We are thankful to Dr. Richard Cerione, Dr. Marc Antonyak, and Kelly Sullivan, Cornell University, for providing technical advice, and to Dr. Gerlinde Van de Walle, Cornell University, for sharing their Olympus CKX41 inverted microscope.

1. הכנה של 3% 2-Hydroxyethyl Agarose <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לתוך בקבוק נקי, יבש 100 מיליליטר זכוכית, להוסיף 0.9 גרם של agarose 2-hydroxyethyl (Agarose VII) ואחריו 30 מיליליטר של מים מזוקקים. </li><li style=";text-align:right;direction:rtl"> מיקרוגל את התערובת במשך 15 שניות ועדינות מערבולת. חזור על שלב זה לפחות עוד שלוש פעמים עד שאבקת agarose מתמוססת באופן מלא. </li><li style=";text-align:right;direction:rtl"> החיטוי הבקבוק המכיל פתרון במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> אפשר פתרון agarose להתקרר לRT לפני שימוש נוסף. אחסן את הפתרון בRT. </li></ol> 2. הכנת השכבה התחתונה: 0.6% Agarose ג&#39;ל <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> טרום חם 5 מיליליטר ו -10 מיליליטר בטפטפות 37 ° C חממה כמה למנוע agarose לחיזוק בפיפטה בעת טיפול. </li><li style=";text-align:right;direction:rtl"> חלקית לשחרר את מכסה הבקבוק ומיקרוגל 3% פתרון agarose 2-hydroxyethyl שהוכן מראש, במשך 15 שניות. ואז, בעדינות מערבולת הפתרון ומיקרוגל לעוד 15 שניות. זהירות: היזהרי בעת מתערבלת agaroפתרון se כי הפתרון עולה כאשר נחשף לאוויר ויכול לגלוש. </li><li style=";text-align:right;direction:rtl"> אם יש ג&#39;ל השייר המוצק בבקבוק, מיקרוגל לעוד כמה שניות. </li><li style=";text-align:right;direction:rtl"> שמור את הבקבוק המכיל את פתרון agarose באמבט מים C ° 45 במהלך השלבים הבאים כדי למנוע את פתרון agarose מהגיבוש בטרם עת. </li><li style=";text-align:right;direction:rtl"> תקשורת MCF10DCIS חמה באמבט מים 37 מעלות צלזיוס. הערה: תקשורת MCF10DCIS מורכבת DMEM / F12, 5% בסרום סוס, 5% סטרפטומיצין פניצילין. </li><li style=";text-align:right;direction:rtl"> העברה 3 מיליליטר של פתרון agarose 3% באמצעות טפטפות המחוממות מראש לתוך צינור חרוטי 50 מיליליטר סטרילי. </li><li style=";text-align:right;direction:rtl"> מייד להוסיף 12 מיליליטר של תקשורת MCF10DCIS החמה ועדינות להפוך את צינור חרוטי לערבב agarose עם התקשורת. נסה לא ליצור בועות כפי שיפריע למושבה ספירה מאוחר יותר. </li><li style=";text-align:right;direction:rtl"> בעדינות להוסיף 2 מיליליטר של תערובת זו לכל גם צלחת תרבות 6 היטב בלי להרכיב את כל בועות אוויר. </li><li style=";text-align:right;direction:rtl"> דגירה horizonta צלחת תרבות 6 היטבlly על משטח שטוח ב 4 מעלות צלזיוס במשך שעה 1 כדי לאפשר את התערובת לביסוס. </li><li style=";text-align:right;direction:rtl"> לאחר מתמצק התערובת, מניח את הצלחת לתוך 37 ° C חממה למשך 30 דקות. השכבה התחתונה היא מוכנה לשימוש. </li></ol> 3. הכנת השכבה המכיל הנייד: 0.3% Agarose ג&#39;ל <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תאי Trypsinize MCF10DCIS ולדלל אותם לריכוז תא של 4 x 10 4 מיליליטר /. </li><li style=";text-align:right;direction:rtl"> קח 2 מיליליטר של agarose 3% באמצעות טפטפות מחוממות מראש ולהעביר לתוך צינור חרוטי 50 מיליליטר סטרילי. </li><li style=";text-align:right;direction:rtl"> מייד להוסיף 8 מיליליטר של תקשורת