Johan Ides

Chemoradiation interactions under reduced oxygen conditions: Cellular characteristics of an in vitro model

Hypoxic tumour regions often contain viable cells that are more resistant to chemotherapy and/or radiotherapy, making it of key importance to analyse new combination treatments under both normoxic and hypoxic conditions. In this study, the impact of moderate hypoxia and anoxia on cellular characteristics was investigated in isogenic A549 cells differing in p53 status. VEGF expression, doubling time, cell cycle distribution, induction of apoptosis and p53 protein expression were evaluated. Radiation survival curves yielded an oxygen enhancement ratio of 1.16-1.67. In conclusion, an in vitro hypoxia model that will be highly useful to analyse chemoradiation interactions is presented.

COUNTING CLONOGENIC ASSAYS FROM NORMOXIC AND ANOXIC IRRADIATION EXPERIMENTS MANUALLY OR BY USING DENSITOMETRIC SOFTWARE

The clonogenic assay is the method of choice to determine cell reproductive death after in vitro irradiation treatment. Traditionally, colony quantification has been performed by manual counting, a very laborious, time-consuming and rather subjective task. In this study, we compared manual counting by two skilled investigators with automated counting using the freely available ClonoCounter program. Five human tumour cell lines were irradiated under normoxia (21% O(2)) or anoxia (<0.1% O(2)), after 24 h or 6 h anoxic preincubation. Colonies were quantified manually or using the ClonoCounter software. A positive correlation between the absolute number of colonies counted manually and automatically was shown. Though there was a general trend of underpredicting the absolute number of cell colonies when counting automatically, survival curves were very similar, and in none of the cell lines were significant differences in radiobiological parameters such as mean inactivation dose, surviving fraction at 2 Gy and oxygen enhancement ratio detected. Our results suggest that the ClonoCounter provides sufficient reliability to be implemented for counting human tumour colonies in in vitro irradiation experiments. In contrast to several previously reported computer-aided colony-counting methods, it is a freely available program, requiring only minimal instrument costs.

The role of apoptotic cell death in the radiosensitising effect of gemcitabine

Background:The aim of this study was to evaluate the radiosensitising effect of gemcitabine, in terms of cell-cycle progression, induction of apoptosis, and to investigate the molecular events regulating apoptosis.Methods:Tumour cells were treated with gemcitabine, radiation, or the combination. 0–72 h after treatment, cells were collected for cell-cycle analysis and apoptosis determination. Caspase 8 and 9, Bid and tBid expression were determined by western blot. The mitochondrial membrane potential was determined using flow cytometry. An RT2 Profiler PCR Array for human apoptotic genes was performed after the combination or TRAIL treatment.Results:Gemcitabine and radiation resulted in an early S-phase block immediately after treatment, after which the cells moved synchronously through the cell cycle. When cell-cycle distribution returned to pre-treatment levels, an increased induction of apoptosis was observed with activation of caspase 8 and 9 and a reduction of the mitochondrial membrane potential. Gene expression after treatment with radiosensitising conditions was comparable with expression after the TRAIL treatment.Conclusion:A role for the cell-cycle perturbations and the induction of apoptosis could be attributed to the radiosensitising effect of gemcitabine. Apoptosis induction was comparable with the apoptotic pathway observed after the TRAIL treatment, that is the involvement of the extrinsic apoptosis pathway.

