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Role of RUNX3 in suppressing metastasis and angiogenesis of human prostate cancer.
PUBLISHED: 01-01-2014
RUNX3 (runt-related transcription factor-3) has been reported to suppress tumor tumorigenesis and metastasis in different human cancers. In this study, we used tissue microarray (TMA) to determine the significance of RUNX3 in prostate cancer progession. Our results showed ectopic expression of RUNX3 in prostate cancer tissues when compared with tumor adjacent normal prostate tissues, and reduced RUNX3 staining was significantly correlated with TNM stage. Moreover, we demonstrated that RUNX3 overexpression inhibited prostate cancer cell migration and invasion resulting from the elevated upregulation of tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), which subsequently inhibited metalloproteinase-2 (MMP-2) expression and activity in vitro. Knock down of RUNX3 expression broke up the balance of TIMP-2/MMP-2, whereas silence of TIMP-2 resulted in the inhibition of MMP-2 expression in prostate cells. We also showed that restoration of RUNX3 decreased vascular endothelial growth factor (VEGF) secretion and suppressed endothelial cell growth and tube formation. Strikingly, RUNX3 was demonstrated to inhibit tumor metastasis and angiogenesis in vivo. Altogether, our results support the tumor suppressive role of RUNX3 in human prostate cancer, and provide insights into development of targeted therapy for this disease.
Authors: Janet Pavese, Irene M. Ogden, Raymond C. Bergan.
Published: 09-18-2013
Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.
23 Related JoVE Articles!
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MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)
Authors: Aida Gordanpour, Robert K. Nam, Linda Sugar, Stephanie Bacopulos, Arun Seth.
Institutions: University of Toronto, Sunnybrook Health Sciences Centre, Toronto, Canada, Sunnybrook Health Sciences Centre, Toronto, Canada, Sunnybrook Research Institute.
MicroRNAs (miRNAs) are single-stranded, 18–24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets. Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8. Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.
Cancer Biology, Issue 63, Medicine, cancer, primer assay, Prostate, microRNA, tumor, qPCR
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Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion
Authors: Alimatou M. Tchafa, Arpit D. Shah, Shafei Wang, Melissa T. Duong, Adrian C. Shieh.
Institutions: Drexel University .
The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression. Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 μm s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20. The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.
Biomedical Engineering, Issue 65, Bioengineering, Biophysics, Cancer Biology, Cancer, interstitial fluid flow, invasion, mechanobiology, migration, three-dimensional cell culture, tumor microenvironment
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MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression
Authors: Mansoureh Sameni, Arulselvi Anbalagan, Mary B. Olive, Kamiar Moin, Raymond R. Mattingly, Bonnie F. Sloane.
Institutions: Wayne State University , Wayne State University .
We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.
Medicine, Issue 60, Immunology, Breast, cancer, extracellular matrix, invasion, proteolysis, tumor microenvironment
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Detection of Functional Matrix Metalloproteinases by Zymography
Authors: Xueyou Hu, Christine Beeton.
Institutions: Baylor College of Medicine.
Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11. Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.
Basic Protocols, Issue 45, Protease, enzyme, electrophoresis, gelatin, casein, fibrin
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Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound
Authors: Tobias Bäuerle, Dorde Komljenovic, Martin R. Berger, Wolfhard Semmler.
Institutions: German Cancer Research Center, Heidelberg, Germany, German Cancer Research Center, Heidelberg, Germany.
Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques. For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US). MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7. In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.
Cancer Biology, Issue 66, Medicine, Physiology, Physics, bone metastases, animal model, angiogenesis, imaging, magnetic resonance imaging, MRI, volumetric computed tomography, ultrasound
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Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia
Authors: Tristan M. Nicholson, Kristen S. Uchtmann, Conrad D. Valdez, Ashleigh B. Theberge, Tihomir Miralem, William A. Ricke.
Institutions: University of Wisconsin-Madison, University of Rochester School of Medicine & Dentistry, University of Wisconsin-Madison.
New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.
Medicine, Issue 78, Cancer Biology, Prostatic Hyperplasia, Prostatic Neoplasms, Neoplastic Processes, Estradiol, Testosterone, Transplantation, Heterologous, Growth, Xenotransplantation, Heterologous Transplantation, Hormones, Prostate, Testosterone, 17beta-Estradiol, Benign prostatic hyperplasia, Prostate Cancer, animal model
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Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy
Authors: Esra Güç, Manuel Fankhauser, Amanda W. Lund, Melody A. Swartz, Witold W. Kilarski.
Institutions: École Polytechnique Fédérale de Lausanne, Oregon Health & Science University.
Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.
Bioengineering, Issue 86, Intravital imaging, epifluorescence, two-photon imaging, Tumor matrix, Matrix remodeling
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Bioluminescent Orthotopic Model of Pancreatic Cancer Progression
Authors: Ming G. Chai, Corina Kim-Fuchs, Eliane Angst, Erica K. Sloan.
Institutions: Monash University, University of Bern, University of California Los Angeles .
Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.