MCF10DCIS לצינור חרוטי ועדינות להפוך לערבב agarose עם התקשורת. הימנע יצירת בועות. </li><li style=";text-align:right;direction:rtl"> קח 2 מיליליטר של תאי MCF10DCIS (4 x 10 4 / מיליליטר) ולטפל עם BB-CLA (0 מיקרומטר (DMSO) או 1 מיקרומטר). </li><li style=";text-align:right;direction:rtl"> בדילול 1: 1, לערבב את התאים עם agarose 0.6%. </li><li style=";text-align:right;direction:rtl"> קח 1 מיליליטר של תערובת התא-agarose ועדינות להוסיף על השכבה התחתונה של עמ &#39;תרבות 6 היטב(2 x 10 4 תאים / מיליליטר) מאוחר. </li><li style=";text-align:right;direction:rtl"> מניחים את צלחת תרבות 6 היטב אופקי על משטח שטוח ב 4 מעלות צלזיוס במשך לפחות 15 דקות, כדי לאפשר את השכבה העליונה כדי לחזק. </li><li style=";text-align:right;direction:rtl"> לאחר מתמצק התערובת, מניח את הצלחת לתוך 37 ° C חממה במשך שבוע לפני הוספת שכבת ההאכלה. </li></ol> 4. הכנת שכבת המזין: 0.3% Agarose ג&#39;ל <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מיקרוגל 3% פתרון agarose 2-Hydroxyethyl שהוכן מראש, במשך 15 שניות. מערבולת בעדינות את הפתרון ומיקרוגל לעוד 15 שניות. </li><li style=";text-align:right;direction:rtl"> לאזן את בקבוק פתרון agarose באמבט מים 45 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> לחמם את תקשורת MCF10DCIS באמבט מים 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> מערבבים 1 מיליליטר של 3% פתרון agarose עם 9 מיליליטר של תקשורת MCF10DCIS החמה לתוך צינור חרוטי 50 מיליליטר ועדינות להפוך לערבב agarose עם התקשורת. הימנע יוצר בועות אוויר. </li><li style=";text-align:right;direction:rtl"> פנק את התערובת עם BB-CLA (0 מיקרומטר (DMSO) או 1 מיקרומטר). </li><li style=";text-align:right;direction:rtl"> בעדינות להוסיף 1 מיליליטר של תערובת זו (בלי למינג בועות) לבאר כל צלחת תרבות 6 היטב המכילה את החלק התחתון ושכבות רכות. </li><li style=";text-align:right;direction:rtl"> מניחים את צלחת תרבות 6 היטב אופקי על משטח שטוח ב 4 מעלות צלזיוס במשך לפחות 15 דקות, כדי לאפשר את התערובת לביסוס. </li><li style=";text-align:right;direction:rtl"> לאחר שכבת המזין מתמצקת, למקם את הצלחת לתוך 37 ° C חממה. </li><li style=";text-align:right;direction:rtl"> חזור שבועי הליך זה על ידי האכלה כיסה 1 מיליליטר של 0.3% פתרון agarose / בינוניים / טיפול על שכבת המזין הקיימת כדי לחדש את התאים עם תקשורת חדשה עד הקמת מושבה הוא ציין. הערה: אגר בשכבות הרכות ומזין היא רכה מאוד, ולכן, חומרים מזינים הגיעו משכבת ​​המזין יהיו מוכנים לפזר את השכבה המכיל תא כדי להגיע לתאים. </li></ol> 5. איסוף נתונים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחר 2.5 שבועות של צמיחת תאים באגרו הרך, לספור את מספר המושבות בכל טוב באמצעות מיקרוסקופ אור. כדי להקל על כימות, להדפיס רשת על שקיפות ולצרף את הרשת ל6-גם צלחת לסיוע באיתור שבו התאים נמצאים בספירה. מאז גודל מושבה (כלכמת ידי הקוטר של כל מושבה) ישתנה, להגדיר מראש גודל מושבה התייחסות כדי לקבוע אילו מושבות יהיה כבש. לדוגמא, כוללים גדלי מושבה של 70 מיקרומטר או גדולים יותר בניתוח הנתונים. </li><li style=";text-align:right;direction:rtl"> אחסן את הדגימות ב 4 ° C כדי למנוע היווצרות מושבה נוספת ולספירת עתיד. חותם את צלחת תרבות 6 היטב עם Parafilm כדי למנוע את ג&#39;לי מהתייבשות. </li></ol>

