ESTRO 2023 - Abstract Book

S2007

Digital Posters

ESTRO 2023

PO-2230 Proposing a radiobiological assay using videomicroscopy to dynamically track single cell response

F. Paris 1

1 Inserm, Cancer Research Center, Nantes, France

Purpose or Objective Cellular assays modeling tumor radiosensitization or normal tissue radiotoxicity commonly support knowledge in radiobiology. Despite worldwide use, outcomes from those assays remain biologically limited. The next generation of radiobiological tests must include high throughput dynamic biological inputs at a multiscale cellular level from a single cell to the whole population. In this way, knowledge of tumor radiobiology will be improved, as will the related mathematical models. Here, we proposed to tackle some of those current bottlenecks with the development of EPICeA (for Encoding Phenotype for Isolated Cell Assay), a novel integrated radiobiological tool able to dynamically track 6 independent cellular observables and 55 related features,. Materials and Methods First, U251 glioblastoma cells were stained with the nuclear marker sirDNA and then monitored by videomicroscopy for 5 days after exposure to dose escalation (0, 2, 4, 8, 10, and 15 Gy. After collecting a large video bank representing from 3 independent sets of experiments representing at least 9 videos and 600 cells per irradiation condition, the brightfield films for overall cell fate and fluorescent films for nucleus fate were used to annotate six cellular observables (cell number, normal division, abnormal mitosis, cell fusion and death) as a function of time. This annotation was performed directly for 5 consecutive days. From these six observables (cell number, normal division, abnormal mitosis, cell fusion and death), 55 related features were then extracted using internal C++ and Python computation algorithms allowing an overall radiobiological picture. Results First, cladograms illustrating the overall radiation-induced behaviors of each irradiated mother cell and their related out- springs were set up. Then, the population means and single-cell distributions for different radiobiological features (cell count, percentage of normal division or abnormal division, time between 2 mitosis, percentage of death and death) were computed and showed discrepancy in the strength and incidence as a function of time and dose. Finally, Pearson correlation coefficients for those 55 features derived from those observables and relevant in radiobiology were established, showing singular correlations. Conclusion EPICeA assay represents a promising and robust approach for the study and the evaluation of tumor cell response after radiotherapy. Complementary to standard clonogenic assays, EPICeA provides integrated and dynamic mechanistic insights incrementing the knowledge about radiobiology response to radiation towards achieving a better radiotherapy efficacy. I. Lauwers 1 , K. Pachler 2 , G. Verduijn 1 , A. Sewnaik 3 , J. Hardillo 3 , D. Monserez 3 , S. Keereweer 4 , D. van Gent 2 , M. Hoogeman 1,5 , M. Capala 1 , S. Petit 1 1 Erasmus MC Cancer Institute, University Medical Center Rotterdam, Department of Radiotherapy, Rotterdam, The Netherlands; 2 University Medical Center Rotterdam, Department of Molecular Genetics, Rotterdam, The Netherlands; 3 University Medical Center Rotterdam, Department of Otorhinolaryngology, Rotterdam, The Netherlands; 4 University Medical Center Rotterdam, Deparment of Otorhinolaryngology, Rotterdam, The Netherlands; 5 HollandPTC, Department of Medical Physics and Informatics, Delft, The Netherlands Purpose or Objective Locoregional failure rate remains high in oropharyngeal squamous cell carcinoma (OPSCC) patients, while long-term toxicity is substantial. Therefore, OPSCC patients would benefit from personalized treatments. The first step towards personalization is patient stratification in responders and non-responders, before or early during treatment. We hypothesize that ex vivo tumor irradiation response could be predictive for clinical radiotherapy response. However, it is not clear which ex vivo parameter best reflects clinical response. A logical candidate is apoptosis induction measured 5 days after radiation.[1] However, for personalized treatment selection a faster read out would be desirable. Therefore, the aim of this study was to investigate earlier ex vivo irradiation response markers, using apoptosis induction at 5 days post-irradiation as benchmark. Materials and Methods OPSCC patients of the BIO-ROC study (NL8450) treated between 14/3/2022 and 18/8/2022 were included. Fresh OPSCC biopsy samples derived at the outpatient clinic were sliced, cultured, irradiated (5Gy), and fixed after 24h or 5d post- irradiation. Apoptosis (TUNEL) was imaged using fluorescence microscopy and quantified as the percentage of the total nuclear area (DAPI). DNA repair kinetics were analyzed by residual 53BP1 foci quantification in tumor nuclei (immune- stained with p63) using fluorescence confocal microscopy. The foci count per nuclear tumor volume was automatically determined using a deep learning model.[2] Automatic images analysis enabled analysis of multiple field of views and over 100 nuclei per condition. Apoptosis and DNA repair kinetics at 24h were considered as potential fast readout response markers and apoptosis at 5d was used as benchmark. To determine the effect of radiation, the outcome of the treated condition relative to control condition was calculated. Since two year follow up was not achieved yet for all patients, as surrogate for treatment response, the benchmark apoptosis at 5d was compared between HPV positive and negative patients, of which the latter are known to have unfavorable clinical outcomes.[3] PO-2231 Ex vivo response markers for radiotherapy treatment personalization of oropharyngeal cancer patients

Results