We thank all residents and attending physicians, notably Dr. A.F. Haney (the University of Chicago, Department of Obstetrics and Gynecology) for collecting omental biopsies. Also, we thank Stacey Tobin and Gail Isenberg for carefully editing this manuscript. This work was supported by Bears Care, the charitable beneficiary of the Chicago Bears Football Club, the National Institute of Neurological Disorders and Stroke (NINDS) R21 NS075702, and the National Cancer Institute grant R01 CA111882 to E.L.

Tarif edilen tüm araştırma protokolleri Chicago Kurumsal Değerlendirme Kurulu Üniversitesi&#39;nde (KİK) tarafından incelenmiştir. Aydınlatılmış onam ameliyattan önce her hastadan alındı ​​ve çalışma Chicago IRB Üniversitesi tarafından onaylanmıştır. Korunması için insan dokusu taşıma ve hücrelerin kontamine riskini azaltmak için ne zaman bir biyolojik güvenlik kabini tip 2 ve eldiven kullanılmalıdır. Birincil dönüştürülmemiş Stromal Hücreleri 1. İzolasyon ve Kültür <ol><li> İnsan doku toplama ve hazırlama. <ol><li> Abdominal operasyon esnasında alınan insan omentum, 2 cm3, örneklerini elde edilir ve hemen RT fosfat tamponlu tuzlu su (PBS) (Şekil 2A), dokuyu bırakın. X 2 cm omentum 3 cm&#39;lik bir parçası, tipik olarak 1.000.000 birincil insan mezoteliyal hücreleri (HPMC) ve 200,000 primer insan fibroblastları ve normal omental fibroblastlar (NOF) verir. </li><li> Doku 3 için 0.5 xg&#39;de dalmış PBS aşağı Spindak kısa sürede sonra toplanması (&lt;2 PBS içinde saat) ve 50 ml konik bir tüp içinde 20 ml taze PBS doku aktarın. 15 cm çapında, steril kültür çanak (Şekil 2B) neşter ile kıyma ile 5mm 3 parçaya doku yukarı kesti. </li></ol></li><li> Birincil insan mezotel hücre izolasyonu 8 <ol><li> HPMC izole etmek için, 50 ml konik tüpler içine kıyılmış doku transferi ve katı parçaları üst (Şekil 2C-D) float için 1 dakika bekleyin. HPMC ve kırmızı kan hücrelerinin (RBC) alt kısmında sıvı içinde mevcut olacaktır. Borunun alt ve yeni bir konik tüp içine sıvının çıkması için bir pipet kullanın. 3 dakika boyunca 0.5 xg sıvıyı aşağı Spin ve HPMC / RBC pelet süpernatant aspire. <ol><li> , Kıyılmış dokuya 20 ml PBS eklemek doku, aşağı spin, yükselmeye bir pipet ile ve HPMC / RBC tüpüne alttan sıvıyı çıkarmak için izin ve kaldırın: işlemini üç kez tekrarlayınsüpernatant. Tüm spin tamamlanır ve süpernatan aspire edildikten sonra, pelet HPMC ve RBC&#39;yi içerecektir. </li></ol></li><li> Plaka% 10 cenin sığır serumu [FBS],% 1 MEM vitamin [93 mg / L],% 1 MEM esansiyel olmayan amino asitler [81,4 tam büyüme ortamı, 15 ml DMEM (75 cm 2 şişe içine pelet tüm hücrelerin mg / L],% 1 penisilin streptomisin [Pen-strep, 100 U / ml penisilin ve 100 ug / ml streptomisin]). </li><li> Ek HPMCs izole etmek için ikinci bir PBS yıkama yapın. <ol><li> Doku üstüne yüzen ikincil PBS yıkama için, 200 rpm&#39;de, 20 ml PBS içinde 10 dakika süreyle 37 ° C &#39;de geri kalan katı doku sallayın, daha sonra 1 dakika bekleyin. 