Biz hücre hatları için reaktifler ve Fred Miller (Barbara Ann Karmanos Kanser Intitute, Detroit, ABD) Ken Iwata (OSI Pharmaceuticals, New York, ABD) teşekkür ediyoruz.

1. Methocel çözüm Hazırlama (500 mL) <ol><li> Bir manyetik karıştırıcı içeren 500 ml şişe metilselüloz Otoklav 6 gr. </li><li> 20 dakika, önceden ısıtılmış DMEM/F12 250 ml serum (60 ° C) olmadan metilselüloz çözülür. </li><li> DMEM/F12 (RT),% 10 at serumu içeren 250 ml ekleyin. Mix gecede (4 ° C). </li><li> Santrifüj (5000g, 2h, 4 ° C) çözüm temizleyin. </li><li> Yeni bir tüp (stok solüsyonu yaklaşık% 90-95) açık oldukça viskoz bir çözüm atın. </li><li> Mağaza, 4 ° C kullanana kadar. </li></ol> 2. Sfero kültür <ol><li> % 20 methocel solüsyonu (10 ml methocel ve 40 ml besi) hazırlayın. </li><li> Hücreler PBS ile iki kez yıkayın, 37 yaşında% 0.1 tripsin ve inkübe hücreleri eklemek ° C </li><li> Hücrelerin büyüme ortamında süspanse edin ve hücre sayımı. </li><li> % 20, 10 000 ml başına hücrelerinin süspansiyon hazırlamak methocel. </li><li> 96 iyi yuvarlak alt hücre süspansiyonuuspension levhalar (ortalama 100 ul). </li><li> Ertesi gün, süspansiyon plakalar sfero oluşumunu kontrol edin. Tanecikler ise 1-3 gün sonra kullanıma hazır olacaktır. </li></ol> 3. Kollajen hazırlanması <ol><li> Buz üzerinde 8 ml kollajen, 1 ml PBS 10x, 1 ml 0.1 N NaOH ve 3 damla 0,1 N HCl çözeltisi hazırlayın. PH indikatörü şeritler çözümün bazı tespit ederek pH 7.4 olup olmadığını kontrol edin. PH aralığı olmadığını ayarlayın. </li><li> 96 plaka kuyularda nötralize kollajen çözüm 50 ul ekle </li><li> 37 inkübe ° C kollajen (90 dk) katılaşan kadar. </li><li> Buz üzerinde methocel-kollajen-ligand çözümleri hazırlayın: kollajen çözüm her ligand pipet 1 parçası. Kollajen TGF-β 20 ng ml başına ekleyin. Farklı katmanlar üzerinde methocel ve difüzyon ile seyreltme sonra, TGF-β nihai konsantrasyonu 5 ng / ml Mix tarafından vorteks olacaktır. 1 methocel çözümün parçası ekleyin. Vorteks ile karıştırın. </li></ol> 4. Sfero embedding <ol><li> 200 ul pipet ucu kesilmiş yaklaşık 5 mm, 200 ul ucu yerleştirin. Pipet ile bir sfero emmek ve orta boş bir tabak içine dikkatlice dağıtmak. Sfero ucu içinde kalırsa izleyin. Sfero, beyaz bir nokta olarak görülebilir. Piston bırakmadan, 100 ul kollajen methocel karışımı emmek ve kollajen kaplı iyi 96 kollajen karışımı sfero dağıtmak. 