一种用于改善细胞存活和相对生物学效应（RBE）测量的时间分辨克隆形成试验。

A time-resolved clonogenic assay for improved cell survival and RBE measurements.

作者信息

Koch Robin A, Boucsein Marc, Brons Stephan, Alber Markus, Bahn Emanuel

机构信息

Department of Radiation Oncology, Heidelberg University Hospital, Im Neuenheimer Feld 672, 69120 Heidelberg, Germany.

Heidelberg Institute of Radiation Oncology (HIRO), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

出版信息

Clin Transl Radiat Oncol. 2023 Jul 22;42:100662. doi: 10.1016/j.ctro.2023.100662. eCollection 2023 Sep.

DOI:10.1016/j.ctro.2023.100662

PMID:37576069

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10412889/

Abstract

PURPOSE

The in vitro clonogenic assay (IVCA) is the mainstay of quantitative radiobiology. Here, we investigate the benefit of a time-resolved IVCA version (trIVCA) to improve the quantification of clonogenic survival and relative biological effectiveness (RBE) by analyzing cell colony growth behavior.

MATERIALS & METHODS: In the IVCA, clonogenicity classification of cell colonies is performed based on a fixed colony size threshold after incubation. In contrast, using trIVCA, we acquire time-lapse microscopy images during incubation and track the growth of each colony using neural-net-based image segmentation. Attributes of the resulting growth curves are then used as predictors for a decision tree classifier to determine clonogenicity of each colony. The method was applied to three cell lines, each irradiated with 250 kV X-rays in the range 0-8 Gy and carbon ions of high LET (100 keV/μm, dose-averaged) in the range 0-2 Gy. We compared the cell survival curves determined by trIVCA to those from the classical IVCA across different size thresholds and incubation times. Further, we investigated the impact of the assaying method on RBE determination.

RESULTS

Size distributions of abortive and clonogenic colonies overlap consistently, rendering perfect separation via size threshold unfeasible at any readout time. This effect is dose-dependent, systematically inflating the steepness and curvature of cell survival curves. Consequently, resulting cell survival estimates show variability between 3% and 105%. This uncertainty propagates into RBE calculation with variability between 8% and 25% at 2 Gy.Determining clonogenicity based on growth curves has an accuracy of 95% on average.

CONCLUSION

The IVCA suffers from substantial uncertainty caused by the overlap of size distributions of delayed abortive and clonogenic colonies. This impairs precise quantification of cell survival and RBE. By considering colony growth over time, our method improves assaying clonogenicity.

摘要

目的

体外克隆形成试验（IVCA）是定量放射生物学的主要方法。在此，我们通过分析细胞集落生长行为，研究时间分辨IVCA版本（trIVCA）在改善克隆存活定量和相对生物学效应（RBE）方面的优势。

材料与方法

在IVCA中，细胞集落的克隆形成分类是在孵育后基于固定的集落大小阈值进行的。相比之下，使用trIVCA时，我们在孵育期间获取延时显微镜图像，并使用基于神经网络的图像分割跟踪每个集落的生长。然后将所得生长曲线的属性用作决策树分类器的预测因子，以确定每个集落的克隆形成能力。该方法应用于三种细胞系，每种细胞系分别接受0 - 8 Gy范围内的250 kV X射线和0 - 2 Gy范围内的高传能线密度（100 keV/μm，剂量平均）碳离子照射。我们比较了trIVCA和经典IVCA在不同大小阈值和孵育时间下确定的细胞存活曲线。此外，我们研究了检测方法对RBE测定的影响。

结果

流产集落和克隆集落的大小分布始终重叠，使得在任何读出时间通过大小阈值进行完美分离都不可行。这种效应是剂量依赖性的，系统地夸大了细胞存活曲线的陡度和曲率。因此，所得的细胞存活估计值显示出3%至105%的变异性。这种不确定性传播到RBE计算中，在2 Gy时变异性在8%至25%之间。基于生长曲线确定克隆形成能力的平均准确率为95%。