This research was supported by research grants RO1CA109545 and RO1CA086984 to M.S.S. by the National Institutes of Health/National Cancer Institute and by an award from the Leo and Ann Albert Charitable Trust (to M.S.S.).

Bütün in vitro çalışmalar, Notre Dame Hayvan Bakımı ve Kullanımı Komitesi Üniversitesi tarafından onaylanan ve dişi C57 / BL6J fareler kullanıldı. 1. Fare Yumurtalık Kanseri Hücre Kültürü <ol><li> şöyle ID8 murin yumurtalık kanseri hücre kültürü kullanım sağlar:% 4 fetal sığır serumu (FBS),% 1 Penisilin / Streptomisin, 5 ug / ml insülin, 5 mg / ml transferrin ile takviye edilmiş Dulbecco Modified Eagle Medium (DMEM) 1 L ve 5 ng / mL sodyum selenit. </li><li> RFP ve bir seçim işaretleyici (geni blasticidin) ifade ticari olarak temin lentiviral parçacıklar kullanılarak kırmızı flöresanlı proteinin (RFP) ifade etmek için ID8 fare yumurtalık kanseri hücreleri 13 transdüksiyonu. Yeşil floresan proteini, ancak daha zayıf bir flüoresans sinyali sağlanır unutmayın. <ol><li> Hemen önce transdüksiyon için, kültür hücreleri 50 -% 70 Final 1 ml polybrene (5 mg / ml içeren izdiham ve (penisilin / streptomisin olmadan) taze orta ekleyinkonsantrasyon). </li><li> Yavaşça pipet RFP-lentivirüs (20 ul) kabına damla damla ve 72 saat süreyle 37 o C&#39;de inkübe edin. Ortamı çıkarın ve 24 saat boyunca taze ortam ile inkübe edilir. Kültür ortamında (5 ug / ml) blastisidin eklenerek kalıt aktarımlı hücrelerin seçimi başlayın ve floresan mikroskobu (Şekil 1) kullanılarak, kırmızı hücreler için izler. </li></ol></li><li> Kültür bir doku kültürü çanak içine yaklaşık 10 6 hücre tohumlama ve hücre tek tabaka konfluent kadar bir ışık mikroskobu kullanarak günlük görselleştirerek ID8 ortamında RFP-etiketli hücreler. </li><li> Konfluent, tripsin (% 0.25, 3 dk) kullanarak hücrelerin hasat ve hücreler müstakil kadar 37 o C inkübatör şişeyi geri döndüğünüzde. </li><li> serum içeren kültür ortamı ID8 6 ml tripsin nötralize. yukarı pipet ve çanak alt karşı aşağı, sonra 15 ml konik tüp transfer. 2 dakika boyunca 1.200 rpm&#39;de santrifüj, ardından GL kullanılarak ortam aspireeşek pipet. </li><li> hafifçe sıcak PBS 6 ml yeniden süspanse yukarıda 1.5 gibi santrifüjleme ile fosfat tamponlu tuz (PBS) hücreleri yıkayın. 5 x 10 6 hücre / ml&#39;lik bir son konsantrasyona PBS içinde yeniden askıya. Enjeksiyona kadar ılık hücreleri (37 ° C) saklayın. </li></ol> 2. içi periton ID8 Hücrelerinin Enjeksiyon ve in vivo Imaging <ol><li> Intra-peritoneal (ip) enjeksiyon yoluyla 10.000.000 hücre toplam sıcak ID8-PBS çözeltisi 2 mL her fare enjekte edilir. Tümör hücreleri haftada canlı görüntüleme yaklaşık 10 hafta boyunca in vivo olarak büyümeye olanak sağlar. </li><li> Hemen (0.5 L / dk O 2 akışında% 2,5 debisi) isofluorane kullanarak her fare uyutmak sonra fareler tartmak ve bundan sonra enjeksiyon ve her hafta takip. Not: farelere olmayan tümör yükünün kantitatif ölçüm değil, çalışma boyunca bir fare, fiziksel ilerlemesi daha genel bir tedbir olarak, tartılmıştır. fareler tartmakts kendi önceki ağırlık değerleri ile karşılaştırıldı. <ol><li> Bir isofluorane odasında koyarak fareler anestezi. işlem boyunca% 2.5 debisi isofluorane maruz kalma burun-koni fare kafasını konumlandırarak tarama sırasında sabit anestezi koruyun. </li></ol></li><li> siyah saçlı floresan sinyal zayıflamasını neden olacağından, her fare gövde ve karın saç kaldırmak için tüy dökücü krem ​​kullanın. Fareler tüy dökücü krem ​​uygulamasından sonra ılık su ile yıkanmış ve RT kağıt havlu ile kurutulur. </li><li> anestezi ederken, küçük bir hayvan optik görüntüleme sistemi kullanılarak her bir farenin tüm vücudu tarar. Her bir zaman noktasında bir grupta tüm fareleri tarayın. Aşağıdaki iki adımlı protokolü (Şekil 2A) kullanın: <ol><li> 1. Adımda, floresan etiketli (RFP) tümör hücrelerini gözlemlemek 440 nm, 460 nm, 4 uyarma filtreler kullanılarak 5 görüntülerin toplam toplamak için çok bantlı satın yürütmek80 nm, 520 nm ve 540 nm ve 600 nm&#39;lik bir emisyon filtresi. 15 saniyelik bir standart pozlama, 2 x 2 binning, 160 mm FOV ve 1.1 f durağı: Aşağıdaki toplama parametrelerini kullanın. </li><li> 2. Adımda, bütün hayvan gözlemlemek açık uyarma filtresi (beyaz ışık), bir açık emisyon filtresi, 2 x 2 binning ile 0.2 sn standart pozlama ve 16 cm FOV kullanarak bir yansıma görüntü elde etmek. Not: Gerçek görüntüleme süresi ~ 1 dk. </li></ol></li></ol> 3. Fare Diseksiyon ve Image Acquisition <ol><li> Canlı görüntüleme (karın şişliği ile kanıtlandığı gibi) asitli geniş intra-peritoneal hastalık ya da hayvan sergi birikimi, kayıp veya vücut ağırlığının% 20&#39;den fazla, uyuşukluk ya da sıkıntı diğer belirtileri kazanç varlığını gösterdiğinde, CO 2 kullanarak her fare euthanize anestezi servikal dislokasyon. </li><li> sivri dissectio kullanarak, fare ventral orta hat boyunca öne deri kesmen makas, göğüs kafesinin alt uyluk üst. Sadece deri tabakasının (Şekil 2BI) kesmek için aşırı özen gösterin. Yavaşça periton duvar delmeyin için emin olmak, periton dokusu cilt ayırmak. </li><li> Derinin iki flep oluşturmak için göğüs kafesinin alt ve uyluk üst kısmında hem yanal kesilmiş sivri diseksiyon makas kullanılarak (Şekil 2B ii, iii). </li><li> Açık cilt flepleri ile ventral tarafta her fare (periton boşluğu aşağı bakacak) çekti yerleştirin ve küçük hayvan optik görüntüleme sistemi kullanılarak yerinde onun organları ile her fare tarama (Şekil 3A, B). <ol><li> 2.4.1.-2.4.2 tarif edildiği gibi aynı görüntü tedarik parametreleri kullanarak. </li></ol></li><li> karaciğer, omentum / pankreas, mide, diyafram, ince bağırsak, kalın bağırsak, sol ve sağ periton, yumurtalıklar, kidne içeren bireysel periton organları çıkartarak fare diseksiyon devam edinys, mezenter ve yağ yastığı. <ol><li> , Gözlemlemek numaralandırmak ve her organ üzerinde görünür tümör kaydedin. Her tümörün çapı ölçmek için bir kumpas kullanın. </li><li> Ortam olarak 2 ve 5 mm kadar büyük daha büyük 5 mm arasında, küçük bir çap olarak 2 mm&#39;den daha az tümör belirtin. </li></ol></li><li> Organ tarama şablonuna her organ için önceden belirlenmiş bir konumunu tanımlar (Şekil 4) üzerine organları yerleştirin. nemli organları tutmak için PBS kullanın. </li><li> Küçük hayvan optik görüntüleme sistemi (Şekil 3C, D) ile organların her sayfayı tarayın. <ol><li> Adım 2.4 açıklandığı gibi aynı görüntü elde etme parametrelerini kullanın. </li></ol></li><li> Taramadan sonra,% 10 formaldehit yer organları histoloji için sonraki işlem sağlamaktır. </li></ol> th Tümör Yükü 4. sayısallaştırılmasıe Floresan Görüntüleri <ol><li> Her bir çok bantlı taramada otomatik floresans miktarının azaltılması amacıyla, ilk küçük hayvan görüntüleme sistemi ile uygun yazılımı kullanarak dosyaları unmix. <ol><li> Vivo spektral dosyasında, Autofluor ardından: İlk RFP yükleyin Karışmamış Görüntüler penceresinde Vivo Kırmızı dosyasında ve Unmix seçin. </li></ol></li><li> 16 bit .tiff (ölçeklenmemiş) biçimi olarak .bip dosyaları aktarın. </li><li> (In situ organları ile ex vivo organ ve ardından tam fare gövdesini) ImageJ yazılımı kullanın. <ol><li> ImageJ organ tarama şablon dosyalarını 16 bit .tiff dosyalarını açmak