3 dakika boyunca 0,5 x g&#39;de santrifüj, yeni bir tüp içine tüpün altındaki sıvıyı çıkarın ve süpernatan aspire. </li><li> Tam büyüme ortamı 15 ml ayrı 75 cm 2 şişe yıkama, ikincil PBS tüm HPMC / RBC Plate. </li></ol></li><li> Herhangi r izole etmek içinhacimce PBS solüsyonu: 1,% 0.25 tripsin, 25 mM EDTA: HPMC emaining, 1, 20 ml, 10 dakika boyunca, 200 rpm&#39;de 37 ° C derece kıyılmış doku sallayın. Doku yükselmeye tüpün alt kısmında sıvı toplamak, yeni bir 50 ml tüp içine ve 0.5 gx 3 dakika aşağı dönmesine izin verin. <ol><li> Süpernatant aspire tam büyüme ortamında 15 ml ayrı 75 cm 2 şişe tüm HPMC / RBC plaka. </li></ol></li><li> Kültür nemlendirilmiş bir ortamda 37 ° C&#39;de ve% 5 CO2 plakalandı hücreleri. Harcanan medya çıkarmadan günlerde 3 ve 5 tam büyüme ortamı 15 ml ekleyerek Yem kaplı hücreler. HPMC bölme önce 5-7 gün süre ile kültüre edilebilir. <ol><li> Orijinal fenotip dediferansiyonunu ve modifikasyonu en aza indirmek için tüm deneylerde (geçit 2 kadar) düşük geçit HPMC&#39;ye kullanın. Faz kontrast capabilitie plastik diferansiyel girişim kontrast yetenekleri veya 4-20x amacı ile 20x objektif ile tercihen geniş alan mikroskobu kullanıns hücrelerinin tüm görüntüleri çekmek için (mikroskop faz kontrast filtreleri) ve 3,3&#39;-diaminobenzidin (DAB) boyama immunohistokimyasal analiz parlak alanında 10x objektif (). </li></ol></li><li> HPMC cuboidal olduğunu onaylayın ve immunohistokimyasal 8 (Şekil 3A, C, E) gerçekleştirerek sitokeratinin 8 ve vimentinin ifade eder. </li></ol></li><li> NOF izolasyonu 8 <ol><li> 1: 100 x Pen-Strep 1 karışımı bir 1: birleştirerek kollajenaz tip III çözeltisi (10 x) &#39;in bir stokunun 10 ml hazırlayın kollajenaz tip III 1500 ünite aktivite / ml PBS içinde hyaluronidaz 714 ünite aktivite / ml olmuştur. </li><li> % 1 MEM vitamin [93 mg / L] ile serumsuz ortam içinde seyreltilmiş 10 kat kollajenaz tip III çözeltisi, 10 ml (DMEM 6 saat süre ile 200 rpm&#39;de HPMC izolasyon sonra kalan kıyılmış omentum doku 37 ° C sallayın, % 1 MEM esansiyel olmayan amino asitler [81,4 mg / L],% 1 penisilin streptomisin [Pen-strep, 100 U / ml penisilin ve 100 ug / ml streptomisin]). Sindirilmiş doku opak bakacağız ve bazı lifli enkaz (Şekil 2E) olabilir. </li><li> Çözelti içeren NOF hücreleri Santrifüj oda sıcaklığında 3 dakika boyunca 0,5 x g &#39;de süspansiyon halinde, süpernatan aspire tam büyüme ortamında 13 ml bir 75 cm&#39;lik 2 şişeye pelet plaka. </li><li> Eski ortamı çıkarın ve 24 saat sonra taze tam büyüme ortamı 15 ml ile değiştirin. NOF bölme önce 1-3 gün süre ile kültive edilebilir. Hücreler konfluense ulaştığınızda NOF notu çoğalması sona erecek. </li><li> Birincil insan fibroblastlar, düz, uzun hücreler olduğunu onaylayın ve vimentinin ifade ama immünohistokimyasal 8 gerçekleştirerek 8 sitokeratin değil (Şekil 3B, D, F). Dediferansiyonunu en aza indirmek için (ideal geçit 3 öncesi) deneyler için düşük geçit NOF kullanın ve orijinal fenotip modifikasyonu. Hücrelerin her aşaması fotoğraf çekmek için plastik DIC yetenekleri ile 20x amacı ile geniş alan mikroskobu kullanınve parlak alanında 10x objektif immunohistokimyasal DAB boyama analiz etmek. </li></ol></li></ol> 2. Organotipik Kültür 8 Kaplama <ol><li> En fazla 5 dakika boyunca 3 ml% 0.25 tripsin / EDTA 25 mM, ardından 10 ml PBS ile çalkalayın 75 cm kültür şişesi Yayın NOF. Tam büyüme ortamının en az 3 kez hacmi ile nötralize tripsin. </li><li> 50 ml konik tüp içine tripsinize hücreleri aktarın ve 0.5 gx 3 dk aşağı doğru döndürün. Süpernatantı ve tam büyüme ortamı 5 ml kadar geri hücreleri getirmek. Kültür şişesi kurtarıldı hücreleri saymak