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Quantification of the Mammary Gland in Mice

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Quantification of the Mammary Gland in Mice QUANTITATIVE ANALYSIS OF MAMMARY GLAND IN MICE: EFFECTS OF PFN-1 KNOCK-OUT Nina Rudolph, Zhijie Ding, Partha Roy Ph.D University of Pittsburgh Center of Bioengineering and Biotechnology INTRODUCTION OBJECTIVE Breast cancer is the second leading cause of death among The objective of this research was to determine the effects women [1]. It is said that one in eight women will be of Profilin-1 knock-out by analyzing and quantifying the diagnosed with breast cancer within their lifetime [1]. Each mammary gland in mice, specifically looking at the longest year it is estimated that over 220,000 women in the United length from lymph node to terminal end bud, number terminal States will be diagnosed with breast cancer and more than end buds, and side branching. 40,000 women will die [1]. Breast cancer is a disease in which malignant cancer cells form in the tissue of the breast [1]. Once SUCCESS CRITERIA these malignant cancer cells reach the lymph or blood vessels, Since Profilin-1 could be a tumor suppressor protein, by this is known as invasive breast cancer. Invasive breast cancer knocking out the Profilin-1 three things should happen: the is cancer that spreads outside the membrane of the lobule or length from the end of the lymph node to the longest branch duct in the breast tissue. Cancer found as a result of this would be longer, there will be more terminal end buds, and migration is known as metastatic breast. Although with early there will be more side branching per 4x field when examined detection and treatment, most people do continue to live a under microscopy. normal life [1]. A protein named Profilin-1 is currently being researched to METHODS investigate how exactly it affects the migration of breast cancer. This study was designed to look the effect on knocking out Profilin-1 (PFN-1) is an actin binding protein that is involved in the PFN-1 protein. First, PFN1 was knocked out in 13 mice, the dynamic turnover and restructuring of the actin and 7 mice were untreated. Once this task was completed, the cytoskeleton [3]. In previous studies, it showed that the mice were sacrificed at six weeks, since that is considered the overexpression of PFN-1 reduces the migration of invasive age at which the mice are mature, and the mammary glands breast cancer [4]. Overexpression of a protein is excessive were extracted. The mammary glands were whole-mounted expression of a protein by producing too much of its effects or and stained using the protocol for carmine alum. Images were products [5]. This guides researchers to believe that PFN-1 is a taking of each of the slides containing the mammary gland helpful protein in preventing breast cancer to metastasize. using a 4x compound microscope. These images were then Another study that was performed brought forward that PFN-1 puzzle pieced together in Microsoft PowerPoint and were then may possibly be a tumor-suppressor [6]. Tumor-suppressor able to be analyzed and quantified. proteins are genes that normally restrain cell growth but, when Each of the mammary glands were measured in ImageJ to missing or inactivated by mutation, all cells to grow find the longest length from the end of the mammary gland to uncontrolled [7]. Pfn1 overexpression was found to sensitize the lymph node. This was done to determine to see PFN-1 MDA-MB-231 cells to apoptosis in response to cytotoxic effects on the overall growth of the mammary gland length. stimulus. This suggested for the first time that survival of The next entity to analyze was the terminal end buds breast cancer cells can also be negatively influenced by Pfn1 (TEB). To be able to find the total number of TEBs, each of up-regulation [6]. These findings may provide novel insights them were numbered in Microsoft PowerPoint. From here, the underlying Pfn1’s tumor-suppressive action [6]. area of each of the numbered TEBs were found using ImageJ. Based on this literature, there was a novel idea to quantify The areas were then divided into small, medium, and large and analyze the mammary gland in mice if the PFN-1 protein TEBs based upon our own scale for better quantifications. A was knocked out instead of overexpressed. Knocking out a small TEB was less than 9,000 pixels in area, a medium TEB protein in mice is known as a genetically engineered mouse was between 9,000 and 16,000 pixels in area, and a large TEB created by protein targeting in which a specific protein is was over 16,000 pixels. deleted or inactivated by homologous recombination, in order Lastly, the side branching within the