This study was supported by a grant from the Baden-W&#252;rttemberg Stiftung (Adult Stem Cells Program II) awarded to C.L. We thank Dr. Martina Konantz for critical input and review of the manuscript. We thank Emmanuel Traunecker and Toni Krebs from the DBM FACS Facility (University Hospital Basel) for assistance with FACS sorting.

1. דור של תאי השחלה אדם OVCAR-3 Carcinoma transduced ביציבות עם Lentiviruses מכיל לבנות כתב Sox2 התקינה האזור <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ליצור חלקיקי lentiviral על ידי transfecting קו תא 293T-אריזת HEK עם לבנות כתב הכרת אזור רגולציה Sox2 כ10,21 תיארו. הערה: הכתב לבנות עוד מכיל תחום יציבות של מערכת חומת ProteoTuner לפני חלבון פלואורסצנטי tdTomato. Shield1 נקשר לתחום היציבות ובכך למנוע פרוטאזום להשפיל חלבון פלואורסצנטי 22. </li><li style=";text-align:right;direction:rtl"> Transduce OVCAR-3 תאים עם חלקיקי lentiviral על פני תקופה של 24 שעות זמן. לאחר מכן, להסיר את supernatant ויראלי ולשטוף את התאים עם ופר פוספט (PBS) ותרבית בינונית שלם (RPMI בתוספת 10% FBS, 100 U / ml פניצילין, 100 מיקרוגרם / מיליליטר סטרפטומיצין). </li><li style=";text-align:right;direction:rtl"> 48 שעות מאוחר יותר, 10 מיקרוגרם / מיליליטר פורmycin נוספו לתרבויות ונשמר במשך 5 ימים, כדי לאפשר בחירה של תאי transduced כראוי. </li></ol> 2. הכנת Cell המיון והציפוי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להוסיף Shield1 ב 1: 1000 שעות דילול 24 לפני מיון תא. השתמש OVCAR-3 תאי transduced ביציבות ללא טיפול Shield1 כפקדים שליליים (איור 1). תקשורת לשאוב מבקבוק, לשטוף תאים עם 1x PBS וtrypsinize תאים עם 0.05% טריפסין- EDTA במשך 3 דקות. </li><li style=";text-align:right;direction:rtl"> להפסיק טריפסין באמצעות מדיום מלא (ראה לעיל), לספור מספרי תא, תאי צנטריפוגות ב 300 XG ב RT (15 - 25 מעלות צלזיוס) במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> למזוג supernatant ותאי resuspend בזהירות ב 0.5 - PBS סטרילי 1 מיליליטר. </li><li style=";text-align:right;direction:rtl"> השתמש במסנן כובע מסננת תא 40 מיקרומטר להשיג השעיה תא בודד. </li><li style=";text-align:right;direction:rtl"> התאם ספירת תאים 5 מיליון תאים לכל מיליליטר. </li><li style=";text-align:right;direction:rtl"> הכן 96-גם צלחות נמוכות מצורפים אולטרה עם 100 μl הישות הזכרית והנקביות בינוניים (MEGM בתוספת גורמי גדילה, ציטוקינים, ותוספי מזון,B-27, heparine-נתרן; או DMEM / F12 בתוספת גורמי גדילה, ציטוקינים, ותוספי מזון, B-27, heparine-נתרן, עם או בלי תוספת של 1% methylcellulose, ראה גם לוח 1). לחלופין להוסיף אנטיביוטיקה למדיום בריכוז של 100 U פניצילין מיליליטר / 100 מיקרוגרם / מיליליטר סטרפטומיצין כדי למזער את הסיכון לזיהום אפשרי. </li><li style=";text-align:right;direction:rtl"> מיין + RFP ותאי RFP- ל96-גם צלחות שהוכנו מלמעלה, תא 1 בכל טוב (assay מבוסס תאים בודדים תחומים) וכל גם 100 תאים (assay תחומים מבוססי תאים רב), בהתאמה. בצע סדרן תא מין על זמין מסחרי (ראה חומרים) שימוש במצב תא בודד, מיין התקנה: 100 μ זרבובית, לחץ נדן 20 psi, ומסכת תשואת 0, מסכת טוהר 32, מסכת שלב 16. </li><li style=";text-align:right;direction:rtl"> להעריך את היעילות על ידי ציפוי מיקרוסקופי הבקיע בארות המכילות תאים (עבור assay מבוסס התאים הבודד) ועל ידי ספירת מספר תאים בבארות בודדות (לassay מבוסס תאים הרב; איור 2 </stronז&gt;). </li><li style=";text-align:right;direction:rtl"> דגירה תאים בתנאים סטנדרטיים בתחומים בינוניים (להרכב ראה שלב 2.6) על 37 מעלות צלזיוס, 5% CO 2. להשלים bFGF היומי (20 ng / ml) וEGF (מיליליטר / 20 ng). </li><li