）。照射6 Gy后，所有样本在24小时时辐射诱导的DNA损伤增加，7个样本中有5个在96小时时仍增加，这通过照射细胞中γH2AX/53BP1焦点数量增多得以检测（24小时时平均为3.7；96小时时平均为0.6）。
从小活检标本中获取的角质形成细胞的增殖是可行的。辐射会损害细胞迁移和增殖，新描述的扩散试验能够对细胞放射抗性进行排名。角质形成细胞模型还支持克隆形成存活和DNA双链修复等经典功能试验。其临床相关性有待进一步研究。
