Ralph Graeser

Spheroid-based engineering of a human vasculature in mice.

The complexity of the angiogenic cascade limits cellular approaches to studying angiogenic endothelial cells (ECs). In turn, in vivo assays do not allow the analysis of the distinct cellular behavior of ECs during angiogenesis. Here we show that ECs can be grafted as spheroids into a matrix to give rise to a complex three-dimensional network of human neovessels in mice. The grafted vasculature matures and is connected to the mouse circulation. The assay is highly versatile and facilitates numerous applications including studies of the effects of different cytokines on angiogenesis. Modifications make it possible to study human lymphangiogenic processes in vivo. EC spheroids can also be coimplanted with other cell types for tissue engineering purposes.

EphB4 Promotes Site-Specific Metastatic Tumor Cell Dissemination by Interacting with Endothelial Cell–Expressed EphrinB2

The tyrosine kinase receptor EphB4 interacts with its ephrinB2 ligand to act as a bidirectional signaling system that mediates adhesion, migration, and guidance by controlling attractive and repulsive activities. Recent findings have shown that hematopoietic cells expressing EphB4 exert adhesive functions towards endothelial cells expressing ephrinB2. We therefore hypothesized that EphB4/ephrinB2 interactions may be involved in the preferential adhesion of EphB4-expressing tumor cells to ephrinB2-expressing endothelial cells. Screening of a panel of human tumor cell lines identified EphB4 expression in nearly all analyzed tumor cell lines. Human A375 melanoma cells engineered to express either full-length EphB4 or truncated EphB4 variants which lack the cytoplasmic catalytic domain (ΔC-EphB4) adhered preferentially to ephrinB2-expressing endothelial cells. Force spectroscopy by atomic force microscopy confirmed, on the single cell level, the rapid and direct adhesive interaction between EphB4 and ephrinB2. Tumor cell trafficking experiments in vivo using sensitive luciferase detection techniques revealed significantly more EphB4-expressing A375 cells but not ΔC-EphB4–expressing or mock-transduced control cells in the lungs, the liver, and the kidneys. Correspondingly, ephrinB2 expression was detected in the microvessels of these organs. The specificity of the EphB4-mediated tumor homing phenotype was validated by blocking the EphB4/ephrinB2 interaction with soluble EphB4-Fc. Taken together, these experiments identify adhesive EphB4/ephrinB2 interactions between tumor cells and endothelial cells as a mechanism for the site-specific metastatic dissemination of tumor cells. Mol Cancer Res; 8(10); 1297–309. ©2010 AACR.

Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono- and stromal co-cultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented.

The Potency of Refined Mouse Models: Implications for Clinical Trials

Many promising anti-cancer compounds fail in the clinical phase despite extensive testing in animal models. Reasons for this failure are varied, ranging from inadequate mouse models to dosing schemes that cannot be applied in humans. The apparent shortcomings of exisiting pre-clinical studies suggests a need for improved mouse models. Fur- thermore, the development of an increasing number of targeted drugs requires that biomarkers and/or the in vivo imaging of the tumor or its environment need to be established, ideally before the clinical candidate is selected. In this review, we focus on recent progress in mouse models, emphasizing imaging techniques, biomarker development, the tumor microen- vironment and orthotopic metastatic tumor models. We discuss the application of the latter models to drug testing, making comparisons with classical subcutaneous xenograft models, and also models utilizing transgenic animals. Imaging modali- ties used to detect orthotopically implanted cells in the mouse, e.g. fluorescence or luciferase, which allow relatively high throughput measurements, will be compared to PET, CT, or MRI, which are closer to the clinical application. Examples for a combination of the latter imaging systems with luciferase or fluorescence in the mouse to develop or improve clini- cal imaging will be presented. Finally, the value of biomarkers in mouse models and strategies to identify novel target- specific biomarkers in mouse models will be discussed.