12 parçacıklarının her bir durum için bunu yapın. </li><li> Let en az 30 dakika katılaşmasını kollajen 37 ° C </li><li> 200 ul ucu ile hava kabarcıklarını çıkarın. </li><li> % 1.6 serum ve DMSO içinde çözülmüş inhibitörleri ile 50 ul orta ekleyin. SB-431.542 için 4 ul stok solüsyonu (10 mM) orta 1 ml ekleyin. Difüzyon sonra inhibitörü son konsantrasyonu 10 mcM olacaktır. TGF-β ve inhibitörleri, deney boyunca kalacaktır. 40X büyütme ile mikroskop altında parçacıklarının fotoğraflar çekin. </li><li> 4 sonraTGF-β ve / veya inhibitörleri ile stimülasyon 8h, 40X büyütme ile mikroskop altında parçacıklarının fotoğraf çekmek. </li><li> 2 gün sonra işgalci parçacıklarının alanı ölçün ve başlangıç ​​alanına çıkarmak. Ölçüm alanında bir sfero: Adobe Photoshop Extended (Şekil 2A) sfero resim ve Hızlı Seçim aracı (Şekil 2B) seçeneğini açın. Bir &quot;artı&quot; (+) işareti (şekil 2C) ile bir daire içine fare işaretçisi değişir. Sfero üzerine imleci sürüklerken, Photoshop, sfero sınırları tanıyacak. Sfero dışında bir alan seçili ise araç çubuğu (-) işareti, bu bölgede Alt tuşuna basarak veya tarafından belirtilen olumsuz bir seçim aracını kullanarak seçim dışında olabilir. (-) &#39;Eksi&#39; ile bir daire içine imleç işareti ve yanlış seçilmiş bir alan üzerinde imleci sürükleyerek, bu alanda seçim (Şekil 2D) kaldırılır. Eğer gerekirse, birden fazla bölgede, Sh basarak seçilebilirift düğmesi. Daha doğru bir şekilde çalışmak için, fare işaretçisi boyutu araç çubuğunda fırça çapı (Şekil 2E) değiştirerek ayarlanabilir. Tüm sfero seçildiğinde, seçimin alan menü çubuğunda ANALİZ-TEDBİR aracılığıyla veya Ctrl + Shift + M (Şekil 2F) tuşuna basarak hesaplanır. Dosya adı ve piksel cinsinden seçim alanı yanı sıra diğer verileri (Şekil 2G) gösteren ölçüm kayıt penceresi görünecektir. Eğer birden çok seçim ölçülür, ilk satırı tüm alanları toplamını gösterir. Aşağıdaki satırlarda, bireysel seçimleri ölçümleri sunulmaktadır. Tüm ölçümlerin verileri bir metin dosyasına ihraç ve Excel&#39;de açılabilir. </li></ol> 5. Temsilcisi sonuçları: MCF10A1 (M1) normal meme epitel hücreleri ile sfero testinin bir örneği, H-RAS MCF10AneoT (M2) hücrelerin dönüştürdü ve M2-kökenli MCF10CA1a (M4) Şekil 3&#39;te verilmiştir. M1 hücreleri olmadan sadece zayıf işgali gösterdistimülasyon, ancak önemli ölçüde işgal TGF-β tarafından uyarılması üzerine daha iyi. Buna karşılık, türev M1-M2 uyaranlara ek olmadan verimli bir şekilde zaten işgal RAS-dönüştürülmüş ve bu konuda daha fazla TGF-β tedavisi üzerine 4-5 kat arttı. M4 hücreleri hem de TGF-β ek olmadan, güçlü işgali gösterdi. Sonra, biz