style=";text-align:right;direction:rtl"> אחרי שבוע אחד, לספור מספרים של מתעוררים תחומי גידול באמצעות מיקרוסקופ רגיל עם הגדלה 4X או 10X ומיקרוסקופ פלואורסצנטי כדי לזהות אותות הקרינה ממערכת הכתב המשולבת. רוזן התחומים בקוטר העולה על כתחומים &quot;גדולים&quot; 100 מיקרומטר, ותחומים בקוטר 100 - 50 כתחומים &quot;קטנים&quot; מיקרומטר (איור 3). להיות בטוח שאתה לספור תחומים אמיתיים ולא תא אשכולות. הערה: במבחנים מבוססי תאים בודדים היווצרות כדור קלה יותר להבקיע מיקרוסקופי לאחר 10 (לעומת 7) ימים של התרבות. </li><li style=";text-align:right;direction:rtl"> חשב את חלקם של תאי יוצרי כדור ב+ RFP ותאי בהתאמה RFP- במבחנים מבוססי תאים בודדים (96-גם צלחת אחת לכל ניסוי בודד) או תא-ba הרבמבחני SED תחומים (טוב אחד עבור כל ניסוי בודד) כפי שהוצג על ידי et al שו. 16 הערה: חלקם של תאי תחום יצירה (%) = (מספר תחומים) / (מספר תאי זרע) x 100 </li></ol> 3. Passaging הסידורי של Spheres <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח את התוכן של כל אחד גם בצינור סטרילי מתאים וצנטריפוגות ב XG 300 עבור 10 דקות ב RT. עבור מבחני מבוססי תאים רב התחומים, לאסוף יחד את התחומים מאחד גם. לשטוף היטב 3-5 פעמים עם PBS ו צנטריפוגות 2 דקות יותר. עבור מבחני מבוססי תאים בודדים תחומים, לאסוף כדורים בודדים. לאור המספרים הנמוכים של תאים, להשתמש צינורות 1.5 מיליליטר לצעדי צנטריפוגה וכביסה. </li><li style=";text-align:right;direction:rtl"> הסר supernatant וגלול גלול ב200 μl של 0.05% טריפסין- EDTA. </li><li style=";text-align:right;direction:rtl"> על מנת להשיג הפרדת תא אופטימלית, דגירה ההשעיה התא ב 37 מעלות צלזיוס למשך 5 דקות בייקרו רך. לייעל trypsinization זמן לקו הסלולרי שלך לשיעור מוות של תאים תחתון: אם לא גדולהתחומים הוא triturate הגלוי בעדינות באמצעות קצה פיפטה 100 μl. במקרה תחומים גדולים עדיין קיימים, דגירה עם טריפסין עוד 3 דקות ולאחר מכן להמשיך לשלב טחינה דקה. במקרה של יחיד מבחני מבוססי תאים לוודא כדי לייעל את הזמן לתשואת תא אופטימלי של תאי חיים במהלך שלב trypsinization. </li><li style=";text-align:right;direction:rtl"> כדי להשבית טריפסין, להוסיף 500 μl בינוני ו צנטריפוגות מלאים ב XG 300 במשך 10 דקות, במקרה של מבחני תא בודדים, צנטריפוגות במשך 2 דקות נוספות. </li><li style=";text-align:right;direction:rtl"> הסרת תאי supernatant ו resuspend בזהירות במדיום תחומים. </li><li style=";text-align:right;direction:rtl"> שימוש במסנן כובע מסננת תא 40 מיקרומטר לקבל השעיה תא בודד. </li><li style=";text-align:right;direction:rtl"> במקרה של שימוש ב+ RFP ותאי RFP- במבחנים-bassed תא רב, להעריך את אחוז תאי ניאון בכל אחד גם לאחר resuspension באמצעות cytometer זרימת הניתוח. </li><li style=";text-align:right;direction:rtl"> עבור מבחני סידוריים replating של תאים בודדים, זרע תא 1 לכל גם לתוך צלחת אולטרה חדשה נמוכה מצורף 96-גם הכינו כמפורט לעיל. מכדור אחד בודד, זרע כ 20 בארות בודדות. עבור מבחני replating תחומים עיקריים המבוסס על תאים רב, תאי זרע המתקבלים מכן אחד מתחומים עיקריים לצלחת 96-היטב חדשה ולספור את מספר התאים למחרת על ידי מיקרוסקופ. </li><li style=";text-align:right;direction:rtl"> להעריך את חלקם של תאי יוצרי כדור במבחני תחומים משניים באמצעות הנוסחא המתוארת בשלב 2.11. </li></ol> ניתוח 4. תוצאה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לנתח את התוצאות מניסויים שבוצעו בtriplicates העצמאי ולהשתמש במבחן t דו-צדדי לנתח ערכים מתפלגים נורמלי ואחר Mann-Whitney-בדיקות לניתוח סטטיסטי. </li></ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-small+cell+lung+cancer+cells+expressing+CD44+are+enriched+for+stem+cell-like+properties.">Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties.</a> PLoS One. 5 (11), e14062 (2010).</li><li>Bertolini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+tumorigenic+lung+cancer+CD133++cells+display+stem-like+features+and+are+spared+by+cisplatin+treatment.">Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment.</a> Proc Natl Acad Sci U S A. 106 (38), 16281-16286 (2009).</li><li>Ma, L., Lai, D., Liu, T., Cheng, W., Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem-like+cells+can+be+isolated+with+drug+selection+in+human+ovarian+cancer+cell+line+SKOV3.">Cancer stem-like cells can be isolated with drug selection in human ovarian cancer cell line SKOV3.</a> Acta Biochim Biophys Sin (Shanghai). 42 (9), 593-602 (2010).</li><li>Wang, H., Zhang, Y., Du, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+and+breast+cancer+spheres+are+similar+in+transcriptomic+features+and+sensitive+to+fenretinide).">Ovarian and breast cancer spheres are similar in transcriptomic features and sensitive to fenretinide).</a> Biomed Res Int. 2013, 510905 (2013).</li><li>Du, Y. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+and+mechanisms+of+anti-CD44+monoclonal+antibody+A3D8+on+proliferation+and+apoptosis+of+sphere-forming+cells+with+stemness+from+human+ovarian+cancer.">Effects and mechanisms of anti-CD44 monoclonal antibody A3D8 on proliferation and apoptosis of sphere-forming cells with stemness from human ovarian cancer.</a> Int J Gynecol Cancer. 23 (8), 1367-1375 (2013).</li><li>Xiang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-17+produced+by+tumor+microenvironment+promotes+self-renewal+of+CD133+cancer+stem-like+cells+in+ovarian+cancer.">Interleukin-17 produced by tumor microenvironment promotes self-renewal of CD133 cancer stem-like cells in ovarian cancer.</a> Oncogene. , (2013).</li><li>Liu, T., Cheng, W., Lai, D., Huang, Y., Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+primary+ovarian+cancer+cells+in+different+culture+systems.">Characterization of primary ovarian cancer cells in different culture systems.</a> Oncol Rep. 23 (5), 1277-1284 (2010).</li><li>Soritau, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+chemoresistance+and+tumor+sphere+formation+as+a+laboratory+model+for+peritoneal+micrometastasis+in+epithelial+ovarian+cancer.">Enhanced chemoresistance and tumor sphere formation as a laboratory model for peritoneal micrometastasis in epithelial ovarian cancer.</a> Rom J Morphol Embryol. 51 (2), 259-264 (2010).</li><li>Shi, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cancer+stem+cell-like+cells+from+human+epithelial+ovarian+carcinoma+cell+line.">Identification of cancer stem cell-like cells from human epithelial ovarian carcinoma cell line.</a> Cell Mol Life Sci. 67 (22), 3915-3925 (2010).</li><li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+ovarian+cancer-initiating+cells+from+primary+human+tumors.">Identification and characterization of ovarian cancer-initiating cells from primary human tumors.</a> Cancer Res. 68 (11), 4311-4320 (2008).</li><li>Liang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hypoxic+microenvironment+upgrades+stem-like+properties+of+ovarian+cancer+cells.">The hypoxic microenvironment upgrades stem-like properties of ovarian cancer cells.</a> BMC Cancer. 12, 201 (2012).</li></ol>

The authors have nothing to disclose.

תרבויות תחומים הן שיטה נפוצה לassay תא גזע סרטני פוטנציאלי ולהעשיר לתאי גזע כמו-במגוון רחב של תאים סרטניים אנושיים 15,25,26. בתנאים אלה התרבות, תאי סרטן שאין יכולת התחדשות עצמית צפויים לבדל וסופו של דבר לעבור מוות של תאים. למרות שהם עשויים בתחילה יוצרים צבירי תאים או ?...

There is increasing evidence that ovarian carcinomas are comprised of heterogeneous mixtures of cells and harbor so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for disease initiation, maintenance and relapse after conventional cytotoxic therapies1-3. Therefore, the development of molecular strategies targeting ovarian CSCs is an important goal and promises to improve the therapy of ovarian cancer patients.
A pre-requisite for the understanding of the molecular features of CSCs is their reliable isolation from the non-CSCs. However, identification of ovarian CSCs appears challenging. While CD133 expression and aldehyde dehydrogenase (ALDH) activity4,5 have been reported to mark ovarian CSCs, some data indicate that these markers are unstable6. Consistently, in ovarian cancer, other than for example in breast carcinoma7, expression of ALDH1 associates with favorable outcome8 and expression of the proposed stem cell marker CD44 variant has no prognostic value9. More recently, we have shown that expression of the embryonic stem cell protein SOX2 confers stemness to ovarian carcinoma cells10 and high SOX2 expression associates with clinically aggressive ovarian and breast carcinomas11,12. Therefore, in this report we use a lentiviral reporter construct containing a red fluorescence protein (RFP) whose expression is controlled by a SOX2 regulatory region, as a method to isolate putative ovarian CSCs.
By definition, CSCs can both self-renew and differentiate, giving rise to all tumor cell types. Putative CSC populations need to be analyzed in functional assays performed in vivo. For obvious reasons, in human cells such functional tests are confined to xenograft assays, comprising mostly transplantation of human tumor cells into immuno-compromised mice10,13.
An alternative in vitro method was offered by Brent Reynolds and Sam Weiss who firstly reported the so-called neurosphere assay as a surrogate assay evaluating stem potential in neural cells14. Dontu and colleagues later confirmed the use of this assay for evaluation of stem cell potential in breast cells15,16. Here, human mammary cells were plated in different numbers in serum-free medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), B-27 and heparin and cultured under non-adherent conditions for seven to ten days before sphere formation was scored by microscopy. Following this protocol with some adjustments in cell numbers, growth medium and supplements, several groups have explored in vitro stem cell potential from several cancer types such as breast17, brain18, pancreas19 and colon20 tumors. In ovarian carcinoma, we have recently reported feasibility of the spheres assay and compared its results to those collected in in vivo murine xenograft models10. We found that overexpression of the stem cell protein SOX2 enhanced both in vitro sphere formation as well as in vivo tumorigenicity of human ovarian carcinoma cells10. However, the frequency of sphere-initiating cells was higher than the frequency of tumor-initiating cells measured in vivo10 suggesting that either the sphere assay may lead to false positive results due to technical reasons or, alternatively, the in vivo assay may be inefficient and result in false negative results.
In this report, we analyze multi cell-based ovarian spheres assays in more detail, review the different protocols available in the literature and compare them to a single cell-based assay. We show that the single cell-based assay provides more accurate and reproducible results than multi cell-based assays, which can be highly influenced by the density of plated cells unless methylcellulose is added to the cultures to immobilize cells. However, also in single cell-based assays, in vitro sphere-initiating potential is observed at higher frequency than in vivo tumor-initiating potential.

