123 research outputs found

    Prediction of Proapoptotic Anticancer Therapeutic Response Based on Visualization of Death Ligand-Receptor Interaction and Specific Marker of Cellular Proliferation

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    Emerging targeted therapeutics hold great promise for the treatment of human cancer. However there are still challenges for selecting patients that most likely will benefit from targeted drugs. One of the major limitations of classical imaging methods is the significant delay to provide quantifiable and objective evidence of response to cancer therapy. Molecular imaging may be useful in targeted drug development by assessing the target expression and drug-target interaction, and predicting therapeutic response in both preclinical and clinical settings. The apoptosis pathway triggered by the Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) receptors is a potential target for therapeutic intervention. TRAIL and its proapoptotic receptor agonistic monoclonal antibodies are being developed as targeted therapeutics in the treatment of human cancer. It is our hypothesis that visualization of proapoptotic receptors and binding of their agonists to proapoptotic receptors can noninvasively predict proapoptotic response if the pathway is intact. Hence the objective of this work is to develop efficient multimodality molecular imaging methods to predict proapoptotic anticancer therapy response before or at the very early stage of treatment. Towards this goal, we have labeled proapoptotic receptor agonists (PARAs) with near-infrared (NIR) fluorescent dyes to image PARAs binding to their targets expressed on the cell surface in cultured cells and in human tumor xenografts grown subcutaneously in immunodeficient mice. Both in vitro and in vivo studies demonstrated that imaging PARAs binding to their targets was well correlated with proapoptotic anticancer therapeutic response when TRAIL signaling pathway was intact. To pursue a more general molecular imaging marker that can predict anticancer therapeutic response even when the signaling pathway is impaired, we explored a novel radiotracer for positron emission tomography (PET) imaging [(18)F]-3\u27-fluoro-3\u27-deoxy-L-thymidine ([(18)F]-FLT), an analogue of thymidine and a specific marker of DNA replication and cellular proliferation. Our results suggested that early changes in [(18)F]-PET may not only predict the tumor histological response to anticancer therapeutics but also determine superiority of one treatment regimen over another. In summary our proof-of-concept studies show that multimodality molecular imaging will greatly aid in accelerating anticancer drug approval process and improving survival and response rates in hard-to-treat cancer

    Actively targeted polymersomes for tumor imaging and therapy

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    Hepatocellular Carcinoma

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    Hepatocellular carcinoma (HCC), the most common form of liver cancer, has become a major global health problem and is responsible for a steadily increasing number of cancer-related deaths. Although the risk factors for HCC development are well known and great advances have been made through HBV vaccinations, direct-acting antivirals for HCV treatment, and aflatoxin eradication programs, the overall incidence and mortality rates of HCC are still rising. To tackle the burden of HCC, it is essential to understand the principle molecular and cellular processes as well as fundamental clinical challenges. This book provides an overview on several important disease aspects. Chapter 1 reviews recent studies assessing the potential cellular origins of HCC. Chapter 2 describes the newly discovered regulatory roles of the tumor microenvironment on tumor growth and progression. Chapters 3 and 4 outline the most commonly used in vitro systems and animal models of chronic liver disease and HCC in detail. Chapter 5 provides an overview of metabolic reprogramming and dysregulation of lipid metabolism as a newly recognized hallmark of HCC. Chapter 6 details the currently accepted standards and challenges for the surgical management of HCC, while Chapter 7 provides an overview of the recent developments in the field of tyrosine kinase inhibitors. Chapter 8 discusses multidrug resistance to chemotherapy and potential approaches to overcome this clinical obstacle. The book, written by experts from several countries, addresses each topic in sophisticated detail. It will be a valuable resource for clinicians and investigators who are interested in HCC