için bir hücre sayacı kullanın. </li><li> Bir 96-yuvalı, berrak tabanlı siyah plaka üzerine 100 ul başına 2000-4000 NOF plaka tam büyüme ortamı, uygun hacimde hücreleri ile seyreltilir. Kaplama önce hücre karışıma sıçan kuyruğu kollajen I 0.5 ug / 100 ul ekleyin. Let Hücrelerin en az 4 saat boyunca nemlendirilmiş bir ortamda% 5 CO2 içinde 37 ° C &#39;de oturup, ya da hücrelerin kadarplaka yüzeyine yapışır için. </li><li> Adımlar 2.1 ve 2.2 açıklanan aynı yöntemleri kullanarak kültür şişesi HPMC bırakın. Tam büyüme ortamının uygun miktarda hücrelerin önceden 96 oyuklu plakalar içinde kollajen I kaplama NOF üstünde 50 ul başına 10,000-20,000 HPMC plaka seyreltin. Hücreler deney başlamadan önce en az 18 saat boyunca nemlendirilmiş bir ortamda% 5 CO2 içinde 37 ° C &#39;de bekletin. </li><li> Tam büyüme ortamı HeyA8 yumurtalık kanser hücreleri -expressing Kültür yeşil flüoresan proteini (GFP). HeyA8 yumurtalık kanseri hücreleri, floresan kopepod cGFP (pCDH-CMV-MCS-EF1) eksprese eden bir lentiviral vektör ekspresyonu ile etiketlenir. HeyA8 yumurtalık kanseri hücreleri, kullanım gününde (logaritmik büyüme fazı) üzerine% 80-90 ortak akışlı olmalıdır. Kültür hücreleri plakadan bırakın ve birincil hücreler için adımlar 2,1-2,2 açıklanan aynı yöntemleri kullanarak saymak. </li><li> Yumurtalık kanseri hücreleri, 37 ° C&#39;de 15-20 dakika boyunca tam bir büyüme ortamı içinde kurtarmak için izin verinkapaklı konik tüp gevşek kapatılmış. </li></ol> 3. Yapışma Deneyi 8 <ol><li> İyileştikten sonra 0,5 xg&#39;de 3 dakika boyunca iplik ile yumurtalık kanseri hücrelerini pellet haline getirmek ve daha sonra süpernatant kaldırmak ve 50.000 hücre / 100 ul bir konsantrasyona ulaşmak için serum içermeyen ortam, uygun hacimde hücreleri seyreltin. </li><li> Harcanan ortam kaldırmak için HPMC / NOF Organotipik kültürler ile 96 oyuklu plakalar ters çevirin, ve her bir serumsuz ortam içinde kanser hücre süspansiyonu 100 ul ilave edin. 4 saat 30 dakika boyunca nemlendirilmiş bir ortamda% 5 CO2 içinde 37 ° C &#39;de plaka inkübe edin. </li><li> Ortam ve yapışmayan hücreleri çıkarmak için 96 oyuklu plaka ters çevirin. Dikkatle ve yavaşça her kuyuya PBS 100 ul pipetlemeyin ve tekrar plaka ters düşük güçte bir çok kanallı pipet kullanın. PBS bir kez daha yıkayın tekrarlayın. </li><li> PBS çıkarın ve her bir oyuğa 100 ul% 4 paraformaldehid eklemek için ters çevirin. Plaka 20 mil için bekletinn hücreleri gidermek için. 20 dakika sonra, plaka ters çevirin ve 100 ul PBS ilave edin. </li><li> Toplam kuyu flüoresan kaydedilmemiştir veya hücre sayısı belirlenebilir unutmayın. Ölçümü için, 1.000.000 hücre / ml&#39;lik bir konsantrasyonda HeyA8 hücreleri kullanılarak, iki kopya halinde birinci plaka standart eğrisi. <ol><li> Aşağı 1 milyon hücre iplik medya kaldırarak, ve onları 20 dakika boyunca 1 ml% 4 paraformaldehid oturup izin vererek standart eğri olun. </li><li> Spin hücreleri tekrar PBS (anlattıklarında hücreleri 1000000 / ml konsantrasyon onaylamak için) ml 1&#39;de getirmek. Levha 50.000, 40.000, 30.000, 20.000, 10.000, 5.000, 1.000, ve de her biri çifte biçimde içine 0 hücreleri, ve her çukur 50 ul&#39;lik bir nihai toplam hacim PBS ilave edin. </li></ol></li><li> Alt yüksek kuyu (standart eğri üzerinde 50.000) ölçekleme, bir flüoresan plaka