mammary gland was to study the effects of its absence [8]. This will be done by counted per 4x field. During puberty, tubule formation is studying the mammary gland in mice. One main part of the coupled with branching morphogenesis which establishes the mammary gland is the terminal end buds. TEBs are highly basic arboreal network of ducts emanating from the nipple [9]. proliferated structures that are located on the tips of the One thing that had to be taking into consideration while looking invading ducts [9]. Carcinogen-induced mammary tumors in at the side branching was that a side branch do not bifurcate. mice are initiated in the TEBs. All of the data was implemented in Microsoft Excel for calculations. T-tests were performed on all of the data to assess 1 whether the means of two groups are statistically different from ACKNOWLEDGMENTS each other. Thank you to Dr. Partha Roy for allowing me to have this RESULTS amazing opportunity to work in his lab. Thank you to Zhijie After the T-tests were performed, the p-values were the Ding for being there to answer all of my questions and teaching following: me everything I learned in the lab. Thank you to Marion Joy, William Veon, Chang Jiang, Dave Gau for helping me in the Table 1: T-Test p-values lab anytime I needed it. Thank you to Dr. Wells lab for Length 0.0046 allowing access to your microscope. Thank you to Dr. Lee Total number of TEB’s 0.71 Adrian for your knowledge on the quantification of mammary Side Branching 0.50 glands in mice. Lastly, thank you to Jesse Lowe for all of your help in this class and answering all of my questions. The average length of the wild-type was 6.8 mm with a standard deviation of 1.4 mm and in the knock-out the average REFERENCES length was 9.5 mm with a standard deviation of 1.3 mm. The [1] "Breast Cancer Facts." Www.nationalbreastcancer.org. average number of TEBs in the wild-type was 18.4 with a N.p., n.d. Web. 15 Oct. 2013. standard deviation of 5.3 and the average number of TEBs in <http://www.nationalbreastcancer.org/breast-cancer-facts>. the knock-out was 17.5 with a standard deviation of 3.1. The [2] "Invasive Breast Cancer: Symptoms, Treatments, average number of branching per 4x field in the wild-type mice Prognosis." WebMD. WebMD, n.d. Web. 07 Dec. 2013. was 18.0 with a standard deviation of 6.0 and the average <http://www.webmd.com/breast-cancer/guide/invasive-breast- number of branching per 4x field in the knock-out was 19.8 cancer>. with a standard deviation of 3.3. [3] "Profilin." Wikipedia. Wikimedia Foundation, 12 July 2013. Web. 15 Oct. 2013. DISCUSSION <http://en.wikipedia.org/wiki/Profilin>. Knocking out the PFN-1 protein proved one of the three [4] Roy, Partha, Ph.D, and Et Al. "Overexpression of parts of the success criteria. The longest length from lymph Profilin Reduces the Migration of Invasive Breast Cancer node to terminal end bud was the only p-value was less than Cells." National Center for Biotechnology Information. U.S. 0.05, which is considered a significantly different. This proved National Library of Medicine, Feb. 2004. Web. 15 Oct. 2013. the first part of the success criteria was indeed successfully. <http://www.ncbi.nlm.nih.gov/pubmed/14691948>. This finding confirms that PFN-1 could possibly be tumor- [5] "Overexpression." Merriam-Webster. Merriam- suppressor protein. The mammary gland grew longer when the Webster, n.d. Web. 07 Dec. 2013. <http://www.merriam- protein was knocking out, this shows that when the protein is webster.com/medical/overexpression>. eliminated that the mammary gland are more likely to grow. [6] Roy, Partha, Ph.D, and Et Al. "Profilin-1 Although since the p-values for the total number of TEBs Overexpression Inhibits Proliferation of MDA-MB-231 Breast and side branching per 4x field were both greater than 0.05, the Cancer Cells Partly through P27kip1 Upregulation." US last two parts of the success criteria were not significant. This National Library of Medicine National Institutes of Health. may be due to the fact that PFN-1 does not specifically affect NCBI, 1 June 2010. Web. 15 Oct. 2013. the number of TEBs in the mammary gland, but it may affect <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872929/>. the size of the TEBs, since the TEBs contain stem cell that [7] "Tumor Suppressor Gene." - Glossary Entry. U.S. could one day turn into cancer. The branching may not be National Library of Medicine, 3 Dec. 2013. Web. 7 Dec. 2013. significant because PFN-1 may not directly contribute to the <http://ghr.nlm.nih.gov/glossary=tumorsuppressorgene>. branching but the length of the mammary gland as confirmed [8] "Knockout Mouse (redirected from Knock-out by these results.
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