<table><tbody><tr><td>Low-attachment plate</td><td>Corning</td><td>3474</td><td></td></tr><tr><td>MEGM</td><td>Lonza</td><td>CC-3151</td><td></td></tr><tr><td>Insulin</td><td>Lonza</td><td>CC-4136</td><td>SingleQuots&amp;trade; Kit</td></tr><tr><td>Hydrocortisone</td><td>Lonza</td><td>CC-4136</td><td>SingleQuots&amp;trade; Kit</td></tr><tr><td>EGF</td><td>Lonza</td><td>CC-4136</td><td>SingleQuots&amp;trade; Kit</td></tr><tr><td>EGF</td><td>Sigma</td><td>E9644</td><td>end concentration: 20 ng/ml</td></tr><tr><td>FGF</td><td>PeproTech</td><td>100-18B</td><td>end concentration: 20 ng/ml</td></tr><tr><td>B-27</td><td>Invitrogen/ Gibco</td><td>17504-044</td><td>end concentration: 1X</td></tr><tr><td>Heparin-Natrium-25000 IE</td><td>Ratiopharm</td><td>N68542.02</td><td>dilution 1:1,000</td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>FCS</td><td>Gibco</td><td>10500-064</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>21875-034</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s PBS (1X)</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Shield1</td><td>Clontech</td><td>632189</td><td>dilution 1:1,000</td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>21041-025</td><td></td></tr><tr><td>DMEM/F12 (powder)</td><td>Gibco</td><td>42400-010</td><td></td></tr><tr><td>Methyl cellulose</td><td>Sigma</td><td>M0387</td><td></td></tr><tr><td>Puromycin dihydrochloride</td><td>Applichem</td><td>A2856</td><td></td></tr><tr><td>Cell sorter</td><td>BD</td><td></td><td>Aria III cell sorter</td></tr><tr><td>FACS analyser</td><td>BD</td><td></td><td>Accuri c6 flow cytometer</td></tr><tr><td>Microscope</td><td>Olympus</td><td></td><td>IX50 Osiris</td></tr></tbody></table>

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.

במבחני התחומים קונבנציונליים, כמעט 40% מRFP + OVCAR-3 תאים לעומת 20% מתאי RFP- הולידו תחום גידול בודד בassay תחומים העיקרי (איור 4 א). יתר על כן, תחומים שהוקמו על ידי RFP + תאים היו גדולים יותר בגודל יותר מאלה שהוקמו על ידי תאי RFP-. כאשר מצו?...

Watch this Scientific Journal Video about Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays at JoVE.com

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

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.

הערכה של מאפייני תא גזע בקרצינומה של תאי השחלות אדם באמצעות Multi ומבחני Spheres מבוסס תא בודדים

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

evaluation-stem-cell-properties-human-ovarian-carcinoma-cells-using

Research

JoVE Journal

Medicine

9.5K Views.  University Hospital Basel. 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.

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

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.

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.

הקמת סרטן גזע תא תרבויות מן האדם הקונבנציונלי אוסטאוסרקומה

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

Cancer Research

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

ב Assay עמילות חוץ גופית mesothelial כי מודלים השלבים המוקדמים של גרורות סרטן השחלות

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

החולה הפיזיולוגי הנגזרות 3D Spheroids עבור הקרנת תרופות אנטי-נאופלסטית כדי למקד את תאי גזע הסרטן

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Heterotypic תלת ממדי<em&gt; במבחנה</em&gt; מידול של אינטראקציות stromal-אפיתל בייזום והתקדמות סרטן השחלות

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

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.

קבלת סרטן גזע כדורים מגינקולוגית סרטן השד גידולים

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

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

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 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

בידוד הרחבת האדם multiforme Glioblastoma גידול תאים שימוש Assay Neurosphere

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 ...

Neuroscience

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

הערכה של קובץ מצורף סרטן השחלות אליפטית ופלישה של תאי mesothelial בזמן אמת

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

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.

העשרה לתאי גזע Chemoresistant סרטן השחלות משורות תאים אנושיות

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

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Assay גיבוש Mammosphere משד אדם סרטן רקמות וקווים סלולריים

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

הערכה של מאפייני תא גזע בקרצינומה של תאי השחלות אדם באמצעות Multi ומבחני Spheres מבוסס תא בודדים

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