ESTRO 37 Abstract book

ESTRO 37

S590

Fig 1. Migration and invasion of HNSCC cell lines in scratch assays.

Poster: Radiobiology track: Radiobiology of head and neck cancer

PO-1049 HNSCC Cell Lines with DNA Repair Defects show an Invasive Phenotype in vitro. P. Essers 1 , E. Ploeg 1 , M. Verheij 2 , C. Vens 2 1 Netherlands Cancer Institute, Cell Biology I, Amsterdam, The Netherlands 2 Netherlands Cancer Institute, Department of Radiation Oncology and Department of Cell Biology I, Amsterdam, The Netherlands Purpose or Objective We recently developed a gene expression based DNA repair defect biomarker using an HNSCC cell line panel and employing novel machine learning algorithms, with hypersensitivity to mitomycin C and olaparib as in vitro proxies for distinct functional DNA repair defects. In a patient cohort, we found that tumors with gene expression similar to cell lines which are sensitive to both drugs (SensitiveMO) have increased metastasis rates. The purpose of this study was to validate the observed metastatic potential of SensitiveMO tumors in vitro . Material and Methods From our original cell line panel, we selected three to four cell lines to represent each category: SensitiveMO , SensitiveM (sensitive to mitomycin C but not to olaparib) and Normal (not sensitive to either drug). We determined the in vitro migratory capacity of these cell lines using scratch and transwell assays. In scratch assays, a confluent layer of cells is scratched and the rate at which the remaining cells close the resulting wound is determined. In trans-well assays, the fraction of cells that is able to pass through a porous membrane is measured. To measure the invasive capacity of these cells, these assays were modified by depositing extracellular matrix proteins (matrigel) in the scratch or on the membrane. Results We found that both types of repair defected ( SensitiveM and SensitiveMO ) cell lines migrated significantly more often and faster than Normal classified cell lines in both, scratch assays (see figure; p=0.0058 and p=0.0149 respectively) and trans-well migration assays (p=0.0056 and p=0.00030 respectively). We observed that sensitiveMO cell lines were able to invade through matrigel in both scratch (see figure; p=0.026) and trans- well invasion assays (p=0.0028), while this was not efficient in the sensitiveM or Normal cell lines. To further test whether this is indeed DNA repair defect related, we impaired DNA repair in the repair proficient Normal cell lines, using the RAD51 inhibitor B02 for 24h. Treatment with the RAD51 inhibitor resulted in strong induction of migratory (p=0.026) and invasive behavior (p=0.012).

Values shown are the average of at least 3 experiments containing 5 replicates each .

Conclusion We conclude that DNA repair defected cells are migratory and invasive, supporting our observation of pronounced metastasis incidence in the clinic. Moreover, such migratory behavior can develop within 24 hours after DNA repair is inhibited, suggesting that DNA damage directly regulates migration. PO-1050 Color Coded HNSCC Tumor Cells for Clonal Tracing of Radioresistance K. Ina 1 , D. Salminger 2 , H. Stephan 2 , M. Anger 2 , C. Peitzsch 3 , M. Baumann 1 , A. Dubrovska 2 1 DKFZ, RadioOncology/ RadioBiology E220, Heidelberg, Germany 2 OncoRay - National Center for Radiation Research in Oncology, Biomarker for Individualized Radiotherapy, Dresden, Germany 3 NCT-Dresden, Translational Radiooncology, Dresden, Germany Purpose or Objective Despite significant progress in the diagnosis and treatment, head and neck squamous cell carcinoma (HNSCC) is associated with high mortality and a low five- year survival rate of around 50% for advanced stage disease. Radiotherapy or radio chemotherapy can fail to achieve long-lasting HNSCC cure if they do not eradicate tumor initiating or cancer stem cells (CSCs), which cause tumor recurrence. Tumor heterogeneity and cellular plasticity are the main drivers for tumorigenesis and therapy resistance. These cellular mechanisms can be explained with the cancer stem cell hypothesis in combination with the clonal evolution model. Recently, CSCs in some type of tumors were proposed to be protected from therapy by multiple intrinsic and extrinsic mechanisms. Identification of predictive biomarkers and characterization of those molecular mechanisms by which HNSCC CSCs can survive treatment may ultimately lead to more efficient cancer therapy. In order to unravel and verify novel biomarker for such HNSCC CSC it is indispensable to elucidate the evolution of radioresistant CSC and to track down those tumor cells that harbor the potential to relapse. Up to date, it is unknown whether relapse and radioresistance of tumor cells is due to selection of already resistant cell clones while non- resistant CSCs are killed or due to an induction of radioresistant CSCs out of non-CSCs, or both scenarios play a role in HNSCC radioresistance. Material and Methods In order to track down the inductive or selective nature of the evolution of radioresistant CSC populations we have generated a constitutively color-coded model system based on established tumor cell lines (HNSCC: