Name: establishment of cancer stem cell cultures from human conventional osteosarcoma
Uploaded: 2016-10-14T13:00:00.000+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.

<table><tbody><tr><td>Dulbecco&#039;s Phosphate Buffered Saline with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; (DPBS)</td><td>LONZA</td><td>BE17-513F</td><td>_</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline&amp;nbsp; without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; (DPBS)</td><td>LONZA</td><td>BE17-512F</td><td>_</td></tr><tr><td>Porcine Trypsin 1:250</td><td>BD Difco</td><td>215310</td><td>Solvent: DPBS. Stock concentration: Powder</td></tr><tr><td>Ethylenediamine tetraacetic acid disodium salt dihydrate(EDTA)</td><td>Sigma-Aldrich</td><td>E4884</td><td>Solvent: DPBS. Stock concentration: Powder</td></tr><tr><td>Collagenase from Clostridium histolyticum</td><td>Sigma-Aldrich</td><td>C0130</td><td>Solvent: Buffer Solution, pH 7.4. Stock concentration: Powder</td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>BDH Chemicals-VWR</td><td>10323</td><td>_</td></tr><tr><td>Nutrient Mixture F-12 Ham</td><td>Sigma-Aldrich</td><td>F6636</td><td>Solvent: Ultrapure dH&lt;sub&gt;2&lt;/sub&gt;O. Stock concentration: Powder</td></tr><tr><td>2-Phospho-L-ascorbic acid trisodium salt</td><td>Sigma-Aldrich</td><td>49752</td><td>Solvent: DPBS. Stock concentration: 5 mg/mL</td></tr><tr><td>&amp;beta;-Glycerol phosphate disodium salt pentahydrate</td><td>Sigma-Aldrich</td><td>50020</td><td>Solvent: DPBS. Stock concentration: 1 M</td></tr><tr><td>Insulin. Human Recombinant</td><td>Sigma-Aldrich</td><td>91077</td><td>Solvent: NaOH 0.1 M. Stock concentration: 10 mM</td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma-Aldrich</td><td>I5879</td><td>Solvent: DMSO. Stock concentration: 500 mM</td></tr><tr><td>Indomethacin</td><td>Sigma-Aldrich</td><td>I7378</td><td>Solvent: DMSO. Stock concentration: 200 mM</td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td>Solvent: DMSO. Stock concentration:&amp;nbsp; 1 mM / 100 &amp;micro;M. Store in liquid nitrogen to maintain the biological activity</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>F7524</td><td>_</td></tr><tr><td>Fetal Bovine Serum South America&amp;nbsp;</td><td>EUROCLONE</td><td>ECS0180L</td><td>_</td></tr><tr><td>Penicillin-Streptomycin (PEN-STREP) 10,000 U/mL</td><td>LONZA</td><td>DE17-602E</td><td>_</td></tr><tr><td>Methyl cellulose</td><td>Sigma-Aldrich</td><td>274429</td><td>Solvent: Ultrapure dH&lt;sub&gt;2&lt;/sub&gt;O. Stock concentration: 2%</td></tr><tr><td>Putresceine dihydrochloride</td><td>Sigma-Aldrich</td><td>P5780</td><td>Solvent: Ultrapure dH&lt;sub&gt;2&lt;/sub&gt;O. Stock concentration: Powder</td></tr><tr><td>apo-Transferrin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T-1147</td><td>Solvent: DPBS. Stock concentration: 25 mg/mL</td></tr><tr><td>Human Epidermal Growth Factor (EFGF)</td><td>Sigma-Aldrich</td><td>E5036</td><td>Solvent: DPBS pH 7.4. Stock concentration: 10 &amp;micro;g/mL</td></tr><tr><td>Fibroblast Growth Factor-Basic Human</td><td>Sigma-Aldrich</td><td>F0291</td><td>Solvent: DPBS + 0.2% BSA. Stock concentration: 25 &amp;micro;g/mL</td></tr><tr><td>Selenous Acid</td><td>Sigma-Aldrich</td><td>211176</td><td>Solvent: DPBS. Stock concentration: 30 mM</td></tr><tr><td>Progesterone</td><td>Sigma-Aldrich</td><td>P8783</td><td>Solvent: ETOH. Stock concentration: 10 mM</td></tr><tr><td>Toluidine Blue O</td><td>Sigma-Aldrich</td><td>198161</td><td>Solvent: Ultrapure dH&lt;sub&gt;2&lt;/sub&gt;O. Stock concentration: Powder</td></tr><tr><td>Oil Red O</td><td>ICN Biochemicals</td><td>155984</td><td>Solvent: 2-Propanol. Stock concentration: Powder</td></tr><tr><td>Naphtol AS-MX Phosphate Disodium Salt</td><td>Sigma-Aldrich</td><td>N5000</td><td>Solvent: DMSO. Stock concentration: Powder</td></tr><tr><td>Fast Blue BB Salt</td><td>Sigma-Aldrich</td><td>F3378</td><td>Solvent: Tris HCL, pH 9.0. Stock concentration: Powder</td></tr><tr><td>Fast Red Violet LB Salt</td><td>Sigma-Aldrich</td><td>F3381</td><td>Solvent: Tris HCL, pH 9.1. Stock concentration: Powder</td></tr><tr><td>Bovine Serum Albumin, Fraction V (BSA)</td><td>Sigma-Aldrich</td><td>A-4503</td><td>Solvent: DPBS. Stock concentration: 2%</td></tr><tr><td>Alizarin Red S</td><td>ICN Biochemicals</td><td>100375</td><td>Solvent: Ultrapure dH&lt;sub&gt;2&lt;/sub&gt;O. Stock concentration: Powder</td></tr><tr><td>Formaldehyde solution</td><td>Sigma-Aldrich</td><td>533998</td><td>4%</td></tr><tr><td>Triton-100X</td><td>MERCK</td><td>11869</td><td>Solvent: DPBS. Stock concentration: 0.2%. Danger - Use only under chemical hood</td></tr><tr><td>Calcein</td><td>MERCK</td><td>2315</td><td>Solvent: DPBS . Stock concentration: 200 &amp;micro;g/mL</td></tr><tr><td>2-Propanol</td><td>MERCK</td><td>109634</td><td>Danger - Use only under chemical hood</td></tr><tr><td>Ab-CD105&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Mouse monoclonal [SN6] to CD105 (FITC)</td><td>Abcam</td><td>ab11415</td><td>Liquid. Application:&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Flow Cytometry (Flow Cyt)</td></tr><tr><td>Ab-CD44&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Mouse monoclonal&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; [F10-44-2] to CD44 (PE/Cy7&amp;reg;) )</td><td>Abcam</td><td>ab46793</td><td>Liquid. Application:&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Flow Cytometry (Flow Cyt)</td></tr><tr><td>Ab-CD45&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Mouse monoclonal&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; [MEM-28] to CD45 (PerCP))&amp;nbsp;</td><td>Abcam</td><td>ab65952</td><td>Liquid. Application:&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Flow Cytometry (Flow Cyt)</td></tr><tr><td>Ab-CD105&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Human CD105 Purified Antibody)&amp;nbsp;</td><td>Invitrogen</td><td>MHCD10500</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application: Immunofluorescence staining (IF)</td></tr><tr><td>Ab-CD44&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Anti-CD44 Antibody)&amp;nbsp;</td><td>Abcam</td><td>EPR1013Y(ab51037)</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application: Immunofluorescence staining (IF)</td></tr><tr><td>Ab-Stro-1&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Mouse anti-STRO-1)&amp;nbsp;</td><td>Invitrogen</td><td>398401</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application:&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Flow Cytometry (Flow Cyt)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Immunofluorescence staining (IF)</td></tr><tr><td>Alexa Fluor 488&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Anti-Rabbit IgG (Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L))</td><td>Invitrogen</td><td>A-21206</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application: Immunofluorescence staining (IF)</td></tr><tr><td>FITC Anti-Mouse IG (FITC-Rabbit Anti Mouse IgG (H+L))</td><td>Invitrogen</td><td>61-6511</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application:&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Flow Cytometry (Flow Cyt)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Immunofluorescence staining (IF)</td></tr><tr><td>Alexa Fluor 635 Phalloidin&amp;nbsp;</td><td>Invitrogen</td><td>A34054</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application: Immunofluorescence staining (IF)</td></tr><tr><td>AutoMACS&amp;trade;Running Buffer MACS Separation Buffer</td><td>Miltenyi</td><td>130,091,221</td><td>Liquid. Store at 4 &amp;deg;C</td></tr><tr><td>QIAzol&amp;reg;Lysis Reagent</td><td>QIAGEN</td><td>79306</td><td>Danger - Use only under chemical hood</td></tr><tr><td>QUANTITECT&amp;reg;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Reverse Transcription Kit</td><td>QIAGEN</td><td>205314</td><td>_&amp;nbsp;</td></tr><tr><td>Chlorophorm</td><td>Sigma-Aldrich</td><td>C2432</td><td>Liquid. Danger - Use only under chemical hood</td></tr><tr><td>Laminar flow hood</td><td>GELAIRE</td><td>BSB6A</td><td>_&amp;nbsp;</td></tr><tr><td>Chemical hood</td><td>ARREDI TECNICI&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Villa</td><td>Modello&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; DYNAMICA</td><td>_&amp;nbsp;</td></tr><tr><td>Centrifuge</td><td>EPPENDORF</td><td>5415R</td><td>_&amp;nbsp;</td></tr><tr><td>Laser Scanning Confocal Microscopy LSM 5109 Meta&amp;nbsp;</td><td>ZEISS</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>iCycler PCR Thermalcycler&amp;nbsp;</td><td>BIORAD</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>CyFlow&amp;reg;SPACE&amp;nbsp;</td><td>(PARTEC)</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Inverted Micrposcope Axiovert 200M&amp;nbsp;</td><td>ZEISS</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Freezing container ,</td><td>Nalgene</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Original Pipet-Aid</td><td>pbiBrand</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Micropipettes</td><td>EPPENDORF</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Glass