We would like to thank NIH, LACaTS and Ochsner clinic foundation for the financial support.

Tüm farenin deneyleri Tıp LSU Okulu Kurumsal Hayvan Bakımı ve Kullanımı komite (IACUC) tarafından onaylanan protokollere uygun olarak yapıldı. Tüm cihazlar diseksiyon öncesinde otoklavlanmalıdır gerekir. Fare göğüs tümörlerinden gelen primer hücre hazırlanması 1. <ol><li> Genetik olarak, daha önce tarif edildiği gibi 6 spontan tümörler geliştirmektedir fare meme tümörü virüs Polyoma Orta T-antijen (MMTV PyMT transgenik fare) ile tasarlanmış olan bir fare modeli kullanarak. </li><li> İzofluran (% 99.9) ile fareler kurban ve steril bir ortamı (Şekil 1A) sağlamak için% 75 etanol püskürtün. </li><li> Makasla boyun üretral orifis alanı cildi açın. Organların (Şekil 1B) kolay erişim sağlamak için cilt aşağı pin. </li><li> Meme, meme tümörü izole ve soğuk, steril fosfat tamponlu tuzlu su (PBS) (pH 7.4) (Şekil 1C) ile yıkayın. </li><li> Tümörün aktarınsteril 10cm doku kültürü plakası yaklaşık 1 mm 3 adet steril jilet ve makas ile doğrayın ve. Daha sonraki aşamalar, steril bir laminer akış başlığı içinde yapılmalıdır. </li><li> 37 ° C &#39;de 2 saat süre ile bir rocker 1 mg / ml lik bir nihai konsantrasyonda serumsuz DMEM ortamı içinde çözülmüş kollajenaz ile kesilmiş tümör dokusu inkübe edin. X gr 200&#39;de 3 dakika boyunca tümör dokusu süspansiyon santrifüjleyin. Not: Kolajenaz inkübasyon doku enzimatik ayrışması için gereklidir. </li><li> Süpernatant atılır ve 200 x g&#39;de 3 dakika boyunca santrifüj ile, PBS (pH 7.4) ile Çökelti 3 kez yıkayın. Son yıkamadan sonra, süpernatant süzün. </li><li> Nutating ise 37 ° C &#39;de 5 dakika boyunca tripsin EDTA, 2 ml ile doku yeniden süspanse edin. 200 x g&#39;de 3 dakika boyunca bir süspansiyon santrifüj ve yıkama tekrarlayın. </li><li> 10 cm plaka, normal büyüme ortamında (DMEM,% 10 FBS, 2X pen / strep) doku pelletini. DeğişiklikOrtam Sonraki 3 gün boyunca her gün. İstediğiniz hücre sayısı kadar Kültür hücreleri elde edilir. NOT: Yaklaşık 1x10 7 hücreleri bu prosedür izole edilecektir. MMTV PyMT karşılaştırıldığında örneğin MMTV-Neu gibi diğer fare meme tümör oluşumu modelleri, hücrelerin daha düşük verim verir. Bir düşük değerli ürününü oluşturmak modeller kullanılarak, bu artık bir daha büyük bir tümör ve Kültür hücreleri ile başlamak için faydalıdır. </li></ol> 2. Fare Akciğer Ekstraksiyon Akciğer Metastazı gözünüzde canlandırın için <ol><li> Akciğer çıkarma öncesinde, karaciğer ve diyaframı çıkarın. Akciğerleri rahatsız etmeden kaburga yerinden. </li><li> Makas ince çift, şah damarından daha yakın olan trakea üst kısmını açın. </li><li> Bir kateter kullanılarak, akciğer (Şekil 2A) doldurmak için trakea yoluyla Z-fix ya da Hindistan, mürekkep gibi 1 ml bir madde enjekte edilir. Göm parafin Akciğerler Z-düzeltin. </li><li> Fekete çözümü Hindistan mürekkep enjekte akciğerleri De-leke(100 mi% 70 alkol,% 10 tamponlu formalin 10 ml buzlu asetik asit 5 mi) 5 dakika boyunca. Çapında bir ≥ 0.5 mm ile ikincil bölgelerde tümör nodülleri tanımlayın metastaz olarak nodüller belirler. NOT: tümör tarafından bronşlar ve bronşiyol tıkanmaya, tümör nodülleri% 15 Hindistan mürekkebi absorbe edemez ve bu nedenle normal akciğer dokusu siyah olacak ve tümör nodülleri beyaz renk 7 olacak dolayı. Bu tek tek tümör nodülleri (Şekil 2B) görselleştirme sağlayacaktır. </li></ol> Fare Embriyolar gelen fare embriyonik fibroblastlar (MEFS) hazırlanması 3. <ol><li> İzofluran (% 99.9) ile birlikte hamile fareler kurban ve steril bir ortam sağlamak için% 75 etanol püskürtün. </li><li> Makasla boyun üretral orifis alanı cildi açın ve rahim embriyolar izole. </li><li> Cildi açarken embriyolar rahatsız etmemek için dikkatli olun. </li><li> Rahim boynuzları çıkarın ve soğuk ste onları yıkayınPBS (pH 7.4) içinde çözündürülür. </li><li> Buz soğukluğunda PBS (pH 7.4) içeren 10 cm&#39;lik tabak içinde olan çok dikkatli bir şekilde plasenta ve yer embriyolar embriyo ayırın. </li><li> Embriyo kafa, karaciğer ve gut çıkarın ve PBS (pH 7.4) ihtiva eden bir 12 gözlü doku kültür plakalarında kalan kısmı yerleştirin. </li><li> Başka bir 12 kuyu plaka içeren tripsin EDTA embriyo gövdesini aktarın ve steril bir jilet ve makas kullanarak küçük parçalara (~ 1 mm 3) oyulmuş. 37 ° C&#39;de 5 dakika boyunca tripsin EDTA, 5 ml kıyılmış malzeme inkübe edin. </li><li> Doğranmış embriyolar 1 ml cenin sığır serumu (FBS) eklenerek tripsin inaktive. Santrifüj, 10 dakika boyunca 200 x g hızında süspansiyonu, dikkatli bir şekilde, süpernatant kaldırmak sıcak DMEM ortamı, 1 ml ile pelet tekrar süspansiyon ve 10 mi DMEM (% 20 FBS ve 2X penisilin / streptomisin içeren 10 cm&#39;lik tabak bütün içeriğini aktarmak ). </li><li> İki gün için orta her gün değiştirin. Kültür, istenen hücre kadar hücrelernumara elde edilir. </li></ol>