okuyucusu (uyarım = 488 nm, emisyon = 528 nm) ile kültür plakasına okuyun. Organotyp yapıştırılır kaç yumurtalık kanseri hücrelerini hesaplamak için standart eğri kullanınHer bir oyuğa (Şekil 4A-C) &#39;ic kültürü. </li></ol> 4. Proliferasyon Deneyi 10 <ol><li> Yumurtalık kanseri hücreleri, geri sonra% 1 FBS (DMEM,% 1 FBS ortamı uygun hacimde hücreleri sulandırmak sonra 0.5 x g&#39;de 3 dakika boyunca iplik hücreleri pelet,% 1 MEM (adım 2.5 ve yukarıda belirtilen 2,6 bakınız) vitaminler [93 mg / L],% 1 MEM esansiyel olmayan amino asitler [81,4 mg / L],% 1 penisilin streptomisin [Pen-Strep, 100 U / ml penisilin ve 100 ug / ml streptomisin]) 4.000 hücre konsantrasyonu elde etmek için / 150 ul. </li><li> Her bir oyuğa FBS ortamı harcanan ortamını çıkarın ve 1% kanser hücre süspansiyonu 150 ul eklemek için HPMC / NOF Organotipik kültürler ile 96 oyuklu plaka ters çevirin. 72 saat boyunca nemlendirilmiş bir ortamda% 5 CO2 içinde 37 ° C &#39;de plaka inkübe edin. </li><li> Alt görece proliferasyonunu değerlendirmek için bir kez bir flüoresan plaka okuyucusu günde (uyarım = 488 nm, emisyon = 528 nm) kültür plaka okumahücre. 48 saat (Şekil 5A-C) &#39;de ortamının değiştirilmesi, 96 saat boyunca deney devam edin. (Plaka tercih edilir tersini) ortam değiştirirken kültürünü rahatsız etmemek için dikkatli olun. </li></ol> 5. Invasion Testi 8 <ol><li> 3D kültür kaplama önce, 24 oyuklu bir kültür plakasına parçası (8 um gözenek boyutu) 200 ul PBS içinde 7.5 ug fare kuyruk kolajeni eklemek. Plaka inkübe O / N, daha sonra dikkatlice hemen kollajen kaplı ekler (yukarıdaki adım 2) üzerine HPMC / NOF organotipik kültürünü kaplama önce bir pipet ile PBS kaldırmak edelim. </li><li> Adım 3.1 olarak yumurtalık kanseri hücrelerini hazırlayın. Ekler üzerinde organotipik kültürlerinin üzerine yumurtalık kanseri hücrelerini plaka dikkatle ekler üst fazla olan ortamı çıkarmak için bir pipet kullanın. Her bir oyuğa, serumsuz ortam içinde yumurtalık kanseri hücre süspansiyonu 100 ul ekle. </li><li> Kanser hücresini ikna etmek için iyi bir ekin altındaki içine tam büyüme ortamı 750 ul ekleyinorganotipik kültürlerinin içine vasion. 24 saat boyunca nemlendirilmiş bir ortamda% 5 CO2 içinde 37 ° C &#39;de plaka inkübe edin. Not: Organotipik kültür ekleme çapraz ve ekin alt tarafında kalacaktır istila Yumurtalık kanseri hücrelerini. </li><li> Boş kuyuya insert taşıyın, 24 saat sonra, maşa ile ekleme kavramak ve kısaca PBS beher durulayın. 1 dakikalık bir toplam pamuklu çubuk her iki yanına, her bir ek üst fýrçalayýn. Hızlı bir şekilde her bir 750 ul% 4 paraformaldehid içeren bir plaka içine ekleme yerleştirin. Her insert 750 ul PBS ile dolu kuyu içine ekler aktarmadan önce 10 dakika boyunca paraformaldehid oturup bekleyin. </li><li> 2.5x büyütme mikroskop ile işgal hücreler (ekler dibine yapıştırılır ve sabit) görüntüleyin. Tüm kuyu görüş alanı içinde olduğu zaman, kaçınarak, 10x büyütme ayarlayın ve 5 resim, kuyunun her kadranda merkezine yakın bir ve 1 almakkenarlarına çok yakın görüntüler alarak. Her iyi için aynı desen izleyin. </li><li> Onların protokole göre her bir kuyu (Şekil 6A-C) &#39;de alan başına işgal hücrelerin ortalama sayısını elde etmek için her görüntüdeki hücre sayısını saymak için üreticinin programı kullanın. </li></ol>

