Monika A. Jarzabek

Interrogating open issues in cancer precision medicine with patient-derived xenografts

Patient-derived xenografts (PDXs) have emerged as an important platform to elucidate new treatments and biomarkers in oncology. PDX models are used to address clinically relevant questions, including the contribution of tumour heterogeneity to therapeutic responsiveness, the patterns of cancer evolutionary dynamics during tumour progression and under drug pressure, and the mechanisms of resistance to treatment. The ability of PDX models to predict clinical outcomes is being improved through mouse humanization strategies and the implementation of co-clinical trials, within which patients and PDXs reciprocally inform therapeutic decisions. This Opinion article discusses aspects of PDX modelling that are relevant to these questions and highlights the merits of shared PDX resources to advance cancer medicine from the perspective of EurOPDX, an international initiative devoted to PDX-based research.

In Vivo Bioluminescence Imaging Validation of a Human Biopsy-Derived Orthotopic Mouse Model of Glioblastoma Multiforme

Glioblastoma multiforme (GBM), the most aggressive brain malignancy, is characterized by extensive cellular proliferation, angiogenesis, and single-cell infiltration into the brain. We have previously shown that a xenograft model based on serial xenotransplantation of human biopsy spheroids in immunodeficient rodents maintains the genotype and phenotype of the original patient tumor. The present work further extends this model for optical assessment of tumor engraftment and growth using bioluminescence imaging (BLI). A method for successful lentiviral transduction of the firefly luciferase gene into multicellular spheroids was developed and implemented to generate optically active patient tumor cells. Luciferase-expressing spheroids were injected into the brains of immunodeficient mice. BLI photon counts and tumor volumes from magnetic resonance imaging (MRI) were correlated. Luciferase-expressing tumors recapitulated the histopathologic hallmarks of human GBMs and showed proliferation rates and microvessel density counts similar to those of wild-type xenografts. Moreover, we detected widespread invasion of luciferase-positive tumor cells in the mouse brains. Herein we describe a novel optically active model of GBM that closely mimics human pathology with respect to invasion, angiogenesis, and proliferation indices. The model may thus be routinely used for the assessment of novel anti-GBM therapeutic approaches implementing well-established and cost-effective optical imaging strategies.

Molecular imaging in the development of a novel treatment paradigm for glioblastoma (GBM): An integrated multidisciplinary commentary

Current therapeutic strategies against glioblastoma (GBM) have failed to prevent disease progression and recurrence effectively. The part played by molecular imaging (MI) in the development of novel therapies has gained increasing traction in recent years. For the first time, using expertise from an integrated multidisciplinary group of authors, herein we present a comprehensive evaluation of state-of-the-art GBM imaging and explore how advances facilitate the emergence of new treatment options. We propose a novel next-generation treatment paradigm based on the targeting of multiple hallmarks of cancer evolution that will heavily rely on MI.

Interrogation of gossypol therapy in glioblastoma implementing cell line and patient-derived tumour models

Background:Glioblastoma (GBM), being a highly vascularised and locally invasive tumour, is an attractive target for anti-angiogenic and anti-invasive therapies. The GBM/endothelial cell response to gossypol/temozolomide (TMZ) treatment was investigated with a particular aim to assess treatment effects on cancer hallmarks.Methods:Cell viability, endothelial tube formation and GBM tumour cell invasion were variously assessed following combined treatment in vitro. The U87MG-luc2 subcutaneous xenograft model was used to investigate therapeutic response in vivo. Viable tumour response to treatment was interrogated using immunohistochemistry. Combined treatment protocols were also tested in primary GBM patient-derived cultures.Results:An endothelial/GBM cell viability inhibitory effect, as well as an anti-angiogenic and anti-invasive response, to combined treatment have been demonstrated in vitro. A significantly greater anti-proliferative (P=0.020, P=0.030), anti-angiogenic (P=0.040, P<0.0001) and pro-apoptotic (P=0.0083, P=0.0149) response was observed when combined treatment was compared with single gossypol/TMZ treatment response, respectively. GBM cell line and patient-specific response to gossypol/TMZ treatment was observed.Conclusions:Our results indicate that response to a combined gossypol/TMZ treatment is related to inhibition of tumour-associated angiogenesis, invasion and proliferation and warrants further investigation as a novel targeted GBM treatment strategy.