<ol>
	<li>Minn, A. J., 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=Genes+that+mediate+breast+cancer+metastasis+to+lung.">Genes that mediate breast cancer metastasis to lung.</a> Natur. 436 (7050), 518-524 (2005).</li><li>Poste, G., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+of+cancer+metastasis.">The pathogenesis of cancer metastasis.</a> Natur. 283 (5743), 139-146 (1980).</li><li>Wan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Metastasis+Moving+New+Biological+Insights+into+the+Clinic.">Tumor Metastasis Moving New Biological Insights into the Clinic.</a> Nat Med. 19 (11), 1450-1464 (2013).</li><li>Bergers, G., Benjamin, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumorigenesis+and+the+angiogenic+switch.">Tumorigenesis and the angiogenic switch.</a> Nat Rev Cancer. 3 (6), 401-410 (2003).</li><li>Frese, K. K., Tuveson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+mouse+cancer+models.">Maximizing mouse cancer models.</a> Nat Rev Cancer. 7 (9), 654-658 (2007).</li><li>Guy, C. T., Cardiff, R. D., Muller, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mammary+tumors+by+expression+of+polyomavirus+middle+T+oncogene:+a+transgenic+mouse+model+for+metastatic+disease.">Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease.</a> Mol Cell Biol. 12 (3), 954-961 (1992).</li><li>Wexler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+Identification+of+Experimental+Pulmonary+Metastases.">Accurate Identification of Experimental Pulmonary Metastases.</a> J Natl Cancer Inst. 36 (4), 641-645 (1966).</li><li>Boczkowski, D., Nair, S. K., Nam, J. H., Lyerly, H. K., Gilboa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+tumor+immunity+and+cytotoxic+T+lymphocyte+responses+using+dendritic+cells+transfected+with+messenger+RNA+amplified+from+tumor+cells.">Induction of tumor immunity and cytotoxic T lymphocyte responses using dendritic cells transfected with messenger RNA amplified from tumor cells.</a> Cancer Res. 60 (4), 1028-1034 (2000).</li><li>Vargo-Gogola, T., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+breast+cancer:+one+size+does+not+fit+all.">Modelling breast cancer: one size does not fit all.</a> Nat Rev Cancer. 7 (9), 659-672 (2007).</li><li>Watts, A. M., Kennedy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+Tumor+Foci+in+an+Experimental+Murine+Tumor+Model+Using+Computer-Assisted+Video+Imaging.">Quantitation of Tumor Foci in an Experimental Murine Tumor Model Using Computer-Assisted Video Imaging.</a> Anal Bioche. 256 (2), 217-219 (1998).</li><li>Rosenberg, S. A., 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=Regression+of+Established+Pulmonary+Metastases+and+Subcutaneous+Tumor+Mediated+by+the+Systemic+Administration+of+High-Dose+Recombinant+Interleukin+2.">Regression of Established Pulmonary Metastases and Subcutaneous Tumor Mediated by the Systemic Administration of High-Dose Recombinant Interleukin 2.</a> J Exp Me. 161 (5), 1169-1188 (1985).</li><li>Fantozzi, A., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+breast+cancer+metastasis.">Mouse models of breast cancer metastasis.</a> Breast Cancer Re. 8 (4), 212 (2006).</li><li>Veer, 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=Gene+expression+profiling+predicts+clinical+outcome+of+breast+cancer.">Gene expression profiling predicts clinical outcome of breast cancer.</a> Nature. 415 (6871), 530-536 (2002).</li></ol>