<ol>
	<li>Hamburger, A. W., Salmon, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+bioassay+of+human+tumor+stem+cells.">Primary bioassay of human tumor stem cells.</a> Science. 197, 461-463 (1977).</li><li>Wang, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+signaling+regulating+anchorage-independent+growth+of+cancer+cells.">Molecular signaling regulating anchorage-independent growth of cancer cells.</a> Mt Sinai J Med. 71 (6), 361-367 (2004).</li><li>Vossenaar, E. R., Zendman, A. J., van Venrooij, W. J., Pruijn, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAD,+a+growing+family+of+citrullinating+enzymes:+genes,+features+and+involvement+in+disease.">PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease.</a> Bioessays. 25 (11), 1106-1118 (2003).</li><li>Horibata, S., Coonrod, S. A., Cherrington, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+peptidylarginine+deiminase+enzymes+in+disease+and+female+reproduction.">Role for peptidylarginine deiminase enzymes in disease and female reproduction.</a> J Reprod Dev. 58 (3), 274-282 (2012).</li><li>Mohanan, S., Cherrington, B. D., Horibata, S., McElwee, J. L., Thompson, P. R., Coonrod, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+role+of+peptidylarginine+deiminase+enzymes+and+protein+citrullination+in+cancer+pathogenesis.">Potential role of peptidylarginine deiminase enzymes and protein citrullination in cancer pathogenesis.</a> Biochem Res Int. , 895343 (2012).</li><li>McElwee, J. 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=Identification+of+PADI2+as+a+potential+breast+cancer+biomarker+and+therapeutic+target.">Identification of PADI2 as a potential breast cancer biomarker and therapeutic target.</a> BMC Cancer. 12, 500 (2012).</li><li>Knight, J. 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=Peptidylarginine+deiminase+inhibition+disrupts+NET+formation+and+protects+against+kidney,+skin+and+vascular+disease+in+lupus-prone+MRL/lpr+mice.">Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice.</a> Ann Rheum Dis. , 1-8 (2014).</li><li>Miller, F. R., Santner, S. J., Tait, L., Dawson, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCF10DCIS.com+xenograft+model+of+human+comedo+ductal+carcinoma+in+situ.">MCF10DCIS.com xenograft model of human comedo ductal carcinoma in situ.</a> J Natl cancer Inst. 92, 1185-1186 (2000).</li><li>Fan, D., Morgan, L. R., Schneider, C., Blank, H., Fan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+evaluation+of+human+tumor+chemosensitivity+in+the+soft-agar+assay+and+its+clinical+correlations.">Cooperative evaluation of human tumor chemosensitivity in the soft-agar assay and its clinical correlations.</a> J Cancer Res Clin Oncol. 109, 23-28 (2000).</li><li>Hamburger, A. W., White, C. P., Dunn, F. E., Citron, M. L., Hummel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+human+tumor+colony+growth+in+soft+agar+by+serum.">Modulation of human tumor colony growth in soft agar by serum.</a> Int J Cell Cloning. 1 (4), 216-229 (1983).</li><li>Anderson, S. N., Towne, D. L., Burns, D. J., Warrior, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+soft+agar+assay+for+identification+of+anticancer+compound.">A high-throughput soft agar assay for identification of anticancer compound.</a> J Biomol Screen. 12, 938-945 (2007).</li></ol>

The authors have nothing to disclose.

קצב היווצרות מושבה באגרה רך משתנה בהתאם לסוג התא 9. לכן, מספר התאים להתחיל עם צריך להיות מותאם ובהתאם. מגוון התחלה הציע הוא בין 5 x 02-01 אוקטובר x 10 4 תאים לכל גם באמצעות צלחת 6 היטב. בנוסף, גודל מושבה משתנה בהתאם לקצב הצמיחה של כל תא. לכן, יש צורך בחתך מוגדר ?...