Retention of the In Vitro Radiosensitizing Potential of Gemcitabine Under Anoxic Conditions, in p53 Wild-Type and p53-Deficient Non-Small-Cell Lung Carcinoma Cells

PURPOSE Whereas radiosensitization by gemcitabine is well studied under normal oxygen conditions, little is known about its radiosensitizing potential under reduced oxygen conditions. Therefore, the present study evaluated the impact of anoxia on gemcitabine-mediated radiosensitization. METHODS AND MATERIALS The clonogenic assay was performed in three isogenic A549 cell lines differing in p53 status (24 h, 0-15 nM gemcitabine, 0-8 Gy irradiation, normoxia vs. anoxia). Using radiosensitizing conditions, cells were collected for cell cycle analysis and apoptosis detection. RESULTS Whereas wild-type p53 A549-LXSN cells were more sensitive to radiation than p53-deficient A549-E6 cells, both cell lines showed similar radiosensitization by gemcitabine under normoxia and anoxia. Independent of p53 functionality, gemcitabine was able to overcome anoxia-induced G(0/1) arrest and established an (early) S phase block in normoxic and anoxic cells. The percentage early and late apoptotic/necrotic cells increased with the gemcitabine/radiation combination, with a significant difference between A549-LXSN and A549-E6. CONCLUSIONS This study is the first to show that gemcitabine retains its radiosensitizing potential under low oxygen conditions. Although radiosensitization was observed in both p53 wild-type and p53-deficient cells, p53 status might influence induction of apoptosis after gemcitabine/radiation treatment, whereas no effect on cell cycle progression was noticed.

Synthesis and in vivo preclinical evaluation of an 18F labeled uPA inhibitor as a potential PET imaging agent

INTRODUCTION The urokinase plasminogen activator (uPA) system is a proteolytic cascade involved in tumor invasion and metastasis. uPA and its inhibitor PAI-1 are described as biomarkers for breast cancer with the highest level of evidence. The present study describes the synthesis and first in vivo application of an activity based uPA PET probe. METHODS Based on the design of a small irreversible and selective uPA inhibitor we developed an (18)F-labeled activity based probe for uPA imaging. Human uPA expressing MDA-MB-231-luc2-GFP cells were inoculated in the mammary fat pads of nude mice and treated with the probe once tumors reached a volume of 150mm(3). Scans were performed at 0.25, 0.75, 1.5, 4 and 6h post injection. To evaluate tumor uptake in vivo and ex vivo data were gathered. Biodistribution data of the organs and tissues of interest were collected at all time points. Due to a relatively low tumor uptake, probe stability was further evaluated. RESULTS The uPA targeting PET tracer was produced in high purity and with good specific radioactivity. In vivo PET data showed a maximum tumor uptake of 2,51±0,32 %ID/g at 4h p.i. A significant correlation between in vivo and ex vivo tumor uptake calculation was found (R=0.75; p<0.01). Due to a high blood signal at all time points, probe stability was further examined revealing high plasma protein binding and low plasma stability. CONCLUSIONS In vivo and ex vivo results clearly demonstrate that uPA expressing tumors can be detected with non-invasive PET imaging. Stability tests suggest that further optimization is needed to provide a better tumor-to-background contrast.

Preclinical evaluation of [111In]MICA-401, an activity-based probe for SPECT imaging of in vivo uPA activity

Urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 are key players in cancer invasion and metastasis. Both uPA and PAI-1 have been described as prognostic biomarkers; however, non-invasive methods measuring uPA activity are lacking. We developed an indium-111 (111 In)-labelled activity-based probe to image uPA activity in vivo by single photon emission computed tomography (SPECT). A DOTA-conjugated uPA inhibitor was synthesized and radiolabelled with 111 In ([111 In]MICA-401), together with its inactive, hydrolysed form ([111 In]MICA-402). A biodistribution study was performed in mice (healthy and tumour-bearing), and tumour-targeting properties were evaluated in two different cancer xenografts (MDA-MB-231 and HT29) with respectively high and low levels of uPA expression in vitro, with either the active or hydrolysed radiotracer. MicroSPECT was performed 95 h post injection followed by ex vivo biodistribution. Tumour uptake was correlated with human and murine uPA expression determined by ELISA and immunohistochemistry (IHC). Biodistribution data with the hydrolysed probe [111 In]MICA-402 showed almost complete clearance 95 h post injection. The ex vivo biodistribution and SPECT data with [111 In]MICA-401 demonstrated similar tumour uptakes in the two models: ex vivo 5.68 ± 1.41%ID/g versus 5.43 ± 1.29%ID/g and in vivo 4.33 ± 0.80 versus 4.86 ± 1.18 for MDA-MB-231 and HT-29 respectively. Human uPA ELISA and IHC showed significantly higher uPA expression in the MDA-MB-231 tumours, while mouse uPA staining revealed similar staining intensities of the two tumours. Our data demonstrate non-invasive imaging of uPA activity in vivo, although the moderate tumour uptake and hence potential clinical translation of the radiotracer warrants further investigation. Copyright