Yazarlar herhangi bir açıklama yok.

Bu protokol, taze fare tümör primer hücrelerin izole edilmesi içindir. Bu yöntemle hazırlanan lizatlar protein, DNA ve RNA analizleri için de yararlıdır. Primer meme tümörün kanserli hücreleri genellikle beyin, akciğer ve kemik 1 metastaz. Dahil metastaz saptanması için akciğer çıkarmak için kullanılan bir tekniktir. Şekil 2B&#39;de görüldüğü gibi, bir mürekkep lekesi ile akciğer boyama tümör nodülleri görünüm için izin verir. Z-fix çözeltisi içinde akciğerlere Tespit bize akciğer bölümleri yapmak ve metastatik mekanizmalarını incelemek için uygun antikorlar ile leke sağlayacaktır. Ayrıca fare embriyonik fibroblastlar izolasyonu için bir protokol dahildir. Bu, aynı hayvan türü embriyosu akciğer fibroblast primer tümör hücrelerin gen ekspresyonunu karşılaştırmak için faydalıdır. Birincil hücre ve MEFS hazırlanmasında, en kritik adım malzemenin kıyma olduğunu. Lo uygun arıza olmadanw hücre sayısı elde edilecektir. Düşük hücre sayısı elde edilir, protokol de takip edilebilir, ancak hücre akciğer ekstraksiyonunda .. daha uzun bir dönem için daha kültive edilmesi gerekecektir uygun boyanması ve destaining tümör nodülü görselleştirme için izin vermek için gereklidir. Akciğerlerden RNA / protein ayıklamak için, lekesiz ve sabitlenmemiş olan akciğerler kullanmak iyi olacaktır. Bununla birlikte, RNA sabit dokularda da 8 elde edilebilir. Meme kanseri metastatik evre etkilenen bireysel 2 büyük tehdit bu yana, meme tümör oluşumu tüm bileşenlerini görselleştirmek için önemlidir. Meme kanseri ilerlemesi mekanizmalarını anlamada büyük bir meydan okuma hastalığı 9 ilerlemesini özetlemek modellerin bulunmasıdır. Meme tümör oluşumu ve metastaz moleküler mekanizmaların açıklanması, fare 9, 10 olarak, hayvan modelleri kullanılarak elde edilmiştir. En yaygın yolu mous indüklemee meme tümörleri, genetik, onkogenler veya tümör bastırıcılarının mutasyon öncesi bertaraf kanserojen tanıtan ve son derece invaziv kanser hücrelerini enjekte 10-12 ile fareler mühendis vardır. Elde edilen tümörler daha sonra gen ekspresyonu için analiz edilebilir. Hastanın 13 klinik sonuçlarını tahmin beri meme kanseri araştırmaları için, gen ifadesi profilleme önemlidir. Bazı sınırlamalar meme tümörogenez okuyan fare modeli ile mevcuttur. Fareler insanlarla moleküler ve fizyolojik benzerlikler paylaşmak rağmen, her zaman insan hastalıklarının 5 tüm yönlerini özetlemek yok. Bazı genetik olarak fareler kullanılarak, bu göz önüne alınması gerekmektedir. Genel olarak, fare modelleri gibi doku kültürü ve izole edilmiş insan tümörler gibi diğer mevcut modellere göre fare meme tümörogenez analiz etmek daha gerçekçi bir yol sağlar. Birlikte ele alındığında, bu protokol farklı yönlerini daha fazla araştırma için izin verirMeme tümör oluşum.