için&gt; Dosya Aç kullanın. </li><li> Görüntü&gt; Ayarlama altında&gt; Parlaklık / Kontrast, Minimum değeri 0 görüntülenen ve Maksimum +35353 değer gösterilir ve daha sonra altında yer alan tercihGörüntü&gt; Arama Tabloları, Red Hot seçin. Not: Çeşitli hayvan modelleri, farklı parlaklık seviyeleri gerektirir, ancak belirli bir çalışma boyunca tutarlı parlaklık tutmak önemlidir. </li><li> Serbest Seçim aracını kullanarak, her organın etrafında faiz seçimi (ROI) serbest biçimli bir bölgesini çizmek ve her organın yüzey alanını hesaplamak için Ölçü aracını (CTRL + M) kullanın; Bir e-tablodaki her bir organın yüzey alanı kaydedin. </li><li> Her organın etrafında çizilen ROI ile, sağ ROI içinde tıklayın ve sadece organı dahil etmek için Çoğalt seçeneğini seçin. </li><li> Eşik&gt; Ayarla&gt; Görüntü altında, bireysel organa bakarken, onl seçmek için + 1200 Alt Eşik Düzeyi ve + 1700 Üst Eşik Seviyesi görüntünün eşiğini ayarlayın tercihy en parlak fluoresan bölgeler ve Karanlık Arka Plan seçmek için kontrol edin; Tamam seçeneğini seçin. Not: Daha önce parlak alanlar analiz aşaması için seçilen böylece yukarıda gösterildiği gibi görüntüler, ImageJ eşik sürgü elle manipülasyon gerektirebilir. Farklı hastalık modelleri farklı eşik kısıtlamaları gerektirir, ancak belirli bir çalışma boyunca tutarlı eşik seviyesini düşük tutmak için önemlidir. </li><li> Analiz altında&gt; değiştirmek Boyutu, Parçacıklar Analiz (piksel ^ 2) 10-Infinity, Show tercih: Anahat, sadece &quot;sonuçlarını görüntüle&quot; seçeneğini ve alanları ve bu aydınlık bölgelerin ham entegre yoğunluğu hem ölçmek için Tamam &#39;ı kontrol, sayılır tümörler. </li><li> Yüzey Alanı değerlerini ve görüntülenen her tümör seçimi Ham Entegre Yoğunluk kaydedin <strong&gt; Organ yüzey alanları içeren aynı e-tabloda Sonuçları tablo. </li><li> Analiz altında&gt; Araçlar&gt; Kalibrasyon Bar, organların görüntülere bir kalibrasyon ölçek çubuğu ekleyin. Not: Bu örnekte, bu Üst Hakkı, Siyah Dolgu Rengi, Beyaz Etiket Renk, 5&#39;e Etiketler sayısı, 0 Ondalık, 12 Yazı Boyutu ve Zoom Yer değiştirerek yapıldı 4.0 faktör ve Kalın Metin sağlayan. </li><li> Dosya altında&gt; Farklı Kaydet ... Bir .TIFF ve .jpeg olarak montaj kaydedin. </li><li> .jpeg Organların dosya ve sonra Ekle altında&gt; Metin Kutusunu açmak için&gt; Dosya Aç kullanın </strong&gt;, organların üç sıra her aşağıda bir metin kutusu oluşturarak, organ etiketleri ekleyin. </li><li> Fare sayısının sırasına ImageJ tam vücut taramalarının 16 bit tiff dosyaların her biri açmak için&gt; Dosya Aç kullanın. </li><li> Değeri 0 gösterilir ve Maksimum + 35353 değerini görüntülenen ve ardından Görüntü altında&gt; Arama Tabloları, Red Hot seçin Görüntü altında&gt;&gt; Parlaklık / Kontrast ayarlama Minimum Set seçin. </li><li> Dosya altında&gt; Farklı Kaydet ..., a. TIFF ve .jpeg olarak montaj kaydedin. </li></ol></li></ol> 5. Veri Analizi <ol><li> her bir farenin Her organ için sırasıyla tümörün Seçim alanları ve ham entegre yoğunlukları ekleyin ve her bir fare için toplam organı tümör alanı kaydedilir. </li><li> Kaydedilen alanı ve ham bütünleşik Normaleİlgili Organ yüzey alanı (Tablo 1) toplam tümör alanı ve toplam ham entegre yoğunluk değerleri bölerek her organın boyutu için ed yoğunluk değerleri. </li><li> Hesaplamak ve normalize tümör yüzey alanları ve normalize ham entegre yoğunluk değerleri, her iki ortalamasını arsa (Şekil 5a, b). </li><li> görsel metastatik lezyonlar her organın denetim sırasında çıplak gözle görülebilen tümörlerin sayarak elde edilen bu ortalama değerleri karşılaştırın. </li></ol>

