JoVE Visualize What is visualize?
Related JoVE Video
Pubmed Article
ALDH activity selectively defines an enhanced tumor-initiating cell population relative to CD133 expression in human pancreatic adenocarcinoma.
PUBLISHED: 05-06-2011
Multiple studies in recent years have identified highly tumorigenic populations of cells that drive tumor formation. These cancer stem cells (CSCs), or tumor-initiating cells (TICs), exhibit properties of normal stem cells and are associated with resistance to current therapies. As pancreatic adenocarcinoma is among the most resistant human cancers to chemo-radiation therapy, we sought to evaluate the presence of cell populations with tumor-initiating capacities in human pancreatic tumors. Understanding which pancreatic cancer cell populations possess tumor-initiating capabilities is critical to characterizing and understanding the biology of pancreatic CSCs towards therapeutic ends.
Authors: Zeshaan Rasheed, Qiuju Wang, William Matsui.
Published: 09-26-2010
Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5. Recently, we and others identified CSCs from pancreatic adenocarcinoma based on ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent, a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.
16 Related JoVE Articles!
Play Button
Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines
Authors: Jennifer M. Cole, Stancy Joseph, Christopher G. Sudhahar, Karen D. Cowden Dahl.
Institutions: Indiana University School of Medicine.
Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 µM cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.
Medicine, Issue 91, cancer stem cells, stem cell markers, ovarian cancer, chemoresistance, cisplatin, cancer progression
Play Button
Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue
Authors: Elaine Bigelow, Katherine M. Bever, Haiying Xu, Allison Yager, Annie Wu, Janis Taube, Lieping Chen, Elizabeth M. Jaffee, Robert A. Anders, Lei Zheng.
Institutions: The Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine, Yale School of Medicine, The Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine.
B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8. Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12. In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.
Cancer Biology, Issue 71, Medicine, Immunology, Biochemistry, Molecular Biology, Cellular Biology, Chemistry, Oncology, immunohistochemistry, B7-H1 (PD-L1), pancreatic adenocarcinoma, pancreatic cancer, pancreas, tumor, T-cell immunity, cancer
Play Button
Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting
Authors: Chitra Venugopal, Nicole M. McFarlane, Sara Nolte, Branavan Manoranjan, Sheila K. Singh.
Institutions: McMaster University .
Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres. To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 °C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR. The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.
Cancer Biology, Issue 67, Stem Cell Biology, Medicine, Cellular Biology, Molecular Biology, BTIC (brain tumor initiating cells), tumorspheres, self-renewal, flow cytometry, single cell RT-PCR
Play Button
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
Play Button
Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)
Authors: Loic P. Deleyrolle, Mark R. Rohaus, Jeff M. Fortin, Brent A. Reynolds, Hassan Azari.
Institutions: The University of Florida, Shiraz University of Medical Sciences, Shiraz, Iran .
Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.
Medicine, Issue 62, Label-Retaining Cells, Slow-Dividing Cells, Cancer Stem Cells, Glioblastoma, CFSE
Play Button
Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles
Authors: Jing Xu, Mansoor Amiji.
Institutions: Northeastern University.
More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8 Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.
Bioengineering, Issue 59, Gelatin Nanoparticle, Gene Therapy, Targeted Delivery, Pancreatic Cancer, Epidermal Growth Factor Receptor, EGFR
Play Button
Isolation of CD133+ Liver Stem Cells for Clonal Expansion
Authors: C. Bart Rountree, Wei Ding, Hein Dang, Colleen VanKirk, Gay M. Crooks.
Institutions: Pennsylvania State College of Medicine, Pennsylvania State College of Medicine, University of California Los Angeles, School of Medicine.
Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.
Developmental Biology, Issue 56, CD133, liver stem cell, oval cell, liver cancer stem cell, stem cell, cell isolation, non-parenchymal fraction of liver, flow cytometry
Play Button
Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen
Authors: Dennis Ma, Jonathan Collins, Tomas Hudlicky, Siyaram Pandey.