Abstract 5250: Optimization of an orthotopic mouse model for in vivo fluorescent uPA imaging in breast cancer

The urokinase plasminogen activator (uPA) system is one of the serine protease systems involved in extracellular matrix degradation, which will facilitate tumor cell invasion and intravasation. Recently, the Medicinal Chemistry group of the University of Antwerp (UAMC) developed some very selective covalently binding uPA inhibitors. Besides its function as possible therapeutic target, uPA and its endogenous inhibitor PAI-1 are described as prognostic biomarkers for breast cancer with the highest level of evidence. Commercial ELISA and PCR kits are available for the screening of uPA in breast tumors. These ex vivo assays measure or predict the total uPA content (active and inactive). We present here a more advanced technique, which will be able to monitor the actual enzymatic uPA activity in a non-invasive manner. The technology is based on the modification of the UAMC inhibitors towards activity based probes containing a fluorescent label. Currently, we are evaluating these probes as imaging tools for uPA monitoring. The developed imaging probes will be profoundly tested in an orthotopic model of human MDA-MB-231 and MCF7 breast cancer cells since these cell lines show a high and low uPA expression respectively. We will use these models to study the potential beneficial characteristics of activity based probes in the field of bio-imaging applications targeted to uPA as a validated biomarker. The current protocol, which is still under evaluation, can be described as followed: Cell suspensions (2.106 cells/100μl) of each cell line were implanted in the mammary fat pads at both flanks of 7 mice, alternatively with and without Matrigel. Tumor size was monitored using bioluminescent imaging (BLI) and caliper measurements. When the tumor volume reached 200 - 400mm3, the rhodamine labeled uPA-inhibitor (uPA-probe) was administered (0.3 mg / kg i.v.). Fluorescent images were taken at 1, 2, 4, 6, 8, 12, 24 and 48 hours after injection. At 48 hours, the mice were sacrificed and the tumor, liver and lung were excised for ex vivo bioluminescent or fluorescent imaging and stored for histological examination. In this presentation, we describe the evaluation of the model and the first promising results. Considering the role of uPA in migration and invasion, the uPA-probe will also be tested in the future for the detection of micrometastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5250. doi:1538-7445.AM2012-5250

Abstract 3910: Targeting urokinase plasminogen activator: evaluation of activity-based imaging probes in an orthotopic breast cancer model.