<ol>
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Metastasis. 5, 181-197 (1987).</li><li>Kanemoto, T., Martin, G. R., Hamilton, T. C., Fridman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+synthetic+peptides+and+protease+inhibitors+on+the+interaction+of+a+human+ovarian+cancer+cell+line+(NIH:OVCAR-3)+with+a+reconstituted+basement+membrane+(matrigel).">Effects of synthetic peptides and protease inhibitors on the interaction of a human ovarian cancer cell line (NIH:OVCAR-3) with a reconstituted basement membrane (matrigel).</a> Invasion Metastasis. 11, 84-92 (1991).</li><li>Rieppi, M., 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=Mesothelial+cells+induce+the+motility+of+human+ovarian+carcinoma+cells.">Mesothelial cells induce the motility of human ovarian carcinoma cells.</a> Int. J. Cancer. 80, 303-307 (1999).</li><li>Barbolina, M. V., Adley, B. P., Ariztia, E. V., Liu, Y., Stack, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+membrane+type+1+matrix+metalloproteinase+activity+in+ovarian+carcinoma+cells+via+collagen-induced+EGR1+expression.">Microenvironmental regulation of membrane type 1 matrix metalloproteinase activity in ovarian carcinoma cells via collagen-induced EGR1 expression.</a> J. Biol. Chem. 282, 4924-4931 (2007).</li><li>Burleson, K. M., 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=Ovarian+carcinoma+ascites+spheroids+adhere+to+extracellular+matrix+components+and+mesothelial+cell+monolayers.">Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers.</a> Gynecol. Oncol. 93, 170-181 (2004).</li><li>Suzuki, N., 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=HMOCC-1,+a+human+monoclonal+antibody+that+inhibits+adhesion+of+ovarian+cancer+cells+to+human+mesothelial+cells.">HMOCC-1, a human monoclonal antibody that inhibits adhesion of ovarian cancer cells to human mesothelial cells.</a> Gynecol. Oncol. 95, 290-298 (2004).</li><li>Kishikawa, T., 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=Two+distinct+pattern+of+peritoneal+involvement+shown+by+in+vitro+and+in+vivo+ovarian+cancer+dissemination+models.">Two distinct pattern of peritoneal involvement shown by in vitro and in vivo ovarian cancer dissemination models.</a> Invas. Metast. 15, 11-21 (1995).</li><li>Casey, R. C., 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=Establishment+of+an+in+vitro+assay+to+measure+the+invasion+of+ovarian+carcinoma+cells+through+mesothelial+cell+monolayers.">Establishment of an in vitro assay to measure the invasion of ovarian carcinoma cells through mesothelial cell monolayers.</a> Clin. Exp. Metastasis. 20, 343-356 (2003).</li><li>Niedbala, M. J., Crickard, K., Bernacki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+human+ovarian+tumor+cells+with+human+mesothelial+cells+grown+on+extracellular+matrix.">Interactions of human ovarian tumor cells with human mesothelial cells grown on extracellular matrix.</a> Exp. Cell Res. 160, 499-513 (1985).</li><li>White, E. A., Kenny, H. A., Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+modeling+of+ovarian+cancer.">Three-Dimensional modeling of ovarian cancer.</a> Adv. Drug Deliv. Rev. 79-80, 184-192 (2014).</li><li>Kenny, H. 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=Quantitative+high+throughput+screening+using+a+primary+human+three-dimensional+organotypic+culture+predicts+in+vivo+efficacy.">Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy.</a> Nature Comm. , (2015).</li><li>Sawada, K., 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=c-Met+overexpression+is+a+prognostic+factor+in+ovarian+cancer+and+an+effective+target+for+inhibition+of+peritoneal+dissemination+and+invasion.">c-Met overexpression is a prognostic factor in ovarian cancer and an effective target for inhibition of peritoneal dissemination and invasion.</a> Cancer Res. 67, 1670-1680 (2007).</li><li>Kenny, H. A., Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MMP-2+functions+as+an+early+response+protein+in+ovarian+cancer+metastasis.">MMP-2 functions as an early response protein in ovarian cancer metastasis.</a> Cell cycle. 8, 683-688 (2009).</li><li>Sawada, K., 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=Loss+of+E-cadherin+promotes+ovarian+cancer+metastasis+via+alpha+5-integrin,+which+is+a+therapeutic+target.">Loss of E-cadherin promotes ovarian cancer metastasis via alpha 5-integrin, which is a therapeutic target.</a> Cancer Res. 68, 2329-2339 (2008).</li><li>Mitra, A. K., 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=Ligand-independent+activation+of+c-Met+by+fibronectin+and+&#945;(5)&#946;(1)-integrin+regulates+ovarian+cancer+invasion+and+metastasis.">Ligand-independent activation of c-Met by fibronectin and &#945;(5)&#946;(1)-integrin regulates ovarian cancer invasion and metastasis.</a> Oncogene. 30, 1566-1576 (2011).</li></ol>

