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Pubmed Article
The EnhancedIn Vivo Activity of the Combination of a MEK and a PI3K Inhibitor Correlates with [(18)F]-FLT PET in Human Colorectal Cancer Xenograft Tumour-Bearing Mice.
PLoS ONE
PUBLISHED: 01-01-2013
Combined targeting of the MAPK and PI3K signalling pathways in cancer may be necessary for optimal therapeutic activity. To support clinical studies of combination therapy, 3-deoxy-3-[(18)F]-fluorothymidine ([(18)F]-FLT) uptake measured by Positron Emission Tomography (PET) was evaluated as a non-invasive surrogate response biomarker in pre-clinical models. The in vivo anti-tumour efficacy and PK-PD properties of the MEK inhibitor PD 0325901 and the PI3K inhibitor GDC-0941, alone and in combination, were evaluated in HCT116 and HT29 human colorectal cancer xenograft tumour-bearing mice, and [(18)F]-FLT PET investigated in mice bearing HCT116 xenografts. Dual targeting of PI3K and MEK induced marked tumour growth inhibition in vivo, and enhanced anti-tumour activity was predicted by [(18)F]-FLT PET scanning after 2 days of treatment. Pharmacodynamic analyses using the combination of the PI3K inhibitor GDC-0941 and the MEK inhibitor PD 0325901 revealed that increased efficacy is associated with an enhanced inhibition of the phosphorylation of ERK1/2, S6 and 4EBP1, compared to that observed with either single agent, and maintained inhibition of AKT phosphorylation. Pharmacokinetic studies indicated that there was no marked PK interaction between the two drugs. Together these results indicate that the combination of PI3K and MEK inhibitors can result in significant efficacy, and demonstrate for the first time that [(18)F]-FLT PET can be correlated to the improved efficacy of combined PI3K and MEK inhibitor treatment.
Authors: Yingding Xu, Hongguang Liu, Edwin Chang, Han Jiang, Zhen Cheng.
Published: 11-13-2012
ABSTRACT
In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues. Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET. Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.
21 Related JoVE Articles!
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Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia
Authors: Sandra Christoph, Alisa B. Lee-Sherick, Susan Sather, Deborah DeRyckere, Douglas K. Graham.
Institutions: University of Colorado Anschutz Medical Campus, University Hospital of Essen.
Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.
Medicine, Issue 79, Leukemia, Receptor Protein-Tyrosine Kinases, Molecular Targeted Therapy, Therapeutics, novel small molecule inhibitor, receptor tyrosine kinase, leukemia
50720
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Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT)
Authors: Nehal N. Mehta, Drew A. Torigian, Joel M. Gelfand, Babak Saboury, Abass Alavi.
Institutions: University of Pennsylvania, Perelman School of Medicine, University of Pennsylvania, Perelman School of Medicine, University of Pennsylvania, Perelman School of Medicine.
Conventional non-invasive imaging modalities of atherosclerosis such as coronary artery calcium (CAC)1 and carotid intimal medial thickness (C-IMT)2 provide information about the burden of disease. However, despite multiple validation studies of CAC3-5, and C-IMT2,6, these modalities do not accurately assess plaque characteristics7,8, and the composition and inflammatory state of the plaque determine its stability and, therefore, the risk of clinical events9-13. [18F]-2-fluoro-2-deoxy-D-glucose (FDG) imaging using positron-emission tomography (PET)/computed tomography (CT) has been extensively studied in oncologic metabolism14,15. Studies using animal models and immunohistochemistry in humans show that FDG-PET/CT is exquisitely sensitive for detecting macrophage activity16, an important source of cellular inflammation in vessel walls. More recently, we17,18 and others have shown that FDG-PET/CT enables highly precise, novel measurements of inflammatory activity of activity of atherosclerotic plaques in large and medium-sized arteries9,16,19,20. FDG-PET/CT studies have many advantages over other imaging modalities: 1) high contrast resolution; 2) quantification of plaque volume and metabolic activity allowing for multi-modal atherosclerotic plaque quantification; 3) dynamic, real-time, in vivo imaging; 4) minimal operator dependence. Finally, vascular inflammation detected by FDG-PET/CT has been shown to predict cardiovascular (CV) events independent of traditional risk factors21,22 and is also highly associated with overall burden of atherosclerosis23. Plaque activity by FDG-PET/CT is modulated by known beneficial CV interventions such as short term (12 week) statin therapy24 as well as longer term therapeutic lifestyle changes (16 months)25. The current methodology for quantification of FDG uptake in atherosclerotic plaque involves measurement of the standardized uptake value (SUV) of an artery of interest and of the venous blood pool in order to calculate a target to background ratio (TBR), which is calculated by dividing the arterial SUV by the venous blood pool SUV. This method has shown to represent a stable, reproducible phenotype over time, has a high sensitivity for detection of vascular inflammation, and also has high inter-and intra-reader reliability26. Here we present our methodology for patient preparation, image acquisition, and quantification of atherosclerotic plaque activity and vascular inflammation using SUV, TBR, and a global parameter called the metabolic volumetric product (MVP). These approaches may be applied to assess vascular inflammation in various study samples of interest in a consistent fashion as we have shown in several prior publications.9,20,27,28
Medicine, Issue 63, FDG-PET/CT, atherosclerosis, vascular inflammation, quantitative radiology, imaging
3777
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Murine Bioluminescent Hepatic Tumour Model
Authors: Simon Rajendran, Slawomir Salwa, Xuefeng Gao, Sabin Tabirca, Deirdre O'Hanlon, Gerald C. O'Sullivan, Mark Tangney.