The urokinase plasminogen activator (uPA) system is a proteolytic cascade involved in tumor invasion and metastasis. uPA and its inhibitor PAI-1 are described as biomarkers for breast cancer with the highest level of evidence. For the screening of uPA in breast tumors with commercially available ELISA kits that determine the total uPA content (active and inactive), fresh tumor tissue is required. Molecular imaging with activity-based uPA probes might overcome this limitation. Therefore we have developed some highly selective, non-peptidic and specific uPA activity-based probes based on the irreversible uPA inhibitor UAMC-00150. In this study we describe the evaluation of a fluorescent Cy5 labeled and a 18F labeled PET probe in the high uPA expressing MDA-MB-231 breast cancer mouse model to make the step towards clinical translation of this imaging biomarker. Orthotopic MDA-MB-231 tumors were established in nude mice and subjected to uPA imaging at a tumor volume of 150mm3. For the fluorescent imaging, three test groups were considered (n=5): mice from the first and the second experimental group received a single dose of equimolar amounts (0.23 μMol/kg) Cy5-uPA-probe or its inactivated hydrolyzed variant, respectively. The third group received an injection with unlabeled uPA inhibitor, 30 min before administration of Cy5-uPA probe, in a concentration that was 70-fold higher than the probe concentration. Mice were imaged at different time points for 48 h post injection (p.i.) and were subsequently sacrificed for ex vivo imaging and histology. PET imaging (n=15) was performed after i.v. injection of the radiotracer with a maximum of 0,2ml and/or 0,5mCi per injection, resulting in a range of 0,15-0,5mCi/injection. Animals were scanned at 15, 45, 90, 240 and 360 minutes p.i. At each time point, 3 mice were sacrificed to generate ex vivo biodistribution data. The Cy5-uPA-probe demonstrated good tumor-targeting properties in the high uPA-expressing breast tumor model, with fluorescent intensities reaching a maximum at 24 h post-probe administration in mice treated with Cy5-uPA-probe alone. The groups treated with the inactivated probe or the unlabeled inhibitor showed a significant decrease in the fluorescent tumor signal (p>0,016), which was also confirmed on histology. PET imaging with the 18F-uPA-probe showed a peak tumor uptake of 2.76 ± 0.37 %ID/g at 4 h p.i. Further in vitro PPB and ex vivo HPLC data revealed a high affinity of the tracer for blood proteins and a high metabolisation rate potentially explaining the rather moderate to low tumor uptake of the current PET uPa biomarker. In conclusion, fluorescent imaging data clearly indicate that the Cy5-uPA-probe enables non-invasive NIR-imaging of uPA expression in tumors in vivo. The first PET experiments indicate translational capabilities, for which we are now improving the pharmacokinetics of this and future uPa radiotracers. Citation Format: Johan Ides, Christel Vangestel, Jonas Messagie, Dieter Verzele, David Thomae, Sofie Thys, An Wouters, John-Paul Bogers, Sigrid Stroobants, Jurgen Joossens, Pieter Van der Veken, Marc Peeters, Filip Lardon, Steven Staelens, Koen Augustyns. Targeting urokinase plasminogen activator: evaluation of activity-based imaging probes in an orthotopic breast cancer model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3910. doi:10.1158/1538-7445.AM2013-3910

Abstract 2661: The role of HIF-1 protein on the radiosensitizing effect of difluorodeoxyuridine, the main metabolite of gemcitabine, under normoxic and anoxic conditions

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL It is known that gemcitabine and difluorodeoxyuridine (dFdU) have radiosensitizing properties. It has become evident that solid tumors often contain hypoxic regions and that this is one of the causes of resistance or decreased sensitivity to cancer therapy. In hypoxic conditions, the transcription factor, hypoxia-inducible factor-1 (HIF-1), is upregulated. HIF-1 is responsible for the cellular and adaptive responses of tumors to survive in hypoxic conditions. Recently, it has been demonstrated that gemcitabine retains its radiosensitizing potential under hypoxia. In the present study, the radiosensitizing potential of dFdU under anoxic conditions is investigated as well as the role of HIF-1 protein. In order to explore the role of HIF-1, human tumor cell lines included in this study were MDA-MB-231 (breast adenocarcinoma cell line, wt HIF-1), MDA-MB-231 DN-HIF (transfected with dominant-negative HIF-1α) and MDA-MB-231 EV (empty vector transfected control). Anoxic conditions (<0.1% O2,) were achieved in a Bactron IV anaerobic chamber. To analyze the radiosensitizing effect of dFdU, the clonogenic assay was performed. Cells were exposed to normoxic or anoxic conditions and were simultaneously treated with 0, 2 or 4 microM dFdU for 24h immediately before irradiation (0-8 Gy, room temperature, linear accelerator). Immediately following radiation, anoxic cells were reoxygenated and cells were washed with drug-free medium. Using radiosensitizing conditions, cells were collected for cell cycle analysis by flow cytometry. A clear concentration-dependent radiosensitizing effect of dFdU was observed under both normoxic and anoxic conditions (dose enhancement factor (DEF) under normoxia: 1.74-3.38; under anoxia: 2.08-3.81). Combination index (CI) analysis showed a synergistic to additive interaction between dFdU and radiation under normoxia (CI 0.651 ± 0.175), yet an additive interaction under anoxia (CI 0.784 ± 0.120). Statistical analysis using two-way ANOVA revealed that cell survival was significantly influenced by the concentration of dFdU, the dose of radiation, the oxygen tension and the cell line. Post hoc analysis indicated no significant difference between the three cell lines for DEF, ID50, mean inactivation dose and survival fraction at 2 Gy, suggesting that the functionality of HIF-1 protein has no impact on the radiosensitizing effect of dFdU. Considering the cell cycle distribution after treatment with dFdU, 24h treatment with 2 or 4 microM dFdU established a significant S-phase block in both normoxic and anoxic MDA-MB-231 wt and DN-HIF cells. In conclusion, this study showed that dFdU has a clear concentration-dependent radiosensitizing effect in MDA-MB-231 breast cancer cells under normoxic as well as anoxic conditions. No major role for functional HIF-1 protein in radiosensitization by dFdU could be demonstrated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2661. doi:10.1158/1538-7445.AM2011-2661