Abstract C97: Beyond intratumoural steroidogenesis: abiraterone resistance mediated by AR variants and glucocorticoid receptor signalling

Introduction Castration resistant prostate cancer (CRPC) remains dependent on androgen receptor (AR) signalling, driven by adrenal precursors and potentially de novo steroid synthesis in other organ tissues including prostate. Abiraterone, an inhibitor of the steroidogenic enzyme CYP17A1 and the AR has been demonstrated to prolong survival of CRPC patients. In this study, we created a co-culture model using human prostate and adrenal tumours to study abiraterone resistance. Materials and Methods Human androgen-dependent PC (VCaP) and CPRC clones were cultured with substrates for de novo androgen synthesis or with adrenal androgens, or cultured with human adrenal cells (H295R) and treated with either the CYP17A1 inhibitor abiraterone or the antiandrogen MDV3100. Male mice bearing VCaP tumours and human adrenal H295R xenografts were castrated and treated with placebo or abiraterone. Tumour response was assessed by tumour growth, PSA release, steroid quantitation by (LC/MS-MS), immunohistochemistry and mRNA expression analysis of steroidogenic enzymes and nuclear receptors. Results In vitro, physiological levels of adrenal androgen precursors DHEA and androstenedione induced cell growth in parental and CRPC VCaP sub clones, whereas precursor steroids pregnenolone and progesterone for de novo synthesis did not. In a co-culture model, abiraterone blocked H295R-induced growth of VCaP cells. Likewise, in vivo, H295R tumours stimulated castration-resistant VCaP growth. This stimulative effect was inhibited by abiraterone, reducing - but not fully blocking - growth and PSA production. In the absence of H295R tissue, VCaP xenografts grew slow but became castration resistant nonetheless. In contrast to the observed effects on VCaP growing in castrate animals bearing H295R tumours, abiraterone was unable to inhibit the slow VCaP growth and low PSA production in castrate mice without H295R xenografts. LC/MS-MS analysis of plasma and tumour tissue could not confirm increased de novo production of androgens. Castrate and abiraterone-resistant VCaP tumours were characterised by increased levels of AR, AR variants and glucocorticoid receptor (GR) expression, resulting in equal AR target gene expression levels. Conclusions Our data indicate that AR ligand dependent regrowth of CRPC is predominantly supported via adrenal steroid production. Abiraterone resistant disease of VCaP relies on AR overexpression, expression of ligand independent AR variants and GR signalling. Citation Format: Jan Matthijs Moll, Johannes Hofland, Wilma Teubel, Corrina M.A. de Ridder, Anne E. Taylor, Ralph Graeser, Wiebke Arlt, Guido W. Jenster, Wytske M. van Weerden. Beyond intratumoural steroidogenesis: abiraterone resistance mediated by AR variants and glucocorticoid receptor signalling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C97.

Abstract 5322: Bioluminescence, fluorescence, and biomarker mediated monitoring of an orthotopic model of colon cancer treated with VX-680

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnWith 10% of all newly diagnosed cases per year, colon cancer ranks within the top 5 cancer types within the USA, and, with a similar figure of people succumbing to the disease, is a major cause of tumor-related deaths. Primary tumors may often be removed via surgery, but metastatic spread poses considerable difficulties and chemotherapeutic treatments are rarely curative. Novel drugs, and predictive pre-clinical model systems to better mimic human disease are therefore urgently needed.nnAlthough subcutaneously implanted tumor cells have been used for pre-clinical drug testing for decades now, the predictive value of these models has recently become the subject of controversy. Orthotopically implanted cells reflect the human disease much better in that organo-typical tumor stromal interactions are enabled, and tumor cells spread to other sites with distribution patterns reflecting the human disease.nnOrthotopical implantation requires sensitive imaging modalities, similar to the patient situation. Bioluminescence imaging (BLI) is a well established modality for pre-clinical models, but is not translatable to the clinic. Recently, fluorescence molecular tomography (FTM) imaging has been shown to offer good sensitivity and tissue penetration, and sensitive probes – that may be used in the clinic – have been developed.nnA pre-clinical experiment was performed to compare the growth behaviour of orthotopically implanted, luciferase marked HCT116 wild-type and p53 KO human colon cancer cells and test their sensitivity towards the aurora-inhibitor VX-680. Growth of the tumors, and treatment effects were followed in vivo using BLI, and a final imaging analysis with integrisense, using FMT. The in vivo imaging data were validated with necropsy data, including sensitive luciferase assays to detect metastases in organs.nnIn vivo BLI suggested that p53 KO tumors grew much more aggressive, giving rise to larger, more fragmented signals than their wild-type counterparts. Imaging using integrisense confirmed the presence of much larger tumor masses in the p53 KO than in the wild-type group. VX-680 slightly reduced the BLI signals from both groups. Necropsy results confirmed the tumor size differences between wild-type and p53 KO tumors, also the VX-680 effect against wild-type tumors (−50%, SD 70%). However, the drug appeared ineffective against p53 KO tumors (+120%, SD 70%). Luciferase assays from potential target organs detected an increased metastasation in p53 KO tumor bearing animals compared to those with wild-type tumors. Interestingly, metastasation of the p53 KO tumors to the spleen was sensitive to VX-680 treatment (−90%, SD 20%). The effect of the drug could also be demonstrated in skin biopsies taken from the mice as a surrogate tissue.nnThis study represents a ‘high content’, partially translatable mouse model applicable also for the testing of other targeted therapies.nnCitation 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 5322. doi:10.1158/1538-7445.AM2011-5322