    Liver Cancer: Current and Future Trends Using Biomaterials

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    Hepatocellular carcinoma (HCC) is the fifth most common type of cancer diagnosed and the second leading cause of death worldwide. Despite advancement in current treatments for HCC, the prognosis for this cancer is still unfavorable. This comprehensive review article focuses on all the current technology that applies biomaterials to treat and study liver cancer, thus showing the versatility of biomaterials to be used as smart tools in this complex pathologic scenario. Specifically, after introducing the liver anatomy and pathology by focusing on the available treatments for HCC, this review summarizes the current biomaterial-based approaches for systemic delivery and implantable tools for locally administrating bioactive factors and provides a comprehensive discussion of the specific therapies and targeting agents to effciently deliver those factors. This review also highlights the novel application of biomaterials to study HCC, which includes hydrogels and scaffolds to tissue engineer 3D in vitro models representative of the tumor environment. Such models will serve to better understand the tumor biology and investigate new therapies for HCC. Special focus is given to innovative approaches, e.g., combined delivery therapies, and to alternative approaches—e.g., cell capture—as promising future trends in the application of biomaterials to treat HCC

    ABC TRANSPORTERS IN GLIOBLASTOMA: ANTICANCER DRUG TRANSPORT AND TRANSPORTER REGULATION AT THE BLOOD-BRAIN BARRIER

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    Glioblastoma is one of the deadliest cancers, with a median survival of only one year. Even after aggressive treatment consisting of surgical resection, radiation, and chemotherapy, most glioblastoma patients suffer from tumor recurrence within 6-9 months. One reason for treatment failure of anticancer drugs is the blood-brain barrier that protects the brain by impeding xenobiotic uptake from the blood. To this end, efflux transporters at the human blood-brain barrier, such as P-glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2), prevent many compounds, including anticancer drugs, from entering the brain. Thus far, approaches to deliver anticancer drugs across the blood-brain barrier have been unsuccessful in clinical trials. Therefore, novel therapeutic strategies are needed to overcome the blood-brain barrier for improved glioblastoma treatment. Here, I address this need in 3 independent aims: Elucidate the involvement and cooperation of ABC transporters in anticancer drug transport at the blood-brain barrier Establish and characterize human glioblastoma models Evaluate the impact of dual PI3K/Akt inhibition on brain uptake of anticancer drugs Aim 1: While Abcb1/Abcg2 inhibition improved survival in mouse glioblastoma models, clinical trials had to be terminated due to a lack of efficacy, sparking a discussion that other ABC transporters might be involved in this process. To discern how multiple ABC transporters cooperate in restricting anticancer drug uptake at the blood-brain barrier, I evaluated the effect of several efflux transporters at the blood-brain barrier on the brain level of anticancer drugs using transporter inhibitors or knockout mice. The results from this study suggest that Abcc4 works in concert with Abcb1/Abcg2 in restricting brain access of the tested anticancer drugs in mice. Further experiments are necessary to confirm this cooperation at the human blood-brain barrier. In part, these findings might provide one possible explanation why therapeutic strategies that solely focus on ABCB1/ABCG2 failed to improve treatment outcomes for glioblastoma patients. Aim 2: Successful treatment of glioblastoma requires reliable preclinical animal models to evaluate novel approaches and assess their potential therapeutic benefit. While many different glioblastoma models exist, most are not well characterized and only recapitulate a subset of glioblastoma characteristics. Here, I describe and compare two human glioblastoma models, U87-luc2 and U251-FLuc. While both models behave similarly in vitro, they have different in vivo tumor characteristics, such as invasiveness and blood-brain barrier disruption. Together, the two glioblastoma models recapitulate the tumor characteristics of a majority of patients. Aim 3: Direct transporter inhibition is unsuccessful in improving glioblastoma patient survival due to the low efficacy of inhibitors and adverse effects associated with combination treatment. However, efflux transporter regulation could open a “window-in-time” to allow anticancer drug uptake into the brain. Here, I tested a novel molecular switch approach to overcome Abcb1/Abcg2-mediated efflux at the blood-brain barrier. My data indicate that PI3K/Akt could serve as a molecular switch to transiently turn off Abcb1/Abcg2 at the blood-brain barrier and increase brain levels of anticancer drugs

    Targeting the tumor blood vessels: VEGFR2 as a Biomarker and Therapeutic Target in Non Small Cell Lung Cancer