Metastasis is the final step in the progression of breast cancer 1. It involves a series of steps in which malignant cells are released from the primary tumor and disseminated to other organs 2. The cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system 3. Each step in tumorigenesis is tightly controlled by cells through various genetic and epigenetic changes 4. To study the mechanisms of breast cancer tumorigenesis, mouse model systems have been utilized due to their short lifespan, small size and fast breeding time. Some major advantages of using mouse models in cancer research are the molecular and physiological similarities they share with humans 5. 
Established mouse and human cell lines have been used extensively in breast cancer research. Although we can obtain some information about the steps in metastasis using these cells, genetically manipulated animal models with tumor suppressors/oncogenes will give far more information about tumor development and metastasis starting from the embryonic stage. Thus, with these mouse models, we can obtain genetic and developmental regulation of tumor growth. Furthermore, the tumor microenvironment also plays an important role in tumor cell gene expression and it can be difficult to mimic the same scenario with studies using established cell lines. Primary human mammary tumors can also be used to study mouse mammary tumorigenesis. The main disadvantage of using isolated breast tumors is that this newly implanted tumor does not have the mouse&#8217;s vasculature and lymphatic tissue. Whereas the genetic mouse model systems for studying mammary tumorigenesis will allow us to monitor the steps of metastasis, analyze the tumor microenvironment, and access to the entire body 5.
The overall goal of this protocol is to study breast tumorigenesis in mice. It is known that breast cancers typically metastasize to the brain, lung and bone. This protocol allows for the isolation of primary mammary tumor cells, as well as, studying breast cancer metastasis to the lung. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. Together, these techniques are useful in studying mouse mammary tumorigenesis.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>HyClone</td>
			<td>SH30243.01</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Life&#160; technologies</td>
			<td>25300-062</td>
			<td>Store at &#8211; 20 &#176;C</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td>Store at &#8211; 20 &#176;C</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma-Aldrich</td>
			<td>H3506</td>
			<td>Store at &#8211; 20 &#176;C</td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Life&#160; technologies</td>
			<td>15140-122</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gemini Bio-Products</td>
			<td>100-106</td>
			<td>Store at &#8211; 80 &#176;C</td>
		</tr>
		<tr>
			<td>Z-fix</td>
			<td>ANATECH</td>
			<td>#174</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>India Ink</td>
			<td>Yasutomo</td>
			<td></td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>AERRANE (Isoflurane)</td>
			<td>Baxter</td>
			<td>NDC 10019-773-60</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>EMD</td>
			<td>AX0441-3</td>
			<td>Store at room temperature</td>
		</tr>
	</tbody>
</table>

primary tumor and mef cell isolation to study lung metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

