Radiation and Cancer Biology
PD 11 - Biology 5 - Poster Discussion
1090 - In Vitro Cell-Free DNA Quantification: A Novel Method to Accurately Quantify Clonogenic Survival After Irradiation
Tuesday, October 23
2:45 PM - 2:51 PM
Location: Room 217 A/B
Christopher Deig, MD
Indiana University School of Medicine: Researcher: Employee; The Colorado Health Foundation: Resident: Employee
Indiana University School of Medicine - Department of Radiation Oncology: Departmental Research funding
In Vitro Cell-Free DNA Quantification: A Novel Method to Accurately Quantify Clonogenic Survival After Irradiation
C. Deig1, R. Thowe1, E. Frye1, H. Chin-Sinex1, M. S. Mendonca1, and T. Lautenschlaeger2; 1Indiana University School of Medicine, Indianapolis, IN, 2Department of Radiation Oncology, Simon Cancer Center, Indiana University School of Medicine, Indianapolis, IN
Circulating tumor DNA (ctDNA) and cell-free DNA (cfDNA) analysis is a relatively recent concept, which has been shown to aid in the detection of cancer and evaluation of somatic mutations, and is useful during cancer surveillance to potentially detect minimal residual disease with a clinically significant lead time between ctDNA detection and clinical relapse. Recent evidence suggests that cfDNA concentration in plasma increases in proportion to tumor volume, metabolic activity, tumor necrosis, lymphovascular invasion and cellular proliferation indices. Radiation-induced cell death is known to occur predominantly by two pathways, apoptosis and mitotic catastrophe leading to cellular necrosis. As cancer cells grow, a small percentage of cells consistently die (conceptualized as cell loss factor) and release their intracytoplasmic and nuclear contents into plasma, in vivo,
or into cell culture media, in vitro
. We thus hypothesized that cell line-specific cell-free DNA (cfDNA) can be used as a measure of cell viability and clonogenic survival in cell culture.
Clonogenic assays on NSCLC cell lines H322, A549 and H322 were performed with radiation doses to 0, 4 or 8 Gy. Prior to colony fixation and counting, cfDNA was extracted and quantified from cell culture media. The correlation between cell line-specific cfDNA and number of colonies grown on culture plates was examined. An H1299:A549 coculture model was also used to evaluate the differential release of cell line-specific cfDNA.
The results of this work indicated a strong correlation between CfDNA quantification from cell culture media and clonogenic survival at all radiation doses and in all cell lines tested (R2
range = 0.77 to 0.99). Clonogenic survival curves derived from cfDNA were virtually indistinguishable from matched traditional clonogenic survival data (p > 0.05; no significant difference exists between clonogenic curves). CfDNA quantification also accurately estimates colony count in a two cell-line coculture model.
This novel method of using cell-free DNA quantification from cell culture media can accurately measure clonogenic survival, and appears suitable for development in a high-throughput clonogenic assay and radiosensitizer screening platform. We speculate that this assay successfully estimates surviving fraction, because proliferating cells undergo a fixed amount of cell turnover and cfDNA is actively secreted in constant amounts via exosomes. This cell turnover and active cfDNA secretion consistently releases cfDNA into media in quantities proportional to the number of surviving cells. Further, our data show that this assay is able to accurately determine surviving fraction in very low plating densities; lower than that generally used in 96-well plates. This assay may therefore serve as a breakthrough to enable investigators to more rapidly screen the efficacy of radiotherapeutics in multiple cell lines.
Author Disclosure: C. Deig: Departmental Research funding; Indiana University School of Medicine - Department of Radiation Oncology. R. Thowe: Research Funding; Indiana University School of Medicine - Department of Radiation Oncology. E. Frye: None. H. Chin-Sinex: None. M.S. Mendonca: Independent Contractor; Radiation Research Society. Editor-in-Chief of the RRS journal Radiation Research; Radiation Research Society. Executive Committee & Council Member; Radiation Research Society. Executive committee and Editor-in-Chief; Radiation Research Society. T. Lautenschlaeger: None.