Abstract 3502: Activity of combinations of anticancer agents in pancreatic carcinoma cell lines

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnLong term and successful treatment of pancreatic cancer remains a considerable challenge, and there is an urgent need to develop new therapeutic strategies that improve the response rate and overall survival of this disease that responds poorly to monotherapy with anticancer agents such as gemcitabine or 5-fluorouracil. Instead of combining drugs at their respective maximum tolerated doses, a number of recent studies have shown that the “more is better” paradigm may not be the optimal strategy for maximizing the clinical outcome for combination therapy and rather synergistic effects can be achieved if the drugs are dosed at a defined ratio. Such an approach is currently being investigated clinically with liposomal formulations which contain irinotecan and floxuridine at a 1:1 ratio or cytarabine and daunorubicin at a 5:1 ratio. We therefore tested a number of combinations of chemotherapeutic agents using defined ratios ranging from 10:1 to 1:10 for their efficacy against a luciferase transduced pancreatic cell line, MIAPaCa2 ELN, using a 72 h Luciferase proliferation assay. Using this assay, a combination of camptothecin with gemcitabine showed synergy at a ratio of 1:5 over a cell survival range of 10-80% with combination indexes (CIs) between 0.36 and 0.71.nnIn addition, a synergy was found at a 1:10 ratio for methotrexate with doxorubicin with CI values from 0.36 to 0.32 over a 20-90% cell survival range. A third synergistic combination was observed between cytarabine and gemcitabine at a 1:5 ratio with CIs from 0.16 to 0.21 over a 20-90% cell survival range. None of the other combinations tested, including campthotecin with methotrexate, camptotecin with doxorubicin, or cytarabine with daunorubicin showed synergistic effects at ratios from 5:1 to 1:5.nnThese in vitro results show that the ratio of the respective drug combination is a decisive factor for inducing additive, synergistic or even antagonistic effects.nnWe are currently testing the most promising combinations in additional pancreatic carcinoma cell lines in order to elucidate whether the synergistic effects of these combinations and specific ratios apply to pancreatic cancer cells in general.nnCitation 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 3502. doi:10.1158/1538-7445.AM2011-3502

Abstract 1726: Overcoming multi-drug resistance with Paclitaxel-loaded polymer nanoparticles

Multi-drug resistance against chemotherapeutic agents is a major problem in the treatment of cancer. It is primarily mediated by over-expression of P-glycoprotein (P-gp) encoded by the MDR-1 gene by the tumor cells. Most of the promising anticancer drugs such as doxorubicin, docetaxel, paclitaxel, and mitoxantrone are P-gp substrates. Thus, various ways for overcoming the multi-drug resistance, e.g. combination of an anticancer drug and a P-gp inhibitor or the formulation of nanoparticles in conjunction with anticancer drugs as well as the P-gp inhibitor, have been studied. Anticancer drug-loaded nanoparticles, such as paclitaxel-loaded polymer nanoparticles, are excellent candidates to overcome multi-drug resistance due to their enhanced therapeutic efficacy induced by increased cellular accumulation and sustained intracellular drug delivery. In this work, we developed an ampholytic copolymer that can encapsulate paclitaxel, and the resulting nanoparticles were found to have good water-solubility. In addition, in contrast to native paclitaxel, the paclitaxel-loaded nanoparticles were able to reverse multi-drug resistance in MCF7/ADR and MT-3/ADR cell lines, resulting in drug sensitivities comparable to the parental MCF7 and MT3 cell lines. P-gp expression was confirmed in both ADR cell lines by Western blotting, and its functionality was verified using a rhodamine accumulation and efflux assay. The nanoparticles were taken up by endocytosis in both cell lines and their enhanced therapeutic efficacy is probably due to the different mode of entry into the cell that may avoid the P-gp efflux pump. As a consequence higher concentrations of paclitaxel reach the nucleus and induce cell killing. 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 1726. doi:10.1158/1538-7445.AM2011-1726