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Bu makale Bruker BioSpin sponsorluğunda Multimodal Klinik Öncesi Görüntüleme üzerinde bir özel sayısında, bir parçasıdır.

bağışıklık yetersizliği olan farelerde yapılmalıdır insan yumurtalık kanser hücrelerinin kullanan çalışmaların tersine, protokol, yukarıda tarif edilen imünokompetan C57 / BL6 fareleri ve genetik olarak özdeş fare yumurtalık kanseri hücreleri kullanır. Bu tümör büyümesi ve metastazı bağışıklık infiltrasyonların potansiyel rolünün değerlendirilmesi sağlarken, karın yüzeyinde siyah saçlı varlığı görüntüleme az duyarlı hale getirir. tüy dökücü kullanımı görüntüleme ?...

Epitelyal over kanseri (EOC) 2015 yılında ABD&#39;de tahminen 21.290 yeni tanı ve tahminen 14.180 ölüm 1 ile jinekolojik malignite gelen ölümlerin en sık nedenidir. Kadınların büyük çoğunluğu (&gt;% 75) geç evre hastalığı tanısı (evre III veya IV) yaygın-içi periton metastazı ve kötü prognoz ile karakterizedir. Birinci basamak kemoterapi sonrası periton boşluğuna Hastalık nüks da yaygındır ve mortalite 2,3 önemli bir nedenini oluşturmaktadır. EOC doğrudan komşu periton organların birincil tümörden hem de ayrışma ile uzantı veya tek hücre ya da çok hücreli agregatlar halinde primer tümör yüzeyinden hücre dökülmesi her ikisinin de dahil benzersiz bir mekanizma ile metastaz. Onlar dekolmanı kaynaklı apoptoz 4 direnmek burada Hücreler, periton boşluğuna dökülür. döken tümör hücreleri periton lenfatik drenajı ve t blok olarak periton asit birikimi, yaygınumors vasküler geçirgenliğini değiştirerek büyüme faktörlerini üretir. Onlar çapa ve birden çok yaygın ikincil lezyonlar 3,5 üretmek için çoğalırlar bunun üzerine döken tümör hücrelerinin bir kısmı, bağırsak, karaciğer, omentum ve mezenter dahil periton organ ve yapıların yüzeyine tutunur. Hematojen metastaz nadirdir. Böylece, klinik yönetimi yaygın (kadar küçük olursa olsun) tüm görünür tümör rezeksiyonu olarak tanımlanan &quot;Optimal bir sitoredüksiyon sonrası&quot; olmak üzere sitoredüktif cerrahi oluşur. Komple sitoredüksiyon genel sağkalım 6,7 belirgin bir artış ile ilişkilidir ve tanımlanması ve lezyonların çıkarılması &lt;0.5 cm meydan okuma ile ilişkilidir. Küçük hayvan modelleri prognostik biyolojik ve yeni kemoterapi veya kombine tedavi yaklaşımları test belirlenmesi yanı hastalığın ilerlemesi anlayışımızı geliştirmeye yumurtalık kanseri araştırmalarında yarar kanıtlanmıştır. birincil olarakyumurtalık kanseri insidansı ve metastaz yeri periton boşluğu, EOC metastaz ortotopik modelleri intraperitoneal hastalığın analizi ve karakterizasyonu dahil olduğunu. Görüntü tümör hücrelerine yeteneği son gelişmeler olmasına rağmen, hatta tek hücre düzeyinde, hala EOC metastatik tümör yükü miktarının önemli zorlukları da bulunmaktadır. Bu sorunlar nedeniyle sayısı, büyüklüğü ve metastatik lezyonların anatomik konuma ortaya çıkar. Ayrıca, normal konakçı hücrelerden ayırmak için etiket kanser hücreleri için bir ihtiyaç söz konusudur. Daha önceki çalışmalar, antikor bazlı etiketleme protokolleri ya da lusiferaz 8,9 tümör hücrelerinin transfeksiyonu kullanmışlardır. Kanser hücrelerinin direkt floresan etiketleme ilk olarak 1997 yılında 10 Chishima ve arkadaşları tarafından rapor edilmiştir. Floresan etiketleri kanser metastazı 11,12 izlemek için eksojen substratın ilave edilmesini gerektirebilir ve zarif tümör hücre özgüllük sağlamak, daha etkin bir araç temin yok </sus&gt;. Bu yazıda fare ID8 yumurtalık kanseri hücreleri 13 ve bağışıklık yetkili C57 / BL6 farelerinde kırmızı floresan proteini (RFP) oluşan bir singeneik ortotopik ksenograft modeli kullanılarak metastatik hastalık kantitatif analizi için bir optik görüntüleme yöntemi etiketli açıklanmaktadır. Göreceli tümör yükü ölçümü için yeni bir yöntem, doku otomatik floresans kaldırılması ile in vivo ve ex vivo görüntüleme birleştirerek göstermektedir. Bu yaklaşım yumurtalık kanseri organa özgü metastaz belirli genetik, epigenetik veya mikro-çevre değişiklik ve / veya tedavi yöntemlerinin etkisini değerlendirmek için tasarlanmış çalışmalarda potansiyel yarar vardır.