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    Blood vessels transport oxygen and nutrients within the body. However, blood vessels also nourish cancer. Numerous evidences indicate uniformly towards the fact that tumors cannot grow without access to and recruitment of blood vessels, a process widely known as tumor angiogenesis. It has been well described that endothelial cell migration and proliferation is primarily regulated by VEGF-­‐A binding to its receptor VEGFR2. However molecular mechanisms that control the shift in angiogenic switch in Non Small Cell Lung Cancer remain poorly understood till date. In this PhD thesis we have identified a novel autocrine feed-­‐forward loop active in the tumor where tumor-­‐ cell autonomous VEGF:VEGFR2 feed forward loop triggers signal amplification substantially amplifying the pro-­‐angiogenic signal required for establishing fully angiogenic tumors in lung cancer. In 20% of lung cancer patients this feed forward loop was active as the level of VEGF: VEGFR2 binding in tumor cells and directly correlated with tumor angiogenesis. Disruption of this feed forward loop using inhibitors against VEGFR2 or knockdown was sufficient to prevent tumor growth in vivo. Furthermore, inhibition of tumor cell VEGFR2 induced feedback activation of the IRS/MAPK signalling pathway switching the tumor cells from an angiogenic to a proliferative phenotype. Combined pharmacological inhibition of VEGFR2 with ZD6474 and MEK with PD0325901 resulted in dramatic tumor shrinkage. We thereby propose that high expression of tumor VEGF:VEGFR2 can serve as a predictive biomarker for therapeutic efficacy of dual VEGFR2/MEK inhibition in the patients with NSCLC. Our next project was to investigate the role of VEGFR2 in the tumor microenvironment using cancer cells, which do not have a high expression of VEGFR2. In most cancers, tumor vasculature is leaky, disorganized with a chaotic morphology resulting in a hostile tumor microenvironment characterized by increased hypoxia and high interstitial fluid pressure. These abnormal vessels interfere with effective delivery of drugs and supports tumor progression and resistance to treatment. The traditional concept of using anti-­‐ angiogenic therapy to eradicate tumors by starving them from oxygen and nutrient supply by destroying existing vessels has not seen much success. One reason for this failure can be attributed to the vessel-­‐leakiness hindering homogeneous drug delivery within the tumor. Alternative strong evidences are emerging that transient application of anti-­‐angiogenic agents can normalize the aberrant tumor vasculature and that cytotoxic therapy given during this normalization window might have the best outcome. Yet there remains a lack of clarity about how to optimize scheduling such drug combinations. In this PhD thesis, we observed that short-­‐term treatment with the VEGFR / PDGFR inhibitor PTK787 or VEGFR2 inhibitor ZD6474 initiated a transient window of improved blood flow using [15O] H2O Positron Emission Tomography (PET) in a preclinical mouse model of Non Small Cell Lung Cancer. This improvement was associated with reduced vessel leakiness and enhanced pericyte coverage. Initiation of cytotoxic treatment with erlotinib during this normalization window resulted in improved treatment efficacy. Additionally intermittent PTK787 treatment also facilitated long-­‐term tumor regression. Concisely, our findings offer strong evidence that short-­‐term anti-­‐angiogenic therapy can promote transient vessel normalization that can improve the delivery and efficacy of a targeted cytotoxic drug. In summary, VEGFR2 expressed on tumor cells plays a pivotal role in driving tumor angiogenesis and the same receptor expressed in the tumor microenvironment is relevant for normalization of tumor vasculature. Hence VEGFR2 can serve as an effective therapeutic target, which may lead to eradication of tumors or survival advantage in advanced NSCLC patients in the clinic

    Multimodality Imaging of Tumour Pathophysiology and Response to Pharmacological Intervention