Validation of an imageable surgical resection animal model of Glioblastoma (GBM).

BACKGROUND Glioblastoma (GBM) is the most common and malignant primary brain tumour having a median survival of just 12-18 months following standard therapy protocols. Local recurrence, post-resection and adjuvant therapy occurs in most cases. NEW METHOD U87MG-luc2-bearing GBM xenografts underwent 4.5mm craniectomy and tumour resection using microsurgical techniques. The cranial defect was repaired using a novel modified cranial window technique consisting of a circular microscope coverslip held in place with glue. RESULTS Immediate post-operative bioluminescence imaging (BLI) revealed a gross total resection rate of 75%. At censor point 4 weeks post-resection, Kaplan-Meier survival analysis revealed 100% survival in the surgical group compared to 0% in the non-surgical cohort (p=0.01). No neurological defects or infections in the surgical group were observed. GBM recurrence was reliably imaged using facile non-invasive optical bioluminescence (BLI) imaging with recurrence observed at week 4. COMPARISON WITH EXISTING METHOD(S) For the first time, we have used a novel cranial defect repair method to extend and improve intracranial surgical resection methods for application in translational GBM rodent disease models. Combining BLI and the cranial window technique described herein facilitates non-invasive serial imaging follow-up. CONCLUSION Within the current context we have developed a robust methodology for establishing a clinically relevant imageable GBM surgical resection model that appropriately mimics GBM recurrence post resection in patients.

Interrogating open issues in cancer medicine with patient-derived xenografts.

This corrects the article DOI: 10.1038/nrc.2016.140.

A Novel Positron Emission Tomography (PET) Approach to Monitor Cardiac Metabolic Pathway Remodeling in Response to Sunitinib Malate.

Sunitinib is a tyrosine kinase inhibitor approved for the treatment of multiple solid tumors. However, cardiotoxicity is of increasing concern, with a need to develop rational mechanism driven approaches for the early detection of cardiac dysfunction. We sought to interrogate changes in cardiac energy substrate usage during sunitinib treatment, hypothesising that these changes could represent a strategy for the early detection of cardiotoxicity. Balb/CJ mice or Sprague-Dawley rats were treated orally for 4 weeks with 40 or 20 mg/kg/day sunitinib. Cardiac positron emission tomography (PET) was implemented to investigate alterations in myocardial glucose and oxidative metabolism. Following treatment, blood pressure increased, and left ventricular ejection fraction decreased. Cardiac [18F]-fluorodeoxyglucose (FDG)-PET revealed increased glucose uptake after 48 hours. [11C]Acetate-PET showed decreased myocardial perfusion following treatment. Electron microscopy revealed significant lipid accumulation in the myocardium. Proteomic analyses indicated that oxidative metabolism, fatty acid β-oxidation and mitochondrial dysfunction were among the top myocardial signalling pathways perturbed. Sunitinib treatment results in an increased reliance on glycolysis, increased myocardial lipid deposition and perturbed mitochondrial function, indicative of a fundamental energy crisis resulting in compromised myocardial energy metabolism and function. Our findings suggest that a cardiac PET strategy may represent a rational approach to non-invasively monitor metabolic pathway remodeling following sunitinib treatment.

Modelling tumour cell proliferation from vascular structure using tissue decomposition into avascular elements.