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:183px;">Corning</td>
			<td style="width:117px;">10-014-CM</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">penicillin/streptomycin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Insulin-transferrin-sodium selenite media supplement</td>
			<td>Sigma</td>
			<td>I-1884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bruker Xtreme small animal imaging system</td>
			<td>Bruker Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bruker Multispectral software</td>
			<td>Bruker Corp</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">lentiviral particles with Red fluorescent protein</td>
			<td>GenTarget, Inc.</td>
			<td>LVP023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trypsin for cell culture</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">depilatory cream (such as Nair Hair Remover Lotion)</td>
			<td>purchases from drugstore&#160;</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ software&#160;</td>
			<td>http://imagej.nih.gov/ij/&#160;</td>
			<td></td>
			<td>free download</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dissecting tools (forceps)</td>
			<td>Roboz Surgical Instrument</td>
			<td>&#160;RS 5130</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dissecting tools (Scissors)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS 5910</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitation of intra-peritoneal ovarian cancer metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

yumurtalık kanseri, metastatik bir mekanizma çok küçük (&lt;2 mm) lezyonlar da dahil olmak üzere, farklı büyüklüklerde, çok sayıda lezyon arasında oluşan yüksek yaygın intra-peritoneal metastaz ile karakterize edilir. Bu nedenle, RFP etiketli tümör hücrelerinin kullanımı (Şekil 1) ve optik görüntüleme kılavuzu sayım ve lezyon boyutu ölçümü için alternatif bir yöntem sağlar. Zamanla tümör yükünün gelişmesi haftada asit potansiyel va...

Watch this Scientific Journal Video about Quantitation of Intra-peritoneal Ovarian Cancer Metastasis at JoVE.com

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

İçi periton Yumurtalık Kanseri Metastaz kantitasyonu

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

quantitation-of-intra-peritoneal-ovarian-cancer-metastasis

Research

JoVE Journal

Medicine

10.8K Görüntüleme.  University of Notre Dame. Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Video: Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Deneysel metastaz Testi

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Tıp

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

Kuş Embriyo Model kullanarak Kanser metastaz Kantitatif Analiz

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

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

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

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

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

In vitro Mezotelyal Gümrükleme Assay o Yumurtalık Kanseri Metastaz Erken Adımlar modeller

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

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Kemik Metastaz Modelleri

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

İnsan Prostat Kanser metastaz Ortotopik fare modeli

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Katı Örneklerinin From İlköğretim Yumurtalık Kanseri Hücreleri alma için yöntem

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

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

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

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

Real Time Mezotelyal Hücrelerinin Yumurtalık Kanseri Sferoid Ek ve Invasion Değerlendirilmesi

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

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Non-invaziv Görüntüleme için fare modeli Algılama ve Monitör Yumurtalık Kanseri nüks için

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

İnsan Hücre Lines Kemorezistans Yumurtalık Kanser Kök Hücreleri Zenginleştirme

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

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Yumurtalık Kanseri Tümör başlatan Hücrelerinin in vitro Zenginleşmeden içinde

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...