kimyasal inhibitörleri kullanarak bu sfero model TGF-β-kaynaklı işgali etkileyebilecek olmadığını inceledi. Bu amaçla biz SB-431.542, bir ATP analog ve TβRI kinaz aktivitesini seçici inhibitörü gibi aktivin tip IB reseptör (ActRIB) ve aktivin kinaz-benzeri reseptör (ALK) 7 12 kullanılır. Beklendiği gibi, SB-431.542 kuvvetli M1 TGF-β indüklenen işgali, TGF-β indüklenen bu işgali belirten M2 ve M4 hücreleri (Şekil 4), inhibe TβRI-kinaz bağımlı. Buna ek olarak, bazal parçacıklarının işgali de güçlü bir şekilde inhibe, bu bazal işgali düşündüren de aynı zamandaTGF-β sarkık. <img alt="Şekil 1" src="/files/ftp_upload/3337/3337fig1.jpg" /> Şekil 1: 3D sfero testinin akış çizelgesi. İlk olarak, hücreler, U-şekilli süspansiyonlar plakalar yapışmaz koşullar altında kaplanmıştır. Hücreler multcellular parçacıklarının aggregrate ve kollajen matriks içine gömülü olabilir. Bu kollajen matriks içine, onlar işgal mümkün. <img alt="Şekil 2" src="/files/ftp_upload/3337/3337fig2.jpg" /> Şekil 2. Genişletilmiş Adobe Photoshop kullanarak parçacıklarının alan Kantitasyonu. (A) sfero resmi kullanarak wongly yer alan sfero (D) Kaldırma alanı seçmek için sfero üzerine imleci sürükleme Hızlı Seçim aracı Adobe Photoshop Extendend (B) Seçim (C) açıldı daha doğru bir alanı seçmek için Hızlı Seçim aracı fırça boyutunu olumsuz Hızlı Seçim aracı (E) Ayarı (F) Seçimiölçüm komutu bir ölçüm kayıt örneği kaydetmek için (G). <img alt="Şekil 3" src="/files/ftp_upload/3337/3337fig3.jpg" /> Şekil 3 kendiliğinden ölümsüzleştirdi MCF10A1 (M1) (A) parçacıklarının TGF-β indüklenen işgali, MCF10AneoT (M2 (B) ve metastatik türevi MCF10CA1a (M4) (C). M1, M2 ve M4 parçacıklarının gömülü RAS dönüştürülmüş belirtildiği gibi 48 saat için kollajen içine TGF-β 5ng/ml ile tedavi edildi. AC Temsilcisi resim gömme gün alınmış ve iki gün sonra D Bağıl işgali gün 2 gün 0 eksi alan farkı gibi sayısal sonuçlar ifade edilmiştir. olarak ortalama ± SD 13 &#39;den uyarlanmıştır. <img alt="Şekil 4a" src="/files/ftp_upload/3337/3337fig4a.jpg" /> <img alt="Şekil 4b" src="/files/ftp_upload/3337/3337fig4b.jpg" /> Şekil 4. Bazal ve TGF-β-kaynaklı sfero işgali b inhibe ediliry SB-431.542. M1, M2 ve M4 parçacıklarının kollajen içine gömülü ve 5ng/ml TGF-β ve / veya 10 belirtildiği gibi mcM SB-431.542 ile tedavi edildi. Temsilcisi resimler 2 gün sonra alınan (A). Bağıl işgali gün 2 gün 0 (B) eksi alan farkı olarak belirlendi. Sonuçlar, ortalama ± SD 13 den uyarlanmıştır olarak ifade edilir .

<ol>
	<li>Akhurst, R. J., Derynck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+signaling+in+cancer--a+double-edged+sword.">TGF-&#946; signaling in cancer--a double-edged sword.</a> Trends. Cell. Biol. 11, S44-S51 (2001).</li><li>Massagu&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+in+Cancer.">TGF-&#946; in Cancer.</a> Cell. 134, 215-215 (2008).</li><li>Ghellal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+significance+of+TGF-&#946;1+and+TGF-&#946;+in+human+breast+carcinoma.">Prognostic significance of TGF-&#946;1 and TGF-&#946; in human breast carcinoma.</a> Anticancer. Res. 20, 4413-4413 (2000).</li><li>Sheen-Chen, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+levels+of+transforming+growth+factor-&#946;1+in+patients+with+breast+cancer.">Serum levels of transforming growth factor-&#946;1 in patients with breast cancer.</a> Arch. Surg. 136, 937-937 (2001).</li><li>ten Dijke, P., Hill, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+TGF-&#946;-Smad+signaling.">New insights into TGF-&#946;-Smad signaling.</a> Trends Biochem. Sci. 29, 265-265 (2004).</li><li>Moustakas, A., Heldin, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-Smad+TGF-&#946;+signals.">Non-Smad TGF-&#946; signals.</a> J. Cell Sci. 118, 3573-3573 (2005).</li><li>Soule, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+a+spontaneously+immortalized+human+breast+epithelial+cell+line,+MCF-10.">Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10.</a> Cancer Res. 50, 6075-6075 (1990).</li><li>Strickland, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+premalignant+MCF10AT+generates+heterogeneous+malignant+variants+with+characteristic+histologic+types+and+immunohistochemical+markers.">Progression of premalignant MCF10AT generates heterogeneous malignant variants with characteristic histologic types and immunohistochemical markers.</a> Breast Cancer Res. Treat. 64, 235-235 (2000).</li><li>Santner, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+MCF10CA1+cell+lines+derived+from+premalignant+human+breast+epithelial+MCF10AT+cells.">Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells.</a> Breast Cancer Res. Treat. 65, 101-101 (2001).</li><li>Smalley, K. S., Lioni, M., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+isn't+flat:+taking+cancer+biology+to+the+next+dimension.">Life isn't flat: taking cancer biology to the next dimension.</a> In Vitro Cell Dev. Biol. Anim. 42, 242-242 (2006).</li><li>Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquired+multicellular-mediated+resistance+to+alkylating+agents+in+cancer.">Acquired multicellular-mediated resistance to alkylating agents in cancer.</a> Proc. Natl. Acad. Sci. U. S. A. 90, 3294-3294 (1993).</li><li>Inman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SB-431542+is+a+potent+and+specific+inhibitor+of+transforming+growth+factor-b+superfamily+type+I+activin+receptor-like+kinase+(ALK)+receptors+ALK4,+ALK5,+and+ALK7.">SB-431542 is a potent and specific inhibitor of transforming growth factor-b superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7.</a> Mol. Pharmacol. 62, 65-65 (2002).</li><li>Wiercinska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TGF-&#946;/Smad+pathway+induces+breast+cancer+cell+invasion+through+the+up-regulation+of+matrix+metalloproteinase+2+and+9+in+a+spheroid+invasion+model+system.">The TGF-&#946;/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system.</a> Breast Cancer Res. Treat. 128, 657-657 (2011).</li><li>Tang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+switches+from+tumor+suppressor+to+prometastatic+factor+in+a+model+of+breast+cancer+progression.">TGF-&#946; switches from tumor suppressor to prometastatic factor in a model of breast cancer progression.</a> J. Clin. Invest. 112, 1116-1116 (2003).</li><li>Provenzano, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+density+promotes+mammary+tumor+initiation+and+progression.">Collagen density promotes mammary tumor initiation and progression.</a> BMC. Med. 6, 11-11 (2008).</li><li>Ramaswamy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+signature+of+metastasis+in+primary+solid+tumors.">A molecular signature of metastasis in primary solid tumors.</a> Nat. Genet. 33, 49-49 (2003).</li><li>Debnath, J., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+glandular+epithelial+cancers+in+three-dimensional+cultures.">Modelling glandular epithelial cancers in three-dimensional cultures.</a> Nat. Rev. Cancer. 5, 675-675 (2005).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nat. Rev. Mol. Cell. Biol. 8, 839-839 (2007).</li><li>Lee, G. Y. <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+culture+models+of+normal+and+malignant+breast+epithelial+cells.">Three-dimensional culture models of normal and malignant breast epithelial cells.</a> Nat. Methods. 4, 359-359 (2007).</li><li>Petersen, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+with+basement+membrane+serves+to+rapidly+distinguish+growth+and+differentiation+pattern+of+normal+and+malignant+human+breast+epithelial+cells.">Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells.</a> Proc. Natl. Acad. Sci. U. S. A. 89, 9064-9064 (1992).</li><li>Debnath, J., Muthuswamy, S. K., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+oncogenesis+of+MCF10A+mammary+epithelial+acini+grown+in+three-dimensional+basement+membrane+cultures.">Morphogenesis and oncogenesis of MCF10A mammary epithelial acini grown in three-dimensional basement membrane cultures.</a> 30, 256-256 (2011).</li><li>Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p21-Activated+kinase+1+coordinates+aberrant+cell+survival+and+pericellular+proteolysis+in+a+three-dimensional+culture+model+for+premalignant+progression+of+human+breast+cancer.">p21-Activated kinase 1 coordinates aberrant cell survival and pericellular proteolysis in a three-dimensional culture model for premalignant progression of human breast cancer.</a> 10, 314-314 (2011).</li><li>Li, Q., Chow, A. B., Mattingly, R. R. <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+overlay+culture+models+of+human+breast+cancer+reveal+a+critical+sensitivity+to+mitogen-activated+protein+kinase+kinase+inhibitors.">Three-dimensional overlay culture models of human breast cancer reveal a critical sensitivity to mitogen-activated protein kinase kinase inhibitors.</a> J. Pharmacol. Exp. Ther. 332, 821-821 (2010).</li></ol>

