Name: establishment of cancer stem cell cultures from human conventional osteosarcoma
Uploaded: 2016-10-14T13:00:00
Duration: 9 min 25 s
Description: Watch this Scientific Journal Video about Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma at JoVE.com

This study was supported by ITT (Istituto Toscano Tumori) Grant Proposal 2010.

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The authors declare that they have no competing financial interest.

CSCs have several properties that permit the identification of this particular cellular subset in the tumor bulk. On the basis of these characteristics, such as acquired resistance to cytotoxic chemotherapy agents for the overexpression of ATP-binding cassette multidrug efflux transporters28, 32, 33, or for the upregulation of the expression of detoxification enzymes such as ALDH32, for the expression of a particular surface marker, such as CD133, CD44, CD34, CD90, and others30, 34, 35, 41, several different methods to isolate CSCs have been developed42-44. One of these techniques is the sphere formation assay, which is based on the capacity of CSCs to grow under non-adherent conditions.
The ability of tissue stem cells and CSCs to form spheres was first described in studies on the identification of neural stem cells by Reynolds et al.37. Subsequently, Gibbs et al.38 used these studies to begin isolating CSCs from solid tumors, in particular, from bone sarcomas. We have decided to use the sphere formation assay method illustrated by Gibbs et al. to isolate CSCs from OSA cell lines obtained from conventional OS biopsies. We adapted the original method to improve the results of this assay and to facilitate its reproducibility for other cancer cell lines. With reference to the establishment of the sphere formation assay, we verified that plating 40,000 cells/well is a good practice for maintaining cells in isolation at the beginning of the assay. This trick is very important to avoid the possibility that the spherical colonies originate from cellular aggregation and not from the particular and exclusive capacity of a single CSC to grow under non-adherent conditions and form a spherical colony. This ability is a particularly critical point of this assay.
We also certified that to obtain a good rate of sphere formation, it is sufficient to refresh aliquots of growth factors every 3 d&#160;and not every day as described in the original method. In this study, we also established and extensively described a good method for isolating the spherical colonies that formed when cultured under non-adherent conditions. This step is critical in this assay because it is very important to try to isolate as many of the spheres as possible that are formed in each well without damaging them. It is also important to isolate only the spheres and not the single cells, which could remain in suspension for the duration of the assay. To overcome these critical points, we have developed a particular isolation method, which, as shown above, gave good results for CSC isolation. Obviously, there is the possibility that not all the spheres that formed can be recovered, but the loss percentage is very low. Indeed, we also have the possibility to use a filter with 40 &#181;m pores to isolate spheres after they become large (formed approximately&#160;100 - 200 cells).
This isolation stops sphere formation but allows the single cells, part of the methylcellulose residue, and the smallest spheres to be filtered. This elimination is performed by thorough filtration as described in the protocol.
Moreover, the selection of the largest spherical colonies through the 40 &#181;m mesh with the consequent loss of the smallest spherical colonies allows one to select the CSCs with the highest capacity to form spherical colonies and with greater stemness. All of these modifications were performed to improve the assay and to help researchers studying CSCs to understand and reproduce the most critical step of the original method of the sphere formation assay.
Among the studies regarding in vitro methods for isolating CSCs, this study aimed to show how this adapted sphere formation assay could be a good method for isolating CSCs from OSA cell lines. The adaptations to the original method and the detailed isolation technique described improve its efficacy. In a short time, good number of CSCs can be obtained and used for several experiments. Therefore, it is possible to rapidly confirm the stem-like phenotypes and, in particular, to study the double stem-like phenotype that characterizes OS-CSCs. Thus, this modified assay could be a good technique for isolating CSCs and studying their biology. In the future, this method, with additional adaptations, may also be used to isolate CSCs from other finite cancer cell lines obtained by biopsies of rare solid tumors.
The possibility of isolating CSCs from rare solid tumors, such as OS, not only permits the improvement of studies about this particular cancer but also extends to studies of different types of cancer to develop better methods for their isolation and to future studies of the biology of this important cellular subset. Therefore, as we have done in this study, it is important to improve the methods of CSC isolation through the study of CSC biology, with the final goal of finding molecular targets and developing a very specific anticancer therapy directed against this particular cellular subset, which is probably responsible for the maintenance of the primary tumor, the development of its recurrence, and the origin of metastases in several organs. The study of CSC biology is also important for finding therapies that could be incisive in the cure of cancers, such as OS, for which the survival rate after neoadjuvant treatment remains very poor.

