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 border="0" cellpadding="0" cellspacing="0">
	<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...

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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 ...

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28.2K 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

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

אסטרטגיה לזהות מוטציות דה נובו בהפרעות נפוצות כגון אוטיזם וסכיזופרניה

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

חי הדמיה של תגובות סמים בmicroenvironment הגידול במודלים עכבריים של סרטן השד

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

חניכה של סרטן השד גרורתי על ידי מיקוד של האפיתל ductal עם Adenovirus-Cre: דגם רומן מהונדס עכבר של סרטן השד

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

פרופיל של מיקרו RNA המוסדר אסטרוגן בתאי סרטן השד

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

שלוש תרבויות ממדים: כלי ללמוד רגיל אדריכלות acinar לעומת שינוי ממאיר של תאי שד

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

הזרקת orthotopic של תאי סרטן השד לחלב שומן Pad של עכברים לחקר התפתחות גידול.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

<em&gt; Ex vivo</em&gt; דגם לחקר הורמוני פעולה בשד האדם

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

שיטות לExplants רקמות Culturing עצם ירך האדם ללמוד התיישבות תאי סרטן השד גרורתי של הנישה

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

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 ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

In vivo לשעבר vivo גישות לחקר סרטן השחלות גרורתי התיישבות של מבני ספוט החלב בהצפק שומני

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...