Residual γH2AX foci after ex vivo irradiation of patient samples with known tumour-type specific differences in radio-responsiveness

Abstract

known differences in radiation response, i.e. radiosensitive types such as seminomas and resistant types as chondrosarcomas. We hypothesized that the number of residual cH2AX foci corresponds to the expected tumour radiation sensitivity. In addition, in order to enhance clinical practicability for future studies the data were used for simulations to test the robustness of the method when omitting dose levels. Materials and methods Collection and cultivation of patient-derived tumour specimensThe study has been approved by the Ethics Committee of the Medical Faculty of the University of Tübingen (426/2013BO1). All the patients included in the study were untreated prior to surgical procedure. During collection and cultivation tumour tissues were kept in Dulbecco's MEM culture medium supplemented with 2% HEPES, 1% Na-pyruvate, 1% non-essential amino acids, 1% penicillin streptomycin (all Biochrom AG, Berlin, Germany) and 10% FBS (PAN Biotech GmbH, Aidenbach, Germany). Fresh tumour material was retrieved from surgical specimens and placed in 50 ml Falcon tubes (Becton Dickinson International, Heidelberg, Germany) containing 15 ml culture medium. Tumour specimens were transported to the laboratory and subsequently cut manually with the use of surgical forceps (BD027R, B. BRAUN, Aesculap, Tuttlingen, Germany) and scalpels (FEATHER Safety Razor Co., Ltd., Osaka, Japan Number 23) into approximately 2-3 mm slices before being placed in petri dishes (3.5 cm diameter) coated with a 1.5% agarose layer (A9539, Sigma-Aldrich, Germany) containing 3 ml culture media. During all incubation times the petri-dishes were kept in 95% humidified atmosphere at 37 _C and 5% CO2. For the purpose of the study tumour material from a total of 25 patient tumours with 10 different tumour histologies was collected (Table 1; n = 3 seminomas, n = 1 chondrosarcoma, n = 3 urinary bladder carcinomas (Ca), n = 3 colorectal Ca (2 colon Ca + 1 rectum), n = 3 breast Ca, n = 1 hepatocellular Ca, n = 3 renal cell Ca, n = 3 prostate Ca, n = 4 glioblastoma multiforme (GBM) and n = 2 cervix Ca). In one GBM patient, the specimen was removed from the analysis because in the 0 Gy sample there was no viable tumour part (based on morphological criteria and BrdU positivity) and no estimation of the background foci could be performed. Histology and selection of malignant cells for analysis were confirmed by an experienced pathologist (M.S.).Experimental design for evaluation of cH2AX foci in ex vivo irradiated patient-derived tumour specimensThe experimental design has been previously described [45]. Briefly, after initial cultivation for 24 h, the tumour specimens were irradiated typically with 0, 2, 4, 6, 8 Gy single doses (200 kV, RS225 research system Gulmay Medical LTD, Surrey, England; 15 mA; 0.5 mm Cu; dose rate _1 Gy/min). In two tumours, additional doses were delivered to the seminoma sample (#1) doses of 3 and 5 Gy and GBM (#1) 10

Patient-Specific Screening Using High-Grade Glioma Explants to Determine Potential Radiosensitization by a TGF-β Small Molecule Inhibitor

“…This heterogeneous response to RIKI was in agreement with our analysis of TCGA data and immunostaining of FFPE biospecimens for p-Smad2. We conclude that the explant method can distinguish tumors with differential sensitivity to TGF-β inhibition in the context of radioresponse [57] and establishes a proof-of-concept paradigm for patient-specific screening of candidate therapies. Furthermore, the predicted benefit of TGF-β inhibition in most HGGs provides further motivation for their current clinical testing but also underscores the utility of implementing ex vivo assays for personalized efficacy studies.…”

Dynamic In Vivo Profiling of DNA Damage and Repair after Radiotherapy Using Canine Patients as a Model

“…Prior work—including in vivo mouse assays with various cell lines of head and neck carcinoma, as well as in ex vivo assays using different tumor types from patients—has documented radio-responsiveness in sensitive and resistant tumor types to be represented by residual γH2AX-foci [1,2,5]. After antineoplastic treatment, the initiation of DNA repair by the two major responsible pathways is triggered by the phosphorylation of the histone protein H2AX, which leads to formation of γH2AX-molecules at the site of the DSB [4,25,26].…”

“…Monitoring the formation of damage in a cell after radiotherapy (RT) and the evaluation of DNA repair markers may be a means to provide information on intrinsic radiosensitivity and radio-responsiveness [1,2,3]. Ionizing radiation used as a cancer treatment relies on the formation of direct DNA damage or on creating sufficient cellular damage leading to double strand breaks (DSBs), which in turn trigger the activation of cellular death pathways.…”

“…Upon damage, the cells activate a DNA-damage response, consisting of various pathways which will sense the extent of damage and induce an effector pathway either leading to cell death, cell cycle arrest, or DNA damage repair [4]. DNA damage detection from patient samples has been performed for various tumor groups irradiated ex vivo, resulting in robust correlations with radio-responsiveness, as known from clinical behavior [2,5]. Furthermore, monitoring the responses towards damage formation and repair from individual tumors may offer a legitimate chance to monitor cancer treatment and even a possibility to predict clinical response to treatment [6,7].…”

“…While the time-courses in responses to different DNA damaging agents have been well characterized in cultured tumor cells in vitro, little is known about the time-course of DNA damage response in tumor and normal tissues sampled from individual tumor patients [2,5,6,8,9,10]. Specifically, actual time-courses from patients undergoing radiation therapy are lacking.…”