Sarcomas are a heterogeneous group of rare malignant connective tissue tumors originating predominantly from the embryonic mesoderm1. The different types include bone sarcomas and soft tissue sarcomas. Bone sarcomas, a group of relatively uncommon primary tumors, consist of several subtypes, including osteosarcoma (OS). OS, one of the most common primary tumors of the bone, is a mesenchymal malignancy that exhibits extensive clinical, histological, and molecular heterogeneities2, 3. Unfortunately, OS occurs predominantly in children and in young adults4, 5 and represents 60% of the common histologic subtypes of bone sarcoma in childhood6, 7. OS usually affects the skeletal areas, which are characterized by rapid bone growth (e.g., the metaphysis of long bones). Among the histologically different subtypes of OS, conventional OS, also called medullary or central OS, has a high grade of malignancy and a quota share of 80%8. This 80% is composed of 60% conventional osteoblastic OS, 10% chondroblastic OS, and 10% fibroblastic OS6, 8-10. Other OS subtypes include anaplastic, telangiectatic, giant cell-rich and small cell OS. Despite advances in combined surgery and chemotherapy in OS management, the outcome remains poor, with a long-term survival rate of 65-70% in patients without metastasis11, 12. Distant recurrences frequently occur as pulmonary metastases or, less frequently, as metastases to distant bones and local recurrences13. Metastases are often resistant to conventional treatments. This resistance is the reason why the 10-year disease-free survival is approximately 30% in patients with metastatic disease at diagnosis14, 15.
As with normal tissue, cancer tissue is composed of a heterogeneous collection of cell types. Cells within the tumor seem to correspond to different stages of development. Within any normal tissue resides a subpopulation of cells with the ability to selfrenew, thus providing progenitors and mature cells for tissue homeostasis. Similarly, cancer is composed of a similar heterogeneous population of cells at different stages of development, with different degrees of proliferation and tumorigenic potential. A subset of these cancer cells, termed Cancer stem Cells (CSCs), constitutes a reservoir of selfsustaining cells with the exclusive ability to selfrenew and maintain the malignant potential of tumors, thus generating the different cell lineages that constitute the tumor bulk16. In the 1990s, studies on acute myeloid leukaemia provided the first compelling evidence for the existence of CSC subpopulations17, 18. CSCs have since been isolated from a great number of solid tumors19, thereby becoming one of the most researched topics in cancer research. CSCs may indeed arise from normal stem cells by mutations in genes that make the normal stem cells cancerous20-23. Multiple transforming mutations and interactions with the microenvironment could also contribute to&#160;healthy progenitors and mature cells acquiring the selfrenewal capacity and immortality that typify CSCs. There are several hypotheses about this transformation. Healthy progenitors, healthy mature cells, and cancer cells, can dedifferentiate to stem cells, obtaining a stem-like phenotype by activating selfrenewal-associated genes24-28. Despite several recent studies, the origins of CSCs have yet to be discovered.
A particular characteristic of CSCs is that their capacity to resist the multi-therapy approach, which consists of combined surgery and chemotherapy with different drugs. Recent studies have shown that CSCs may also acquire resistance to cytotoxic chemotherapy agents. Possible explanations for this resistance include the overexpression of ATP-binding cassette (ABC) multidrug transporter (i.e.,&#160;MDR1 and BCRP1), overexpression of chemotherapy metabolizing enzymes such as aldehyde dehydrogenase 1 (ALDH1), and/or changes in cell cycle kinetics30-33. The direct consequence of all these concepts that have been described thus far is that a cancer therapy would be efficient only if the CSC subpopulation were completely eliminated, while local recurrence or distant metastasis could occur if even a single CSC survived.
The discovery of CSCs in human sarcomas34, particularly OS35, or in any other bone and soft tissue cancers, has great clinical importance because it offers a possible explanation as to why many treatments seem to be effective initially, but the patients later relapse. Therefore, the hope for the future battle against conventional OS is to find new and specific targeted therapies based on the development of innovative drugs directed at OS-CSCs&#160;thanks to the molecular characterization of this subpopulation and to the study of CSC biology.
In 1992, Reynolds and colleagues, who were investigating whether a subset of stem cells was present in the adult mammalian brain, developed a method to isolate cells suspected to be stem-like cells36, 37. This method is based on the particular ability of these cells to form spherical colonies when grown under nonadherent conditions. Similar techniques were employed by Gibbs and colleagues in 2005 to study a subpopulation of stem-like cells in bone sarcomas38. To isolate and characterize OS-CSCs from primary cell cultures of different types of conventional OS, we decided to adapt this technique for OS cell lines.
Here, we describe this adapted method of the sphere formation assay, termed &#34;sarcosphere assay&#34;, which can be used to isolate OS-CSCs from finite primary cell lines derived from human biopsies of conventional OS. We also describe all the techniques used to validate the stem-like CSC phenotype of the cell lines isolated by this assay: 1) evaluation of the expression of genes that characterize pluripotent Embryonic Stem Cells (ESCs) and of the CD133 gene, which is a marker of CSCs; 2) colony-forming unit (CFU) assay; 3) evaluation of the ability of these cells to differentiate into osteoblasts and adipocytes under appropriate differentiation conditions; 4) study of the surface markers of Mesenchymal Stem Cells (MSCs) (i.e.,&#160;CD44, CD105 and Stro-1) by immunofluorescence staining and by flow cytometric analysis; 5) evaluation of the ALDH activity of these cells.

establishment of cancer stem cell cultures from human conventional osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

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Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

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Cancer Research

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

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Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

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Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

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In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

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Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

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Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

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Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Expansion and Enrichment of Gamma-Delta ÃƒÅ½Ã‚Â³ÃƒÅ½Ã‚Â´ T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...