The authors have nothing to disclose.

Periton mikroçevresinin bir Organotipik modeli yumurtalık kanseri yayılmasına mikroçevresinin hücresel ve doğuştan bileşenlerin hem bireysel ve kolektif işlev (ler) değerlendirmek için kurulmuştur. Yumurtalık kanseri hücre yapışması, proliferasyon ve istila araştırmak için 3D Organotipik kültürü kaplama ve uyarlama için özel protokolleri temin edilmektedir. Primer insan omental mezotel hücreleri ve fibroblastlar hastalardan izole edilen ve normal morfoloji ve hasta varyasyonu korumak için erk...

Ovarian cancer is the deadliest gynecologic malignancy1. The majority of patients are diagnosed after the cancer has disseminated throughout the peritoneal cavity. Once the cancer has spread throughout the peritoneal cavity, cytoreductive surgery and chemotherapy are often not sufficient treatment to prevent cancer recurrence and chemoresistance, resulting in a less than 30% 5-year survival rate. Ovarian cancer metastasis is predominantly limited to the peritoneal cavity, and several other cancer types, including gastric, pancreatic, and colon cancers, metastasize to the same anatomic sites in the peritoneal cavity. In general, ovarian cancer cells detach from the in situ carcinoma in the fallopian tube or the primary ovarian tumor, travel in peritoneal fluid as single cells or spheroids, and attach to mesothelium-lined surfaces of the omentum, bowel, and abdominal wall2. 
The tumor microenvironment plays an important role in disease progression and chemoresistance in many cancers3-6. The peritoneal cavity is a unique microenvironment, with a mesothelial cell monolayer covering the majority of surfaces (Figure 1A)7. The mesothelial lining acts as a barrier that creates a low-friction surface, which tends to be protective against cancer cell adhesion8. Immediately underneath this mesothelial-lined surface is a layer made predominantly of fibroblasts and extracellular matrix (ECM), which promote cancer cell adhesion and invasion8. Ovarian cancer cells secrete factors that induce changes in the mesothelial cell lining that enhance ovarian cancer cell adhesion, invasion, and metastasis9,10. Ovarian cancer cells adhere to the mesothelial surface via integrin and CD44-mediated mechanisms (Figure 1B)11-16. 
Historically, several 3D models have been developed to investigate ovarian cancer interactions with the microenvironment. Some of the first models studied ovarian cancer-ECM interface17-21, ovarian cancer-mesothelial cell communication13,14,21-24, or both25 (reviewed by us 26). Niedbala et al. discovered that ovarian cancer cells display a quicker and firmer adhesion to ECM than to mesothelial cells or to plastic alone25. However, these models did not histologically resemble the peritoneal microenvironment. Therefore, we established a 3D organotypic model to more thoroughly replicate the ovarian cancer microenvironment. In order to better understand the role of the microenvironment and the interaction between cancer and peritoneal cells in the peritoneal dissemination of ovarian cancer, we have developed a 3D organotypic in vitro culture model of the peritoneal cavity lining (schematic in Figure 1C). The proposed model is composed of primary human fibroblasts and ECM, covered with a layer of primary human mesothelial cells-each cell type is isolated from human omentum. Histologically, this model resembles the normal peritoneal or omental lining, and provides a surface on which we can study the tumor microenvironment, the interaction between cancer cells and normal tissue, and the processes of cancer cell adhesion, invasion, and proliferation8.