Both non-transformed (normal) and transformed cells can readily proliferate in a 2D monolayer culture. This form of adherent cell growth is quite dissimilar from that which occurs in vivo where, in the absence of mitogenic stimulation, cells do not often rapidly divide within their microenvironment. The soft agar assay on the other hand is distinct from 2D culture systems because it quantifies tumorigenicity by measuring a cell&#8217;s ability to proliferate and form colonies in suspension within a semi-solid agarose gel1. In this setting, non-transformed cells are unable to rapidly propagate in the absence of anchorage to the extracellular matrix (ECM) and undergo apoptosis, a process known as anoikis. In contrast, cells that have undergone malignant transformation lose their anchorage dependence due to activation of signaling pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt and Rac/Cdc42/PAK. Therefore, these cells are able to grow and form colonies within the semi-solid soft agar matrix2.
A common use of the soft agar assay is to test whether specific compounds, such as PADI inhibitors, are able to suppress tumor growth in vitro. In general, colony count or colony sizes are quantitative read-outs from the assay that can be compared between control and treatment groups to assess differences in cellular tumorigenicity. Therefore, if one finds that colony formation is inversely correlated with increasing drug concentration, then a conclusion could be drawn that the drug is an effective inhibitor of tumorigenicity in vitro. On the other hand, if the drug does not affect colony formation, the drug is either not at the appropriate dosage or it is not an effective tumorigenic inhibitor. Aside from using a soft agar assay to test the anti-tumor effect of a drug, this assay can also be used to probe the relationship between a specific gene and tumorigenesis. For example, the effect of suppressing PADI2 expression on tumorigenicity can be addressed by PADI2-specific siRNA treatment.
PADIs are calcium-dependent enzymes that post-translationally modify proteins by converting positively charged arginine residues into neutrally charged citrulline in a process known as citrullination or deimination3-5. We have recently found that peptidylarginine deiminase 2 (PADI2) may function as a novel breast cancer biomarker and that PADI inhibitors represent candidate therapies for early stage breast cancers6. For example, we have previously demonstrated that a &#8220;pan-PADI&#8221; inhibitor, Cl-amidine, suppresses the proliferation of breast cancer cells using 2D monolayers and that the inhibitor suppressed the growth of 3D tumor spheroids6. In this report, we extend these studies, and highlight the utility of the soft agar assay, by testing the efficacy of a new PADI inhibitor, BB-CLA, in suppressing the growth of MCF10DCIS breast cancer colonies7. We note that we used MCF10DCIS cells for this experiment because they are oncogenic derivatives of non-transformed human MCF10A cells and because they contain high steady state levels of PADI2 protein8. We hypothesize that PADI2 enzymatic activity plays a key role in the tumorigenicity of this cell line and that BB-CLA-mediated inhibition of PADI2 activity will suppress cancer progression.

<table><tbody><tr><td>Zeiss Axiopot</td><td>Carl Zeiss Microscopy</td><td>1021859251</td><td></td></tr><tr><td>Inverted Microscope</td><td>Olympus</td><td>CKX41</td><td></td></tr><tr><td>DMEM/F-12</td><td>Lonza BioWhittaker</td><td>12-719F</td><td></td></tr><tr><td>HyClone Donor Equine Serum</td><td>Fisher Scientific</td><td>SH30074.03</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>2-Hydroxyethylagarose: Type VII, low gelling temperature</td><td>Sigma-Aldrich</td><td>39346-81-1</td><td></td></tr></tbody></table>

utilization of the soft agar colony formation assay to identify inhibitors of tumorigenicity in breast cancer cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Assay הקמת המושבה אגר הרך יכול לשמש למגוון רחב של יישומים המתעדים את tumorigenicity של תאים סרטניים. יתרון עיקרי של שיטה זו הוא שמטריצת חצי מוצקה סלקטיבי מעדיפה את הצמיחה של תאים שיכולים להתרבות באופן עצמאי מעגן-. תכונה זו היא בעיקר הפגינה תאים סרטניים אך לא בתאים נורמלים. אנחנ...

Watch this Scientific Journal Video about Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells at JoVE.com

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

ניצול של assay כינונה רך אגר מושבה לזיהוי מעכבי tumorigenicity בתאי סרטן השד

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

utilization-soft-agar-colony-formation-assay-to-identify-inhibitors

Research

JoVE Journal

Medicine

28.5K Views.  Cornell University. Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro. 

Video: Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&#954;B Activity and Breast Cancer Stem Cells

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an anti-inflammatory drug to standard cancer regiments may improve patient outcome. One such drug, aspirin (acetylsalicylic acid, ASA), has been explored for cancer chemoprevention and anti-tumor activity. Besides inhibiting the cyclooxygenase 2-prostaglandin axis, ASA&#39;s anti-cancer activities have also been attributed to nuclear factor &#312;B (NF&#312;B) inhibition. Because prolonged ASA use may cause gastrointestinal toxicity, a prodrug strategy has been implemented successfully. In this prodrug design the carboxylic acid of ASA is masked and additional pharmacophores are incorporated.
This protocol describes how we synthesized an aspirin-fumarate prodrug, GTCpFE, and characterized its inhibition of the NF&#312;B pathway in breast cancer cells and attenuation of the cancer stem-like properties, an important NF&#312;B-dependent phenotype. GTCpFE effectively inhibits the NF&#312;B pathway in breast cancer cell lines whereas ASA lacks any inhibitory activity, indicating that adding fumarate to ASA structure significantly contributes to its activity. In addition, GTCpFE shows significant anti-cancer stem cell activity by blocking mammosphere formation and attenuating the cancer stem cell associated CD44+CD24- immunophenotype. These results establish a viable strategy to develop improved anti-inflammatory drugs for chemoprevention and cancer therapy.