Institutions: University of Windsor, Brock University.
Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells. We have reported selective induction of apoptosis in cancer cells by the natural compound pancratistatin (PST). Recently, a novel PST analogue, a C-1 acetoxymethyl derivative of 7-deoxypancratistatin (JCTH-4), was produced by de novo synthesis and it exhibits comparable selective apoptosis inducing activity in several cancer cell lines. Recently, autophagy has been implicated in malignancies as both pro-survival and pro-death mechanisms in response to chemotherapy. Tamoxifen (TAM) has invariably demonstrated induction of pro-survival autophagy in numerous cancers. In this study, the efficacy of JCTH-4 alone and in combination with TAM to induce cell death in human breast cancer (MCF7) and neuroblastoma (SH-SY5Y) cells was evaluated. TAM alone induced autophagy, but insignificant cell death whereas JCTH-4 alone caused significant induction of apoptosis with some induction of autophagy. Interestingly, the combinatory treatment yielded a drastic increase in apoptotic and autophagic induction. We monitored time-dependent morphological changes in MCF7 cells undergoing TAM-induced autophagy, JCTH-4-induced apoptosis and autophagy, and accelerated cell death with combinatorial treatment using time-lapse microscopy. We have demonstrated these compounds to induce apoptosis/autophagy by mitochondrial targeting in these cancer cells. Importantly, these treatments did not affect the survival of noncancerous human fibroblasts. Thus, these results indicate that JCTH-4 in combination with TAM could be used as a safe and very potent anti-cancer therapy against breast cancer and neuroblastoma cells.
Cancer Biology, Issue 63, Medicine, Biochemistry, Breast adenocarcinoma, neuroblastoma, tamoxifen, combination therapy, apoptosis, autophagy
Play Button
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
Play Button
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
Play Button
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
Play Button
Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in Clinically Relevant Animal Models and 3D Immunochemistry
Authors: Laura A. Hasselbach, Susan M. Irtenkauf, Nancy W. Lemke, Kevin K. Nelson, Artem D. Berezovsky, Enoch T. Carlton, Andrea D. Transou, Tom Mikkelsen, Ana C. deCarvalho.
Institutions: Henry Ford Hospital.
Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor. Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.
Medicine, Issue 83, Primary Cell Culture, animal models, Nervous System Diseases, Neoplasms, glioblastoma, neurosphere, surgical specimens, long-term self-renewal
Play Button
Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma
Authors: Yvonne Welte, Cathrin Davies, Reinhold Schäfer, Christian R.A. Regenbrecht.
Institutions: Charité - Universitätsmedizin Berlin, Free University Berlin, Charité - Universitätsmedizin Berlin, Charité - Universitätsmedizin Berlin.
Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression. Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin. Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.
Cancer Biology, Issue 73, Medicine, Stem Cell Biology, Cellular Biology, Molecular Biology, Biomedical Engineering, Genetics, Oncology, Primary cell culture, melanoma, MACS, cancer stem cells, CD133, cancer, prostate cancer cells, melanoma, stem cells, cell culture, personalized treatment
Play Button
A Preclinical Murine Model of Hepatic Metastases
Authors: Kevin C. Soares, Kelly Foley, Kelly Olino, Ashley Leubner, Skye C. Mayo, Ajay Jain, Elizabeth Jaffee, Richard D. Schulick, Kiyoshi Yoshimura, Barish Edil, Lei Zheng.
Institutions: The Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine, University of Colorado Anschutz Medical Campus.
Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.
Medicine, Issue 91, Pancreatic Neoplasms, Immunotherapy, Hemispleen, Hepatic Metastases, Pancreatic Cancer, Liver, Preclinical Model, Metastatic, Murine
Play Button
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
Play Button
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
Copyright © JoVE 2006-2015. All Rights Reserved.
Policies | License Agreement | ISSN 1940-087X
simple hit counter

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.