hayvan kurban etmek tümör ilerleme aşaması, belirli çalışma ve IACUC kurallarına bağlıdır. Şekil 1A, fare meme tümörü virüs Polyoma Orta T-antijen (MMTV PyMT) ile 100 günlük bir C57BL / 6 farede üç ayda kurban edilmiştir. cilt makas ile boyun üretral orifis alanı açılmış ve cilt Şekil 1B gösterildiği gibi organlara kolay erişim sağlamak için aşağı tuş oldu. Bu durum, Şekil 1C&#39;de elde edildi tümör kolay ulaşılmasına izin vermektedir. tümörlerden elde edilen birincil hücreler Şekil 1D görselleştirilmektedir. Birçok genin akciğer 1 meme kanseri metastazı aracılık yana, metastatik süreci incelemek için akciğerlere (Şekil 2A) ekstre edildi. Akciğer Z-fix ile şişirilir ve tümör nodülleri görselleştirmek için çini mürekkebi (Şekil 2B) ile boyandı. Şekil 3 fare embriyonik fibr gösterir<html> FVB suşu fare embriyoları izole oblast. <img alt="Şekil 1" src="/files/ftp_upload/52609/52609fig1.jpg" /> Meme Tümörlerinde Primer Hücreleri 1. Hazırlık Şekil. (A) Polyoma Orta T-Antijen (PyMT) ile 100 günlük dişi fare kurban edildi. (B) cilt meme tümör kolay erişim sağlamak için aşağı tuş oldu. (C) göğüs tümörü hayvan kesildi . (100 uM ölçek çubuğu), (D) izole edilmiş primer hücrelerde Dulbecco Modifiye Edilmiş Eagle Ortamı,% 10 fetal sığır serumu ve antibiyotikler ile takviye edilmiş (DMEM) &#39;de kültürlenmiştir. görüntüler 3. günde hücreleri görüntüler <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52609/52609fig1large.jpg" target="_blank">bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a> / 52.609 / 52609fig2.jpg &quot;upload /&gt; Şekil 2. Fare akciğer çıkarma akciğer metastazı görselleştirmek için. (Ölçek çubuğu: 500 uM) (A) Akciğer Z-fix ve tümör nodülleri görselleştirmek için çini mürekkebi ile boyandı (B) ile şişirildi. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52609/52609fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 3," src="/files/ftp_upload/52609/52609fig3.jpg" /> . Fare Embriyolar gelen Fare Embriyonik Fibroblastlar (MEF&#39;ler) hazırlanması Şekil 3. MEF hücreleri FVB fare embriyo (Ölçek çubuğu: 100 uM) çıkarıldı. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52609/52609fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about Primary Tumor and MEF Cell Isolation to Study Lung Metastasis at JoVE.com

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

Primer Tümör ve Akciğer Metastazı Eğitim için MEF Hücre İzolasyonu

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

primary-tumor-and-mef-cell-isolation-to-study-lung-metastasis

Nanyang Technological University

Research

JoVE Journal

Medicine

15.7K Görüntüleme.  Louisiana State University Health Sciences Center. The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

Video: Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Bir In Vitro</emMetastatik Büyüme Tümör Dormansi ve Switch Eğitim> Sistem

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

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Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Sıralama Kök Hücre Tahliller ve Akış Primer Beyin Tümörü Doku İşleme

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

İnsan Nöroendokrin Tümör Terapi Çalışmaları Üç Boyutlu Model İnsan nöroendokrin tümör hücre hatlarında

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

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

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

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

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

Hasta Türetilen Hücre Kültürü ve CD133 İzolasyonu<sup&gt; +</supMelanom dan&gt; sağlayan olası Kanser Kök Hücreleri

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

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

Yeni Bir Yüksek çözünürlüklü<em&gt; In vivo</emTümör Büyüme ve Tedavi için İntrakranial Yapıların Dinamik Tepki Eğitim&gt; Görüntüleme Tekniği

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

Noninvaziv Entübasyon Fare Akciğer Patolojisi ve Fizyolojisi Eğitim için

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Fare Akciğer Fibrin Gel implantasyonu Akciğer özel Anjiogenez Eğitim için

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Bowditch Efekt incelenmesi: Basınç Hacim İlişkisi Tedbir Fare Kardiyak Kateterizasyon

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Tümör spesifik Endotel Hücreleri İzolasyon ve Kültürü Genişleme

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Kantitatif gerçek zamanlı PCR ile fare meme tümör modellerinde akciğer metastazı Değerlendirilmesi

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...