Abstract 2671: Study of the role of functional HIF-1 in the in vitro radiosensitizing effect of gemcitabine in normoxic and anoxic breast adenocarcinoma cells

Introduction: It has been well documented that the efficacy of both chemotherapy and radiotherapy is directly linked with an adequate oxygen tension, and that hypoxic regions in solid tumors often contain viable cells that are intrinsically more resistant to anticancer treatment. Recently, it has been demonstrated that the cytotoxic drug gemcitabine retains its radiosensitizing potential under low oxygen conditions in lung adenocarcinoma cells. As the transcription factor ‘hypoxia inducible factor 1’ (HIF-1) plays a crucial role in regulating the adaptive responses of tumor cells to survive under hypoxic conditions, the present study investigated the potential influence of HIF-1 status on radiosensitization by gemcitabine. Materials & methods: Three isogenic human breast adenocarcinoma cell lines with different HIF-1 status were included in this study: MDA-MB-231 (breast adenocarcinoma cell line, wt HIF-1), MDA-MB-231 DN-HIF (transfected with dominant-negative HIF-1alpha, abrogating HIF-1 function) and MDA-MB-231 EV (empty vector transfected control, functional HIF-1). Anoxic conditions ( Results: A clear concentration-dependent radiosensitizing effect of gemcitabine was observed under both normoxic and anoxic conditions (dose enhancement factor (DEF) under normoxia: 1.02-1.70; DEF under anoxia: 1.11-2.04). Combination index (CI) analysis showed an additive interaction between gemcitabine and radiation under normal oxygen conditions (CI 0.902-1.148), and a synergistic interaction under reduced oxygen conditions (CI 0.455-0.889). Statistical analysis using two-way ANOVA revealed no significant differences in radiobiological parameters (DEF, ID10, ID50, mean inactivation dose, surviving fraction at 2 Gy, oxygen enhancement ratio) between MDA-MB-231 EV and MDA-MB-231 DN-HIF cells, suggesting no influence of HIF-1 functionality on radiosensitivity. Considering the cell cycle distribution after treatment with gemcitabine, 24h treatment with 4 or 8 nM gemcitabine established a significant S-phase block in both normoxic and anoxic MDA-MB-231 wt and DN-HIF cells. Conclusion: This study showed that the retained radiosensitizing effect of gemcitabine under anoxic conditions was not tumor tissue specific and could be observed in MDA-MB-231 breast cancer cells. No major role for functional HIF-1 protein in radiosensitization by gemcitabine could be demonstrated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2671. doi:10.1158/1538-7445.AM2011-2671