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    This thesis describes the need for imaging the tumour pathophysiological microenvironment in order to understand response to treatment. Specifically looking at tumour vascularisation in in vivo murine xenograft models of disease, response to treatment with vascular disruption is assessed via photoacoustic tomography (PAT) and magnetic resonance imaging (MRI). Photoacoustic imaging is a novel imaging modality based on the detection of ultrasound waves created by the absorption of nano-second pulsed laser energy within tissue chromophores. It has the spectral specificity of optical techniques whilst also achieving the high resolution of ultrasound. Haemoglobin is the main chromophore found in biological tissue and this modality is therefore ideally suited to imaging tumour vascularisation. Using a Fabry-Perot interferometer this thesis demonstrates for the first time the feasibility of using PAT for re-clinical research and the characterisation of typical tumour vascular features in a non-invasive non-ionising manner. Response to different concentrations of a vascular disrupting drug is then demonstrated, with novel insights in to how tumours recover from vascular damage observed. MRI of response to vascular disruption is also presented. As MRI is widely used in the clinic it can serve as a translational tool of novel imaging biomarkers, and serves to further understand the differences in response of pathologically vascularised of tumours. This thesis looks at markers associated with disruption of haemodynamics, using apparent diffusion (ADC) to elucidate onset of necrosis, increase in haemoglobin concentration (R2*) as indication of impaired flow, and arterial spin labelling (ASL) as a marker of tumour blood perfusion. This is shown in both subcutaneous and clinically relevant liver metastasis models. Taken as whole, the results from this thesis indicate that whilst understanding the response of the tumour vasculature to pharmacological intervention is complex, novel imaging techniques can provide invaluable translational information on the pathophysiology of tumours

    Development of Human Liver Extracellular Matrix Hydrogel for Three Dimensional Cell Culture and Cell Transplantation

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    Introduction: It is increasingly evident that the currently available in vivo and in vitro methodologies for disease modelling are sub-optimal in recapitulating the complexity of human pathophysiology, as confirmed by the high failure rate of drug candidates due to lack of efficacy and safety. Moreover, hepatocyte transplantation has been tested as an alternative to liver transplantation for the treatment of liver diseases, but its applicability is hampered by the limited source of hepatocytes and poor hepatocyte engraftment. Aims: to develop human liver ECM hydrogels as novel in vitro platform for target identification/drug screening and for cell transplantation. Methods: Human livers unsuitable for transplantation were decellularized. The resulting ECM scaffold was then lyophilized and the resultant liver ECM powder was solubilised and mixed with three different biomaterials such as agarose, inert bio-ink or a synthetic thermo-responsive copolymer for hydrogel development. Samples were bioengineered with human hepatic cell lines (HepG2, LX2 or SNU-449), stem cells (IPSCs) or human primary hepatocytes. Validation of the hepatocellular carcinoma (HCC) model was investigated through treatment of SNU-449 samples with Sorafenib and TGF-β1. Furthermore, HepG2 bioengineered hydrogels were implanted for 3 weeks in immune-deficient mice. Samples were analysed by histology, immunofluorescence, immunohistochemistry, viability assays, gene expression and metabolic activity. Results: Bioengineered human liver ECM-based hydrogels with human liver cells showed an increase in cell survival, engraftment, proliferation and functionality compared to agarose, inert bio-ink or synthetic thermo-responsive copolymer. Viability assays of SNU-499 cells, upon Sorafenib treatment, revealed differences between 2D and 3D modelling in HCC. Implanted HepG2 ECM-hydrogels, retrieved from mice, showed that cells were still alive and engrafted. In vitro, ECM hydrogels combined with synthetic thermo-responsive copolymer showed the highest cell viability, better reproducibility, required less ECM volume and a smaller number of cells compared to ECM hydrogels combined with agarose or inert bio-ink. Conclusion: This study describes the development and the technical validation of human liver ECM hydrogels for in vitro and in vivo applications

    Estabelecimento e caracterização de modelos resistentes e matastaticos de osteossarcoma humano in vitro e in vivo