<table>
	<tbody>
		<tr>
			<td>1. Isolation and culture of primary cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher Scientific</td>
			<td>SH3001304</td>
			<td></td>
		</tr>
		<tr>
			<td>Single-edged razor blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm culture dishes</td>
			<td>BD Biosciences</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass flask</td>
			<td>?</td>
			<td>?</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>16000044_3616914956</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM with L-Glutamine</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamins</td>
			<td>Corning</td>
			<td>25-020-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Nonessential amino acids</td>
			<td>Corning</td>
			<td>25-025-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker&#160;</td>
			<td>Thermo-Fisher</td>
			<td>MaxQ 4450</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5702</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo-Fisher</td>
			<td>Forma Series II Water Jacketed CO2 Incubator Model 3100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA, 1x (0.25%)</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Worthington Biochemical</td>
			<td>LS002592</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 Flasks</td>
			<td>BD Biosciences</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>T-175 Flasks</td>
			<td>BD Biosciences</td>
			<td>353112</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips</td>
			<td>Rainin</td>
			<td>P2, P10, P20, P200 and P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips</td>
			<td>Corning</td>
			<td>Filtered tips P2, P10, P20, P200 and P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>2. Plating 3D culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counter</td>
			<td>Invitrogen</td>
			<td>Countess</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Invitrogen</td>
			<td>C10313</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I (Rat Tail)</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate, clear bottom, black</td>
			<td>BD Biosciences</td>
			<td>353219</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>3. Adhesion assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipet</td>
			<td>Eppendorf</td>
			<td>Xplorer 300</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution 4% in PBS</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Molecular Devices</td>
			<td>Minimax</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>5. Invasion assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Inserts (8um, 24-well)</td>
			<td>BD Biosciences</td>
			<td>353097</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>Q-tips</td>
			<td>cotton swabs</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td>Axiovert 200m</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Profiler</td>
			<td>public domain</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>BD Biosciences</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>6. Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Integrin &#945;V&#946;3 Antibody, clone LM609</td>
			<td>EMD Millipore</td>
			<td>MAB1976</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta 1&#160;</td>
			<td>Oncosynergy</td>
			<td>OS2966</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha 5 [CD49e]</td>
			<td>ID Pharmingen</td>
			<td>555615</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta 4 [CD104]</td>
			<td>EMD Millipore</td>
			<td>MAB 2058</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling the early steps of ovarian cancer dissemination in an organotypic culture of the human peritoneal cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Organotipik kültürü ilk olarak kollajen tip I primer insan fibroblastları karıştırılması ve sonra mezotelyal hücrelerin 5 kez sayı ile bu kültürü overlaying tarafından toplandı. Yumurtalık kanseri hücreleri, yapışma, istilası veya proliferasyonunu çalışma ilave edilmeden önce, kültür en az 18 saat süreyle inkübe edildi. Her bir deney, çoklu tekrarlanmıştır (n = 5) ve istila deneyler (n = 3) (n = 3-5), farklı hastalara ve çok sayıda kuyu elde edilen 3D kültürleri yapışma her durumd...

