γH2AX assay in ex vivo irradiated tumour specimens: A novel method to determine tumour radiation sensitivity in patient-derived material

Menegakis, Apostolos; Neubeck, Cläre von; Yaromina, Ala; Hering, Sandra; Hennenlotter, Joerg; Scharpf, Marcus; Noell, Susan; Krause, Mechthild; Zips, Daniel; Baumann, Michaël

doi:10.1016/j.radonc.2015.03.026

Cited by 39 publications

(49 citation statements)

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“…The 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).…”

Section: Collection and Cultivation Of Patient-derived Tumour Specimensmentioning

confidence: 99%

“…The staining procedure has been previously described [43][44][45]. In brief, three consecutive 3 lm specimen cross-sections from the paraffin-embedded tumour material were stained for (a) haematoxylin and eosin staining (H&E), (b) BrdU (Clone Bu20a, Dako Deutschland GmbH, Hamburg, Germany) and pimonidazole (Natural Pharmacia International, Belmont, MA, USA; immunohistochemistry, IMH) with ARKTM Kit (animal research kit; Dako Deutschland GmbH, Hamburg, Germany) and (c) 40,6-diamidino-2-phenylindole (DAPI) and cH2AX at Ser139 (Merck Millipore, Upstate, clone JBW301, Darmstadt, Germany; immunofluorescence IMF) with TSATM Kit (T20912, containing goat anti-mouse IgG and tyramide labelled with Alexa 488, Life Technologies GmbH, Molecular probes, Invitrogen, Darmstadt, Germany).…”

Section: Tissue Staining Imaging and Ch2ax Foci Analysismentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

“…Taking together, the cH2AX assay is simple, sensitive and straightforward method to quantify DSBs in cells and tissues and therefore promising for translation into clinical trials. For clinical application, we have developed and pre-clinically validated a novel method using residual cH2AX foci in ex vivo irradiated tumour specimens [40,[43][44][45]. In the present study we analyse data using the optimizedprotocol in 25 patient-derived surgical specimens (including 7 previously published [45]) covering a spectrum of 10 tumour types with Gy (both previously published [45]).…”

mentioning

confidence: 99%

“…For clinical application, we have developed and pre-clinically validated a novel method using residual cH2AX foci in ex vivo irradiated tumour specimens [40,[43][44][45]. In the present study we analyse data using the optimizedprotocol in 25 patient-derived surgical specimens (including 7 previously published [45]) covering a spectrum of 10 tumour types with Gy (both previously published [45]). Prior to irradiation, medium containing 20 lmol pimonidazole (hypoxia marker, Natural Pharmacia International, Belmont, MA, USA) and 10 lmol BrdU (proliferation marker, SERVA electrophoresis, Heidelberg, Germany) was added to the specimens for 4 h (20 h post start of cultivation).…”

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confidence: 99%

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

Menegakis¹,

Colle²,

Yaromina³

et al. 2015

Radiotherapy and Oncology

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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

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Section: Collection and Cultivation Of Patient-derived Tumour Specimensmentioning

confidence: 99%

Section: Tissue Staining Imaging and Ch2ax Foci Analysismentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Menegakis¹,

Colle²,

Yaromina³

et al. 2015

Radiotherapy and Oncology

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Preclinical patient‐derived modeling of castration‐resistant prostate cancer facilitates individualized assessment of homologous recombination repair deficient disease

et al. 2023

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The use of mutation analysis of homologous recombination repair (HRR) genes to estimate PARP-inhibition response may miss a larger proportion of responding patients. Here, we provide preclinical models for castrationresistant prostate cancer (CRPC) that can be used to functionally predict HRR defects. In vitro, CRPC LNCaP sublines revealed an HRR defect and enhanced sensitivity to olaparib and cisplatin due to impaired RAD51 expression and recruitment. Ex vivo-induced castration-resistant tumor slice cultures or tumor slice cultures derived directly from CRPC patients showed increased olaparib-or cisplatin-associated enhancement of residual radiation-induced cH2AX/53BP1 foci. We established patient-derived tumor organoids (PDOs) from CRPC patients. These PDOs are morphologically similar to their primary tumors and genetically clustered with prostate cancer but not with normal prostate or other tumor entities. Using these PDOs, we functionally confirmed the enhanced sensitivity of CRPC patients to olaparib and cisplatin. Moreover, olaparib but not cisplatin significantly decreased the migration rate in CRPC cells. Collectively, we present robust patient-derived preclinical models for CRPC that recapitulate the features of their primary tumors and enable individualized drug

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A functional ex vivo assay to detect PARP1‐EJ repair and radiosensitization by PARP‐inhibitor in prostate cancer

Köcher

Lange

Nordquist

et al. 2019

Intl Journal of Cancer

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Here, we present a functional assay to detect the repair switch to the alternative PARP1‐dependent end joining (PARP1‐EJ) pathway and the associated susceptibility to PARPi‐mediated radiosensitization in freshly collected tumor samples from prostate cancer (PCa) patients, thereby facilitating the selection of patients who should benefit from combined PARPi plus radiotherapy (RT) treatment. Our optimized ex‐vivo approach sustains tumor slices for up to 15 days under culture conditions that maintain proliferation and oxygenation rates, as measured by EdU incorporation and pimonidazole staining, respectively. We present a robust system to analyze DSB repair using, for the first time in an ex vivo tumor slice setting, two DSB‐markers simultaneously i.e. γH2AX and 53BP1. A computer‐based processing method (i) controls variations in DNA content and slicing on the number of repair foci and (ii) measures the PARPi‐mediated enhancement ratio on DSB foci numbers to ensure inter‐patient‐comparability. We validated this approach using a PC3 xenograft model with its previously described repair switch to PARP1‐EJ. More importantly, we show that approximately 30% of the analyzed tumor tissue samples collected from PCa patients display a switch to PARP1‐EJ, as indicated by the enhanced number of residual γH2AX/53BP1 foci exclusively after PARPi+RT. Furthermore, normal prostatic tissues show no repair switch to PARP1‐EJ, indicating that this repair switch and its associated radiosensitizing effect is tumor‐specific. Collectively, we present here a predictive assay for the switch to PARP1‐EJ that enables individualization of anti‐cancer treatment using a combination of RT and radiosensitizing anticancer agents such as PARPi in PCa.

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γH2AX assay in ex vivo irradiated tumour specimens: A novel method to determine tumour radiation sensitivity in patient-derived material

Cited by 39 publications

References 44 publications

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

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

Preclinical patient‐derived modeling of castration‐resistant prostate cancer facilitates individualized assessment of homologous recombination repair deficient disease

A functional ex vivo assay to detect PARP1‐EJ repair and radiosensitization by PARP‐inhibitor in prostate cancer

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