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    Doutoramento em BiologiaOsteossarcoma é uma doença rara, sendo o tipo mais comum de tumor maligno do osso. O pico de incidência ocorre durante a adolescência e desenvolve-se principalmente nos ossos longos. Os tratamentos atuais incluem quimioterapia antes e após a cirurgia e a ressecção cirúrgica de todos os locais envolvidos (tumor primário e metástases quando presente). As metástases, principalmente nos pulmões, são um grande problema no diagnóstico (20-30% dos pacientes) e durante a história natural do osteossarcoma (cerca de 30% de recaída), afetam uma percentagem considerável de pacientes e são considerados os maiores problemas desta doença. Biologicamente, os osteossarcomas são um dos tumores mais complexos observados nas crianças, no que diz respeito à heterogeneidade, anomalias moleculares e cromossómicas e ao seu microambiente específico. A resistência aos agentes quimioterápicos utilizados no tratamento do osteossarcoma também é um fator prognóstico de alto risco de recaída, independentemente da quimioterapia utilizada. É urgente compreender os mecanismos relacionados a esses fenómenos e desenvolver novos quimioterápicos para superar esses problemas e aumentar a taxa de sobrevivência do paciente. O desenvolvimento de novos fármacos requer múltiplos modelos pré-clínicos adequados para mimetizar a complexidade genómica do osteossarcoma que se desenvolve-se num microambiente ósseo e metastático nos pulmões, apesar dos tratamentos quimioterápicos habituais. Nesta tese, foram desenvolvidos e caracterizados diferentes modelos pré-clínicos clinicamente relevantes in vitro e in vivo, incluindo modelos resistentes bioluminescentes, de modo a melhor compreender esta doença e alguns dos mecanismos de resistência relacionados. Desenvolvemos, em primeiro lugar, dois modelos ortotópicos xenotransplantados derivados de linhas celulares (CDX) bioluminescentes (Luc/mKate2), capazes de desenvolver metástases espontaneamente. As células bioluminescentes foram injetadas ortotopicamente, em diferentes contextos: imune (estirpes de ratinhos de laboratório - nude e NSG) e ósseo (intratibial e paratibial com ativação do periósteo). O sistema IVIS SpectrumCT, combinando tomografia computadorizada longitudinal (TC) e bioluminescência, foi utilizado para acompanhar o crescimento primário do tumor e a disseminação metastática em tempo real. O contexto imune murino, o contexto genético dos dois modelos CDX e o contexto ósseo (intratibial ou paratibial) influenciaram o enxerto tumoral, o crescimento primário do tumor e o comportamento agressivo local (osteocondensação e osteólise), bem como a disseminação metastática para os pulmões, ossos e baço (uma localização incomum em seres humanos). Observou-se também que a estirpe de ratinhos NSG e a injeção intratibial apresentam melhores características para o desenvolvimento de modelos que a injeção paratibial ou a estirpe de ratinhos nude. Seguidamente, desenvolvemos modelos resistentes bioluminescentes in vitro, aos principais medicamentos utilizados no osteossarcoma, nomeadamente metotrexato (5modelos) e doxorrubicina (1modelo), por exposição contínua a esses medicamentos. Realizando o mesmo procedimento, não foi obtida resistência à mafosfamida. Investigamos os mecanismos da resistência adquirida relacionados com estas drogas e observamos comportamentos diferenciais in vitro e in vivo (com modelos CDX ortotópicos bioluminescentes) das linhas resistentes e respetivas linhas parentais. Um mecanismo de resistência na linha celular resistente à doxorrubicina foi observado, nomeadamente a indução da proteína PgP. Mostramos diferentes mecanismos de resistência adquirida ao metotrexato de acordo com o backgroud genético das linhas celulares, que afetam a expressão génica e provocam alterações no número de cópias ao nível dos cromossomas. Foram observados diferentes comportamentos dos modelos resistentes bioluminescentes ortotópicos (CDX) in vivo em comparação com as respetivas linhas parentais. Finalmente, utilizando amostras de osteossarcoma humano provenientes de biópsias de pacientes em recidiva após a quimioterapia habitual, foram desenvolvidos modelos resistentes xenotransplantados derivados do paciente (PDX), quer subcutaneamente quer ortotopicamente (no osso). A caracterização desses modelos está em curso, em particular a comparação das características moleculares destes (sequenciamento completo do exoma e sequenciamento do ARN) com as do tumor do paciente na recaída e do mesmo no diagnóstico. Todos esses modelos desenvolvidos em diferentes contextos in vitro e in vivo trazem informações complementares para outros tipos de modelos de osteossarcoma já existentes. Estes modelos são necessários para obter mais informações sobre os