Computer models allow the mechanistically detailed study of tumour proliferation and its dependency on nutrients. However, the computational study of large vascular tumours requires detailed information on the 3-dimensional vessel network and rather high computation times due to complex geometries. This study puts forward the idea of partitioning vascularised tissue into connected avascular elements that can exchange cells and nutrients between each other. Our method is able to rapidly calculate the evolution of proliferating as well as dead and quiescent cells, and hence a proliferative index, from a given amount and distribution of vascularisation of arbitrary complexity. Applying our model, we found that a heterogeneous vessel distribution provoked a higher proliferative index, suggesting increased malignancy, and increased the amount of dead cells compared to a more static tumour environment when a homogenous vessel distribution was assumed. We subsequently demonstrated that under certain amounts of vascularisation, cell proliferation may even increase when vessel density decreases, followed by a subsequent decrease of proliferation. This effect was due to a trade-off between an increase in compensatory proliferation for replacing dead cells and a decrease of cell population due to lack of oxygen supply in lowly vascularised tumours. Findings were illustrated by an ectopic colorectal cancer mouse xenograft model. Our presented approach can be in the future applied to study the effect of cytostatic, cytotoxic and anti-angiogenic chemotherapy and is ideally suited for translational systems biology, where rapid interaction between theory and experiment is essential.

Imaging Targeted Therapy Response and Resistance in Glioblastoma

Glioblastoma (GBM) is the most common and most malignant tumour of the central nervous system. Despite recent advances in understanding the biology of GBM, the disease still remains in desperate need of effective treatment options resulting in long-term improvements in overall patient survival. Molecularly targeted therapies, anti-angiogenic therapy and immunotherapy are promising avenues under investigation as future therapeutic options. Molecular imaging (MI) is an essential tool in the development of these targeted treatments, both preclinically and clinically. MI facilitates the preclinical study and interrogation of potential therapies. MI further supports non-invasive, longitudinal monitoring of therapy response and allows the study of emergence of treatment resistance via an imaging-guided therapeutic approach.

Abstract 2096: Mechanistic insights into the pathogenesis of anti-DLL4-related hepatic sinusoidal dilatation

Background: The Delta-like 4 (DLL4)-mediated NOTCH signaling pathway is an attractive therapeutic target in cancer. However, chronic blockade of DLL4 signalling has been observed to result in vascular toxicities, such as hepatic sinusoidal dilatation. As the underlying pathogenesis is unclear, the current study was undertaken to interrogate gene expression changes and potential safety biomarkers associated with anti-DLL4 related hepatic sinusoidal dilatation. Methods: Formalin-fixed paraffin-embedded (FFPE) liver sections were derived from male and female cynomolgus monkeys administered FDLL8566 (recombinant humanized anti-DLL4 F(ab’)2 antibody) by intravenous injection once weekly for 8 weeks at dose levels of 0, 5, 15 and 50 mg/kg/week (n = 3/sex/group). RNA was extracted from whole liver sections and laser capture-microdissected (LCM) hepatic regions with or without sinusoidal dilatation. mRNA expression was quantified using a high-throughput RT-qPCR approach, with 96 pre-validated, species-specific TaqMan gene expression assays. A non-parametric statistical test was used to assess differential gene expression between sinusoidal dilatation-affected and non-affected liver tissues. The Benjamini-Hochberg multiple testing comparison error-based False-Discovery-Rate (FDR) method was applied to calculate the adjusted p-values for each probe set. Results: Fourteen candidate genes were differentially expressed between sinusoidal dilatation-affected and non-affected cynomolgus monkey livers with an FDR adjusted p value Conclusions: This work highlights the involvement of the NOTCH pathway in the maintenance of hepatic sinusoidal homeostasis in the nonhuman primate (NHP). As preclinical toxicities in NHPs have translated to patients for other anti-DLL4 inhibitors, the changes in candidate genes and potential mechanism of toxicity identified in this study are likely to be relevant to humans. The phenotypic characteristics of hepatic sinusoidal dilatation associated with anti-DLL4 resemble the microscopic and molecular features observed in the liver following oxaliplatin treatment of patients (including involvement of angiogenesis), suggesting that similar molecular mechanisms of hepatic toxicity may exist between anti-DLL4 and oxaliplatin. This work was supported by an EU funded Industry Academia Pathways and Partnerships Marie Curie Award (AngioTox) Grant Number 251528. Citation Format: Monika A. Jarzabek, Rupal Desai, Yuda Zhu, Christina Z. de Zafra, Joe Beyer, Gary Cain, Rajiv Raja, Annette T. Byrne, Priti Hegde, Jacqueline M. Tarrant. Mechanistic insights into the pathogenesis of anti-DLL4-related hepatic sinusoidal dilatation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2096.