Watch this Scientific Journal Video about Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity at JoVE.com

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

İnsan periton boşluğuna bir Organotipik Kültür Yumurtalık Kanseri Yaygınlaştırılması Erken Adımlar Modelleme

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

modeling-early-steps-ovarian-cancer-dissemination-an-organotypic

Research

JoVE Journal

Medicine

26.5K Görüntüleme.  University of Chicago. Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation. 

Video: Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modelleme ve Görüntüleme 3-Boyutlu Toplu Hücre Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

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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 ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Üç boyutlu heterotipik<em&gt; In Vitro</emYumurtalık Kanseri Başlatma ve İlerleme sırasında Stromal-Epitel Etkileşimleri&gt; Modelleme

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Bir olarak Beyin Microslices arasında excitotoxic uyarılması<em&gt; In vitro</em&gt; İnme Model

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

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

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

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

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

Bir<em&gt; Ex vivo</em&gt; Model İnsan Meme Hormon Eylem Eğitim için

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

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

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

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

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

Vivo ve Ex Vivo Periton yağ içinde Samanyolu Nokta Yapıların Yumurtalık Kanseri Metastatik Kolonizasyon Eğitim Yaklaşımlar içinde

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

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Primer Multipl Miyelom Hücrelerinin İlaç Duyarlılık Karakterizasyonu için bir Organotipik Yüksek Verimli Sistemi

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

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.

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 ...

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

Yumurtalık Kanseri Erken Teşhisi İçin Umut Verici Bir Araç Olarak Fallop Tüpünün Tubal Sitolojisi

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Fare retina arteriyoller Ex Vivo reaktivite ölçümü için hazırlık adımları

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...