diferentes processos que envolvem o desenvolvimento inicial, a progressão e a sensibilidade/resistência ao tratamento no osteossarcoma. Permitem ajudar ainda a avaliação de novos quimioterápicos, de modo a encontrar soluções para a atual falta de terapias eficientes no osteossarcoma.Osteosarcoma is a rare disease and the most common type of malignant bone tumor. The peak incidence occurs during the adolescence and the disease develops mainly in long bones. Current treatments include chemotherapy before and after surgery and surgical resection of all the involved sites (primary tumor and metastasis when present). Metastases mainly in the lungs are a major challenge at diagnosis (20-30% of the patients) and during the natural history of osteosarcoma (around 30% of relapse, most being metastatic), affect a considerable percentage of patients with osteosarcoma, being considered the biggest problem of this disease. Biologically, osteosarcomas are one of the most complex tumours in children in regard to tumour heterogeneity, molecular and chromosomal abnormalities, and their specific microenvironment. Resistance to the chemotherapeutic agents used in osteosarcoma is also a prognostic factor of high risk of relapse, whatever the chemotherapy used. It is urgent to understand the mechanisms related with these phenomena and develop new drugs in order to overcome these challenges and increase patient survival. New drug development requires suitable multiple pre-clinical models to better mimic the genomic complexity of osteosarcoma which develops in a bone microenvironment and in a metastatic setting in the lungs, despite usual chemotherapeutic treatments. In this thesis, we developed and characterised different and clinically relevant in vitro and in vivo preclinical models, including bioluminescent resistant models in order to understand better this disease and some of the resistant mechanism related. First, two bioluminescent (Luc/mKate2) cell line derived xenograft (CDX) models were developed in an orthotopic bone setting able to spontaneously metastasize. Bioluminescent cells were injected orthotopically, in different immune (nude and NSG mouse strains) and bone (intratibial and paratibial with periosteum activation) contexts. IVIS SpectrumCT system, combining longitudinal computed tomography (CT) and bioluminescence, was used to follow primary tumor growth and metastatic spread in real-time. The murine immune context, the genetic background of the two CDX-models, and the bone context (intratibial or paratibial) influenced tumor engraftment, primary tumor growth and local aggressive behavior (osteocondensation and osteolysis) as well as metastatic spread to lungs, bone, and spleen (an unusual localization in humans). It was also observed that intratibial injection in NSG mice showed better characteristics for model development than paratibial injection or nude mice recipient. We further developed in vitro bioluminescent models that were resistant to the main drugs used in osteosarcoma, methotrexate (5 models) and doxorubicin (one model), by continuous exposure to these drugs. With the same technique no resistance was obtained for mafosfamide. We explored the mechanism of the acquired resistance to these drugs and observed the differential in vitro and in vivo behaviors (with CDX bioluminescent orthotopic models) of the resistant lines and their parental counterpart. A multidrug phenomenon by PgP induction was observed in the doxorubicin resistant cells. We show different mechanisms of acquired resistance to methotrexate according to the genetic background of the cell lines affecting either gene expression and copy number abnormalities. Different in vivo behavior of the resistant bioluminescent orthotopic CDX models compared to their parental counterparts were observed. Finally, using human biopsy samples of osteosarcoma relapsing after usual anti-osteosarcoma chemotherapy were developed resistant patient-derived xenograft (PDX) models, either in subcutaneous as in orthotopic bone setting. The characterization of these models are still ongoing, in particular the comparison of their molecular characteristics, i.e. using whole exome and RNA sequencing, in comparison with the patient tumor at relapse and with the same patient tumor at diagnosis. All these multiple models developed in different in vitro and in vivo contexts bring complementary information to other types of existing osteosarcoma models. They are needed to get more insight into the different processes involving osteosarcoma initiation, progression and in particular treatment sensitivity/resistance. They will further help drug testing to find solution to the current lack of efficient new drugs in osteosarcoma
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