Institutions: University College Cork, University College Cork, South Infirmary Victoria University Hospital.
This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis.
JoVE Medicine, Issue 41, Cancer, Therapy, Liver, Orthotopic, Metastasis
1977
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Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model
Authors: Tamalee R. Kramp, Kevin Camphausen.
Institutions: National Cancer Institute.
Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 μl every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.
Medicine, Issue 61, Neuroscience, mouse, intracranial, orthotopic, radiation, glioblastoma
3397
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Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells
Authors: Qiao Li, Lin Lu, Huimin Tao, Carolyn Xue, Seagal Teitz-Tennenbaum, John H. Owen, Jeffrey S Moyer, Mark E.P. Prince, Alfred E. Chang, Max S. Wicha.
Institutions: University of Michigan, University of Michigan, University of Michigan.
We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c. tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFNγ and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.
Cancer Biology, Issue 83, Cancer stem cell (CSC), Dendritic cells (DC), Vaccine, Cancer immunotherapy, antitumor immunity, aldehyde dehydrogenase
50561
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Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications
Authors: Lori E. Lowes, Benjamin D. Hedley, Michael Keeney, Alison L. Allan.
Institutions: London Health Sciences Centre, Western University, London Health Sciences Centre, Lawson Health Research Institute, Western University.
The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.
Medicine, Issue 84, Metastasis, circulating tumor cells (CTCs), CellSearch system, user defined marker characterization, in vivo, preclinical mouse model, clinical research
51248
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Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation
Authors: J. Geraldo Valadez, Anuraag Sarangi, Christopher J. Lundberg, Michael K. Cooper.
Institutions: Vanderbilt University Medical Center, Vanderbilt University Medical Center, Veteran Affairs TVHS.
Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.
Medicine, Issue 83, Glioma, Malignant glioma, primary orthotopic xenograft, isocitrate dehydrogenase
50865
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Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts
Authors: Urszula M. Polanska, Ahmet Acar, Akira Orimo.
Institutions: University of Manchester, Juntendo University.
Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3. The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of α-smooth muscle actin (α-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients. Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model. In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11. We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.
Medicine, Issue 56, cancer, stromal myofibroblasts, experimentally generated carcinoma-associated fibroblasts (exp-CAFs), fibroblast, human mammary carcinomas, tumour xenografts
3201
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Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts
Authors: Wolfgang Jäger, Igor Moskalev, Claudia Janssen, Tetsutaro Hayashi, Killian M. Gust, Shannon Awrey, Peter C. Black.
Institutions: University of British Columbia.
Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse. With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period. In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.
Medicine, Issue 84, Bladder cancer, cell lines, xenograft, inoculation, ultrasound, orthotopic model
51123
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Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT
Authors: Xukui Wang, Laurie J. Minze, Zheng-Zheng Shi.
Institutions: The Methodist Hospital Research Institute, Houston, The Methodist Hospital Research Institute, Houston.
Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via β adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of β-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by β3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.
Molecular Biology, Issue 69, Neuroscience, Anatomy, Physiology, Medicine, Brown adipose tissue, mice, 18F-Fluorodeoxyglucose, micro-PET, PET, CT, CT scan, tomography, imaging
4060
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Bioluminescent Bacterial Imaging In Vivo
Authors: Chwanrow K. Baban, Michelle Cronin, Ali R. Akin, Anne O'Brien, Xuefeng Gao, Sabin Tabirca, Kevin P. Francis, Mark Tangney.
Institutions: University College Cork.
This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.