This procedure will demonstrate how we synthesized and characterized the anti-NF&#954;B and anti-cancer stem cell activity of an aspirin-fumarate prodrug.

סינתזה ואפיון של prodrug אספירין-fumarate המעכבת תאי גזע סרטניים פעילות השד NFκB

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an ...

Cancer Research

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

Assay Spheroid למדוד TGF-β-Induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

השימוש של העכבר בתאי סרטן הגידול בתוך שיטת הפלישה מחוץ למבחנה כמדד של התנהגות התא אונגניים

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

Genetics

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug screening assays utilize cytotoxicity as one of the critical characteristics to be profiled for individual compounds. Cells in culture are a useful model to assess cytotoxicity before proceeding to follow up on promising lead compounds in more costly and labor-intensive animal models. We describe a strategy to identify compounds that affect cell growth in a tdTomato expressing human neural stem cells (NSC) line. The strategy uses two complementary assays to assess cell number. One assay works via the reduction of 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as a proxy for cell number and the other directly counts the tdTomato expressing NSCs. The two assays can be performed simultaneously in a single experiment and are not labor intensive, rapid, and inexpensive. The strategy described in this demonstration tested 57 compounds in an exploratory primary screen for toxicity in a 96-well plate format. Three of the hits were characterized further in a six-point dose response using the same assay set-up as the primary screen. In addition to providing excellent corroboration for toxicity, comparison of results from the two assays may be effective in identifying compounds affecting other aspects of cell growth.

It is often necessary to assess the potential cytotoxicity of a set of compounds on cultured cells. Here, we describe a strategy to reliably screen for toxic compounds in a 96-well format.

אסטרטגיה לזיהוי תרכובות המשפיעות על צמיחת תאים והישרדות בתאי מיונקים מתורבתים בתפוקה נמוכה לבינונית

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug ...

Biochemistry

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

הערכה במבחנה של טרנספורמציה אונקוגנית בתאי אפיתל ממ"ר אנושיים

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

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

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

בידוד והערכה תפקודית של תאי גזע מסרטן השד האנושי מדגימות תאים ורקמות

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

<em&gt; במבחנה</em&gt; תרדמת דגם של סרטן השד רגיש לאסטרוגן במוח העצם: כלי ללימודי מנגנון מולקולרי והשערת דור

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Assay גיבוש רך אגר המושבה

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Biology

Soft Agar Colony Formation Assay: A Method to Test Effects of Novel Compounds on Cancer Cell Proliferation

Source: Horibata A. et. al., <a href="https://www.jove.com/v/52727/utilization-soft-agar-colony-formation-assay-to-identify-inhibitors"> Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells.</a> J. Vis. Exp. (2015).
This video describes a soft agar colony formation assay to test the effects of a PADI enzyme inhibitor and BB-Cl-amidine on breast cancer tumorigenicity in vitro. This assay also enables the quantitative assessment of cell transformation potential within the context of genetic perturbations.

בדיקת היווצרות מושבת אגר רכה: שיטה לבדיקת השפעות של תרכובות חדשות על התפשטות תאים סרטניים

Source: Horibata A. et. al.,  Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells. J. Vis. ...

JoVE Encyclopedia of Experiments

Breast Cancer

In vitro studies

Methyl Cellulose-Based CFU Assay: A Method to Determine the Effect of Anti-cancer Compounds on Colony Formation in Leukemic Cells

Source: Kesarwani, M., et al. <a href="https://www.jove.com/t/58194">Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence.</a> J. Vis. Exp. (2019).
This video describes the protocol for methyl cellulose-based colony formation unit or CFU assay, which helps identify and count the number of colonies formed by leukemic cells in response to the treatment with anticancer compounds.

בדיקת CFU מבוססת מתיל תאית: שיטה לקביעת ההשפעה של תרכובות אנטי-סרטניות על היווצרות מושבות בתאים לוקמיים

Source: Kesarwani, M., et al. Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence. J. Vis. Exp. (2019).
This video describes the ...