Çıkar çatışması.

Biz MCF10A1 hücreleri ve malign türevleri TGF-β bağımlı bir şekilde bir kollajen matriks içine işgal sfero bir model oluşturdu. Birincisi, parçacıklarının metilselüloz varlığı 96-kuyular yuvarlak alt süspansiyon plakaları oluşur. Tabii ki, aynı zamanda U-şekilli poli-HEMA kaplı plakaları bu amaç için kullanılabilir. Hücreler metilselüloz yokluğunda ölecek beri Metilselüloz kesinlikle, bu adım gereklidir. Hücreler bu faktör kolajen orta op üst eklendiğinde TGF-β daha iyi cevap verdi...

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Metil selüloz </td><td> Sigma-Aldrich </td><td> M0512 </td><td></td></tr><tr><td> PureCol </td><td> Gelişmiş BioMatrix </td><td> 5005-B </td><td></td></tr><tr><td> pH indikatörü şeritleri </td><td> Merck </td><td> 9533 </td><td></td></tr><tr><td> 96-iyi süspansiyon plakalar </td><td> Greiner Bio-one </td><td> 650 (185) </td><td></td></tr><tr><td> TC yapışma 96-well plakalar </td><td> Greiner Bio-one </td><td> 655 180 </td><td></td></tr></tbody></table>

spheroid assay to measure tgf-&#946;-induced invasion

TGF-β başlangıç ​​aşamasında bir tümör baskılayıcı gibi davranan, ancak daha sonraki bir aşamada 1,2 invazyon ve metastaz uyarıcı, meme kanseri ilerlemesinde rol karşı vardır . Ayrıca, TGF-β meme kanseri sık sık aşırı eksprese ve kendi ifadesini, kötü prognoz ve metastaz 3,4 ile ilişkilidir. TGF-β indükler işgali iyi anlaşılmış değildir hangi mekanizmaları. TGF-β, TGF-β Tip II (TβRII) ve tip I (TβRI) reseptörleri aracılığıyla hücresel yanıtlar ortaya çıkarır. TGF-β indüklenen heteromeric kompleks oluşum üzerine, TβRII TβRI fosforile. Aktif TβRI reseptör Smads fosforilleyerek (R-Smads), yani Smad2 ve Smad3, onun hücre içi kanonik sinyal yolağı başlatır. Bu aktive R-Smads çekirdeğinde birikir ve hedef genlerin 5 transkripsiyonunu düzenleyen Smad4 heteromeric kompleksleri oluşturur. Buna ek olarak, daha önce described smad yolu olmayan bazı smad sinyalizasyon yolaklar aktivasyonu reseptör aktivasyonu sonuçları, örneğin Mitojen Protein Kinaz (MAPK) 6 yolakları. Meme kanseri farklı aşamalarında TGF-β rolünü araştırmak için, biz MCF10A hücre sistemi kullanmak yaptı. Bu sistem kendiliğinden ölümsüzleştirdi MCF10A1 (M1), meme epitel hücreleri 7 oluşur, H-RAS farelerde 8 premalign lezyonları M1 türev MCF10AneoT (M2), ve gelen kurulmuştur M2 türev MCF10CA1a (M4) dönüştürdü M2 xenografts ve formlar 9 akciğer metastaz yeteneği ile yüksek dereceli karsinomlar. Bu MCF10A serisi hücre yanıtları farklı bir genetik arka önyargılı değildir malignite farklı sınıflarda eğitim imkanı sunuyor. TGF-β indüklenen işgalinin analizi için, biz homotypic MCF10A sfero hücre kültürleri embe oluşturulan3D kollajen matriks in vitro (Şekil 1) dded. Bu modelleri yakından sfero 10 iç hatta dış nekrotik ve latent veya hücreleri üzerinde aktif ve invaziv hücrelerinde oksijen ve besin maddelerinin bir degrade kurarak in vivo insan tümörleri andırır. Sfero bazlı çalışmaları da tek tabaka kültürler 11 ilaç direnci daha iyi özetlemek için gösterilmiştir. Bu MCF10 3D model sistem malignite farklı aşamalarında meme hücrelerinin invaziv özelliklerini TGF-β sinyal etkisini araştırmak için izin verdi.