Cancer Biology, Issue 76, Medicine, Molecular Biology, Cellular Biology, Genetics, Biomedical Engineering, Surgery, Neoplasms, Pancreatic Cancer, Cancer, Orthotopic Model, Bioluminescence, In Vivo Imaging, Matrigel, Metastasis, pancreas, tumor, cancer, cell culture, laparotomy, animal model, imaging
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Isolation of Cancer Stem Cells From Human Prostate Cancer Samples
Authors: Samuel J. Vidal, S. Aidan Quinn, Janis de la Iglesia-Vicente, Dennis M. Bonal, Veronica Rodriguez-Bravo, Adolfo Firpo-Betancourt, Carlos Cordon-Cardo, Josep Domingo-Domenech.
Institutions: Icahn School of Medicine at Mount Sinai, Memorial Sloan-Kettering Cancer Center.
The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.
Medicine, Issue 85, Cancer Stem Cells, Tumor Initiating Cells, Prostate Cancer, HLA class I, Primary Prostate Cancer, Castration Resistant Prostate Cancer, Metastatic Prostate Cancer, Human Tissue Samples, Intratumoral heterogeneity
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A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells
Authors: Ralph A. Francescone III, Michael Faibish, Rong Shao.
Institutions: University of Massachusetts, University of Massachusetts, University of Massachusetts.
Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.
Cancer Biology, Issue 55, tumor, vascular, endothelial, tube formation, Matrigel, in vitro
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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
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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
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Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer
Authors: Philippe Pérot, Valérie Cheynet, Myriam Decaussin-Petrucci, Guy Oriol, Nathalie Mugnier, Claire Rodriguez-Lafrasse, Alain Ruffion, François Mallet.
Institutions: Joint Unit Hospices de Lyon-bioMérieux, BioMérieux, Hospices Civils de Lyon, Lyon 1 University, BioMérieux, Hospices Civils de Lyon, Hospices Civils de Lyon.
The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1​​. ‘How to use PSA' remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).
Medicine, Issue 81, Cancer Biology, Genetics, Molecular Biology, Prostate, Retroviridae, Biomarkers, Pharmacological, Tumor Markers, Biological, Prostatectomy, Microarray Analysis, Gene Expression, Diagnosis, Human Endogenous Retroviruses, HERV, microarray, Transcriptome, prostate cancer, Affymetrix
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In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells
Authors: Gunjan Gakhar, Neil H. Bander, David M. Nanus.
Institutions: Weill Cornell Medical College, Weill Cornell Medical College.
Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche. The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines. Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions. Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs. Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions. However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly. CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs. Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques. In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system. This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.
Medicine, Issue 87, E-selectin, Metastasis, Microslides, Circulating tumor cells, PSMA, Prostate cancer, rolling velocity, immunostaining, HUVECs, flow chambers
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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
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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
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Models of Bone Metastasis
Authors: J. Preston Campbell, Alyssa R. Merkel, S. Kathryn Masood-Campbell, Florent Elefteriou, Julie A. Sterling.
Institutions: Vanderbilt University, Vanderbilt University, Tennessee Valley Healthcare System (VISN 9), Vanderbilt University, Vanderbilt University.
Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6. This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.
Medicine, Issue 67, Mouse models of bone metastasis, breast cancer, cancer biology, intracardiac injections, intratibial injections, tumor cells
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A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies
Authors: Inti Zlobec, Guido Suter, Aurel Perren, Alessandro Lugli.
Institutions: University of Bern.
Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a ‘donor’ block into a ‘recipient’ block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 ‘recipient’ blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.
Medicine, Issue 91, tissue microarray, biomarkers, prognostic, predictive, digital pathology, slide scanning
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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
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Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Authors: Yves Molino, Françoise Jabès, Emmanuelle Lacassagne, Nicolas Gaudin, Michel Khrestchatisky.
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
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Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay
Authors: Hassan Azari, Sebastien Millette, Saeed Ansari, Maryam Rahman, Loic P. Deleyrolle, Brent A. Reynolds.
Institutions: University of Florida , Shiraz University of Medical Sciences.
Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 μm in diameter can be observed and are ready for further passaging and expansion.
Neuroscience, Issue 56, Glioblastoma Multiforme, Tumor Cell, Neurosphere Assay, Isolation, Expansion
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RhoC GTPase Activation Assay
Authors: Michelle Lucey, Heather Unger, Kenneth L. van Golen.
Institutions: University of Delaware.
RhoC GTPase has 91% homology to RhoA GTPase. Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed. However, RhoC GTPase is expressed in metastatic cancer cells at relatively low levels. Therefore, few RhoC-specific reagents have been developed. We have adapted a Rho activation assay to detect RhoC GTPase. This technique utilizes a GST-Rho binding domain fusion protein to pull out active RhoC GTPase. In addition, we can harvest total protein at the beginning of the assay to determine levels of total (GTP and GDP bound) RhoC GTPase. This allows for the determination of active versus total RhoC GTPase in the cell. Several commercial versions of this procedure have been developed however, the commercial kits are optimized for RhoA GTPase and typically do not work well for RhoC GTPase. Parts of the assay have been modified as well as development of a RhoC-specific antibody.
neuroscience, Issue 42, brain, mouse, transplantation, labeling
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Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging
Authors: Ed Lim, Kshitij Modi, Anna Christensen, Jeff Meganck, Stephen Oldfield, Ning Zhang.
Institutions: Caliper Life Sciences.
Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location. Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.
Medicine, Issue 50, osteolytic lesions, micro CT, tumor, bioluminescence, in vivo, imaging, IVIS, luciferase, low dose, co-registration, 3D reconstruction
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