Immunology, Issue 69, Molecular Biology, Cancer Biology, Genetics, Gene Therapy, Cancer, Vector, Lux, Optical Imaging, Luciferase
4318
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Electrochemotherapy of Tumours
Authors: Gregor Sersa, Damijan Miklavcic.
Institutions: Institute of Oncology Ljubljana, University of Ljubljana.
Electrochemotherapy is a combined use of certain chemotherapeutic drugs and electric pulses applied to the treated tumour nodule. Local application of electric pulses to the tumour increases drug delivery into cells, specifically at the site of electric pulse application. Drug uptake by delivery of electric pulses is increased for only those chemotherapeutic drugs whose transport through the plasma membrane is impeded. Among many drugs that have been tested so far, bleomycin and cisplatin found their way from preclinical testing to clinical use. Clinical data collected within a number of clinical studies indicate that approximately 80% of the treated cutaneous and subcutaneous tumour nodules of different malignancies are in an objective response, from these, approximately 70% in complete response after a single application of electrochemotherapy. Usually only one treatment is needed, however, electrochemotherapy can be repeated several times every few weeks with equal effectiveness each time. The treatment results in an effective eradication of the treated nodules, with a good cosmetic effect without tissue scarring.
Medicine, Issue 22, electrochemotherapy, electroporation, cisplatin, bleomycin, malignant tumours, cutaneous lesions
1038
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Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics
Authors: Adriana D. Corben, Mohammad M. Uddin, Brooke Crawford, Mohammad Farooq, Shanu Modi, John Gerecitano, Gabriela Chiosis, Mary L. Alpaugh.
Institutions: Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center, Weill Cornell Medical College, Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center.
The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.
Cancer Biology, Issue 92, Ex vivo sectioning, Treatment response, Sensitivity/Resistance, Drug development, Patient tumors, Preclinical and Clinical
52157
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Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Authors: Robert S. McNeill, Ralf S. Schmid, Ryan E. Bash, Mark Vitucci, Kristen K. White, Andrea M. Werneke, Brian H. Constance, Byron Huff, C. Ryan Miller.
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
51763
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Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers
Authors: Rasa Ghaffarian, Silvia Muro.
Institutions: University of Maryland, University of Maryland.
Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200 nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.
Bioengineering, Issue 80, Antigens, Enzymes, Biological Therapy, bioengineering (general), Pharmaceutical Preparations, Macromolecular Substances, Therapeutics, Digestive System and Oral Physiological Phenomena, Biological Phenomena, Cell Physiological Phenomena, drug delivery systems, targeted nanocarriers, transcellular transport, epithelial cells, tight junctions, transepithelial electrical resistance, endocytosis, transcytosis, radioisotope tracing, immunostaining
50638
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Analysis of Cell Migration within a Three-dimensional Collagen Matrix
Authors: Nadine Rommerswinkel, Bernd Niggemann, Silvia Keil, Kurt S. Zänker, Thomas Dittmar.
Institutions: Witten/Herdecke University.
The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.
Bioengineering, Issue 92, cell migration, 3D collagen matrix, cell tracking
51963
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Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?
Authors: Julia Y. Ljubimova, Hui Ding, Jose Portilla-Arias, Rameshwar Patil, Pallavi R. Gangalum, Alexandra Chesnokova, Satoshi Inoue, Arthur Rekechenetskiy, Tala Nassoura, Keith L. Black, Eggehard Holler.
Institutions: Cedars-Sinai Medical Center.
Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.
Chemistry, Issue 88, Cancer treatment, personalized medicine, polymalic acid, nanodrug, biopolymer, targeting, host compatibility, biodegradability
50668
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Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET: Dopamine Movies of Cigarette Smoking
Authors: Evan D. Morris, Su Jin Kim, Jenna M. Sullivan, Shuo Wang, Marc D. Normandin, Cristian C. Constantinescu, Kelly P. Cosgrove.
Institutions: Yale University, Yale University, Yale University, Yale University, Massachusetts General Hospital, University of California, Irvine.