Watch this Scientific Journal Video about Spheroid Assay to Measure TGF-Î²-induced Invasion at JoVE.com

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

Kantitatif büyüme faktörü (TGF)-β indüklenen 3 boyutlu kolajen jel işgali dönüştürme ölçmek için bir test tarif edilir. Bu testte, meme kanseri gelişiminin farklı aşamaları temsil hücre hatlarının MCF10A serisi, yararlanır. Bu yöntem, diğer hücre hatları ile birlikte kullanılmak üzere ve diğer potansiyel aktivatörleri veya işgali inhibitörleri araştırmak için kullanılmış olabilir kabul edilebilir.

TGF-β-kaynaklı Invasion Tedbir Sfero Testi

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

spheroid-assay-to-measure-tgf-induced-invasion

Research

JoVE Journal

Medicine

Spheroid Assay to Measure TGF-&#946;-induced Invasion

21.7K Görüntüleme.  Leiden University Medical Centre. Kantitatif büyüme faktörü (TGF)-β indüklenen 3 boyutlu kolajen jel işgali dönüştürme ölçmek için bir test tarif edilir. Bu testte, meme kanseri gelişiminin farklı aşamaları temsil hücre hatlarının MCF10A serisi, yararlanır. Bu yöntem, diğer hücre hatları ile birlikte kullanılmak üzere ve diğer potansiyel aktivatörleri veya işgali inhibitörleri araştırmak için kullanılmış olabilir kabul edilebilir.

Video: Spheroid Assay to Measure TGF-&#946;-induced Invasion

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

Kanser Hücreleri Endotel Hücre Tek tabakalı Invasion Tedbir Gerçek zamanlı Elektrik Empedans Tabanlı Tekniği

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Tıp

Method to Measure Tone of Axial and Proximal Muscle

The control of tonic muscular activity remains poorly understood. While abnormal tone is commonly assessed clinically by measuring the passive resistance of relaxed limbs1, no systems are available to study tonic muscle control in a natural, active state of antigravity support. We have developed a device (Twister) to study tonic regulation of axial and proximal muscles during active postural maintenance (i.e. postural tone). Twister rotates axial body regions relative to each other about the vertical axis during stance, so as to twist the neck, trunk or hip regions. This twisting imposes length changes on axial muscles without changing the body's relationship to gravity. Because Twister does not provide postural support, tone must be regulated to counteract gravitational torques. We quantify this tonic regulation by the restive torque to twisting, which reflects the state of all muscles undergoing length changes, as well as by electromyography of relevant muscles. Because tone is characterized by long-lasting low-level muscle activity, tonic control is studied with slow movements that produce "tonic" changes in muscle length, without evoking fast "phasic" responses. Twister can be reconfigured to study various aspects of muscle tone, such as co-contraction, tonic modulation to postural changes, tonic interactions across body segments, as well as perceptual thresholds to slow axial rotation. Twister can also be used to provide a quantitative measurement of the effects of disease on axial and proximal postural tone and assess the efficacy of intervention.

We have developed a device (Twister) to study the regulation of tonic muscle activity during active postural maintenance. Twister measures torsional resistance and muscular responses in standing subjects during twisting of the body axis. The device can be flexibly configured to study various aspects of tonic control across the neck, trunk, and/or hips.

Eksenel ve Proksimal kas tonu ölçün Yöntem

The control of tonic muscular activity remains poorly understood.  While abnormal tone is commonly assessed clinically by measuring the passive resistance ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

Gerçeklik ve kesinlik Tedbir Diş Hekimliği, A New Way Doğruluk

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Bir Modifiye<em&gt; In vitro</em&gt; Invasion Deneyi Hormonlar, Açan Kanser Hücre Invasion Sitokinlerin ve / veya Büyüme Faktörleri Potansiyel Rolü belirlemek için

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

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 ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Rahim içi Telemetri Fare Kontraktil Basıncını Ölçmek<em&gt; İn Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Üç boyutlu (3D) Tümör Sfero Yayılması deneyindeki

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

Test Chick Kalp Invasion Testi Kanser hücrelerinin invazivliğini ve Potansiyel Anti-invaziv Bileşikler Etkinlik

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

Bir Kanseri Hücre Sfero Testi 3D ortamda Invasion Değerlendirmek için

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

Kafa ve boyun kanseri Perinevral Invazyonu değerlendirmek için Chick Chorioallantoik membran In vivo model

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...