Pasteur Pipette</td><td>SIGMA</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>VICTOR3&amp;trade;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>PERKIN ELMER</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Conical tubes&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (15 and 50 mL)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD FALCON</td><td>352096 (for 15 mL)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 352070 (for 50 mL)</td><td>_&amp;nbsp;</td></tr><tr><td>24-Well Clear Flat Bottom TC-Treated Multiwell-Cell-Culture-Plate</td><td>BD FALCON</td><td>353047</td><td>_&amp;nbsp;</td></tr><tr><td>6-Well Clear Flat Bottom Ultra Low Attachment Multiple-Well-Plates</td><td>CORNING</td><td>3471</td><td>_&amp;nbsp;</td></tr><tr><td>Serological pipettes&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (5 and 10 mL)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD FALCON</td><td>357543 (for 5 mL)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 357551 (for 10mL)</td><td>_&amp;nbsp;</td></tr><tr><td>Syringe (5mL)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>B|BRAUN</td><td>4617053V</td><td>_&amp;nbsp;</td></tr><tr><td>Petri dish 100X20 mm&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD FALCON</td><td>353003</td><td>_&amp;nbsp;</td></tr><tr><td>R&amp;ouml;hren Tubes&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (3.5 mL, 55x12mm, PS)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>SARSTEDT</td><td>55,484</td><td>_&amp;nbsp;</td></tr><tr><td>Petri dish 60X15 mm&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD FALCON</td><td>353004</td><td>_&amp;nbsp;</td></tr><tr><td>Cryovials 1.5 mL&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>NALGENE</td><td>5000-1020</td><td>_&amp;nbsp;</td></tr><tr><td>Cell-Culture Flasks 25 cm&amp;sup2;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD FALCON</td><td>353014</td><td>_&amp;nbsp;</td></tr><tr><td>Nylon Net Filter, Hydrophilic</td><td>MERCK</td><td>NY4104700</td><td>_&amp;nbsp;</td></tr><tr><td>Swinnex Filter Holder</td><td>MERCK</td><td>SX0002500</td><td>_&amp;nbsp;</td></tr><tr><td>Perry tweezer&amp;nbsp;</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Lancet</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Dounce</td><td>_</td><td>_</td><td>_</td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma at JoVE.com

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 ...

establishment-cancer-stem-cell-cultures-from-human-conventional

Research

JoVE Journal

Cancer Research

18.9K Views.  University of Florence. 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.

Video: Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

Genetics

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

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.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Medicine

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

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.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

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 &#181;M 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.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Protocol for Osteosarcoma PDX Mouse Models: Studying Growth and Drug Resistance

Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, the prognosis for osteosarcoma patients has not significantly improved. Therefore, it is crucial to establish a robust preclinical model with high fidelity. The patient-derived xenograft (PDX) model faithfully preserves the genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient. Consequently, PDX models are considered authentic in vivo models for studying various cancers in transformation studies. This article presents a comprehensive protocol for creating and maintaining a PDX mouse model that accurately mirrors the morphological features of human osteosarcoma. This involves the immediate transplantation of freshly resected human osteosarcoma tissue into immunocompromised mice, followed by successive passaging. The described model serves as a platform for studying the growth, drug resistance, relapse, and metastasis of osteosarcoma. Additionally, it aids in screening the target therapeutics and establishing personalized treatment schemes.

Author Spotlight: Replicating Human Osteosarcoma Progression in Immunodeficient Mice for Cancer Study

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, ...