We describe experimental and statistical steps for creating dopamine movies of the brain from dynamic PET data. The movies represent minute-to-minute fluctuations of dopamine induced by smoking a cigarette. The smoker is imaged during a natural smoking experience while other possible confounding effects (such as head motion, expectation, novelty, or aversion to smoking repeatedly) are minimized. We present the details of our unique analysis. Conventional methods for PET analysis estimate time-invariant kinetic model parameters which cannot capture short-term fluctuations in neurotransmitter release. Our analysis - yielding a dopamine movie - is based on our work with kinetic models and other decomposition techniques that allow for time-varying parameters 1-7. This aspect of the analysis - temporal-variation - is key to our work. Because our model is also linear in parameters, it is practical, computationally, to apply at the voxel level. The analysis technique is comprised of five main steps: pre-processing, modeling, statistical comparison, masking and visualization. Preprocessing is applied to the PET data with a unique 'HYPR' spatial filter 8 that reduces spatial noise but preserves critical temporal information. Modeling identifies the time-varying function that best describes the dopamine effect on 11C-raclopride uptake. The statistical step compares the fit of our (lp-ntPET) model 7 to a conventional model 9. Masking restricts treatment to those voxels best described by the new model. Visualization maps the dopamine function at each voxel to a color scale and produces a dopamine movie. Interim results and sample dopamine movies of cigarette smoking are presented.
Behavior, Issue 78, Neuroscience, Neurobiology, Molecular Biology, Biomedical Engineering, Medicine, Anatomy, Physiology, Image Processing, Computer-Assisted, Receptors, Dopamine, Dopamine, Functional Neuroimaging, Binding, Competitive, mathematical modeling (systems analysis), Neurotransmission, transient, dopamine release, PET, modeling, linear, time-invariant, smoking, F-test, ventral-striatum, clinical techniques
50358
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Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease
Authors: Vivian P. Chou, Novie Ko, Theodore R. Holman, Amy B. Manning-Boğ.
Institutions: SRI International, University of California-Santa Cruz.
Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer's disease, but its effects in Parkinson's disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone. To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder. Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD. The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates. This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.
Medicine, Issue 83, MPTP, dopamine, Iba1, TH, GFAP, lipoxygenase, transgenic, gene-environment interactions, mouse, Parkinson's disease, neurodegeneration, neuroinflammation
50960
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Identification of Disease-related Spatial Covariance Patterns using Neuroimaging Data
Authors: Phoebe Spetsieris, Yilong Ma, Shichun Peng, Ji Hyun Ko, Vijay Dhawan, Chris C. Tang, David Eidelberg.
Institutions: The Feinstein Institute for Medical Research.
The scaled subprofile model (SSM)1-4 is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain image data while rejecting lesser components (Figure 1). Applied directly to voxel-by-voxel covariance data of steady-state multimodality images, an entire group image set can be reduced to a few significant linearly independent covariance patterns and corresponding subject scores. Each pattern, termed a group invariant subprofile (GIS), is an orthogonal principal component that represents a spatially distributed network of functionally interrelated brain regions. Large global mean scalar effects that can obscure smaller network-specific contributions are removed by the inherent logarithmic conversion and mean centering of the data2,5,6. Subjects express each of these patterns to a variable degree represented by a simple scalar score that can correlate with independent clinical or psychometric descriptors7,8. Using logistic regression analysis of subject scores (i.e. pattern expression values), linear coefficients can be derived to combine multiple principal components into single disease-related spatial covariance patterns, i.e. composite networks with improved discrimination of patients from healthy control subjects5,6. Cross-validation within the derivation set can be performed using bootstrap resampling techniques9. Forward validation is easily confirmed by direct score evaluation of the derived patterns in prospective datasets10. Once validated, disease-related patterns can be used to score individual patients with respect to a fixed reference sample, often the set of healthy subjects that was used (with the disease group) in the original pattern derivation11. These standardized values can in turn be used to assist in differential diagnosis12,13 and to assess disease progression and treatment effects at the network level7,14-16. We present an example of the application of this methodology to FDG PET data of Parkinson's Disease patients and normal controls using our in-house software to derive a characteristic covariance pattern biomarker of disease.
Medicine, Issue 76, Neurobiology, Neuroscience, Anatomy, Physiology, Molecular Biology, Basal Ganglia Diseases, Parkinsonian Disorders, Parkinson Disease, Movement Disorders, Neurodegenerative Diseases, PCA, SSM, PET, imaging biomarkers, functional brain imaging, multivariate spatial covariance analysis, global normalization, differential diagnosis, PD, brain, imaging, clinical techniques
50319
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Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice
Authors: Sharif U. Ahmed, Murtuza Zair, Kui Chen, Matthew Iu, Feng He, Oyedele Adeyi, Sean P. Cleary, Anand Ghanekar.
Institutions: University Health Network, University Health Network, University Health Network.
In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.
Medicine, Issue 79, Liver Neoplasms, Hepatectomy, animal models, hepatocellular carcinoma, xenograft, cancer, liver, subcutaneous, intrahepatic, orthotopic, mouse, human, immunodeficient
50544
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