Bu araştırma için fon Cancer Research UK.

1. Floresan proteinleri ile hücre retroviral etiketleme <ol><li> 0.25 x 10 6 hücre ortalama 6-iyi Plaka retroviral ambalaj hücreleri (örneğin Phoenix) % 10 fetal sığır serumu (FBS) / DMEM çanak. </li><li> Retroviral DNA Transfect hücrelerin 48 saat sonra hücrelerin üreticinin talimatlarına göre Effectene kullanarak. </li><li> Kuyu daha sonra orta 24 saat ile iki kez durulayın, daha sonra 1.5 ml% 10 de ortalama FBS / DMEM ekleyin. </li><li> Pipetleme ve 2 ml mikrosantrifüj tüpler transfer orta 48 saat sonra doku kültürü paketlenmiş virüs toplayın. </li><li> Pelet herhangi bir hücre için 5 dakika 1600 rpm&#39;de santrifüj uygulayın. </li><li> Temiz bir tüpe supernatant ve mağaza çıkarın -80 ° C </li><li> Retrovirally transduced hücreleri 6 iyi bir yemeğin başına 1,5 x 10 5 hücre kaplama ve nemlendirilmiş bir inkübatör 37 ° C gecede yerleştirilir . </li><li> Ertesi gün, hücreleri ve reklam ortamı çıkarın.d her kuyuya 4 ul polybrene (2 mg / ml) ile desteklenmiş virüs stokunun 1 ml. Inkübatör plakalarının değiştirin. </li><li> 5-6 saat inkübasyon sonrasında, her bir kuyunun 2 ml% 10 FBS / DMEM eklemek ve plakalar 37 ° C inbubator gecede değiştirilir. </li><li> Ertesi gün, medyanın yerini ve uygun seçici besiyerleri eklemeden önce hücreler 24 saat geçmesini bekleyin. </li><li> Konfluent (hücrelerin büyüme hızını ve verimliliğini retroviral transdüksiyon bağlıdır, ancak genellikle 4-14 gün sürer) Ne zaman,-transduced kullanarak floresan hücreler için bir referans olarak hücre ve sıralama trypsinize, toplamak ve havuz hücreleri floresans, -80 olmayan transduced hücreleri ve dondurma alikotları daha büyük ° C ileride kullanmak için. </li></ol> 2. Ters Matrigel işgali tahlil <ol><li> Yavaşça buz (Şekil 1A) tam Matrigel (yani büyüme faktörleri içeren) bir kısım Çözülme. </li><li> Once buz PBS içinde çözülmüş, seyreltik Matrigel 1:1 (başka herhangi bir ek ile birlikte2x konsantrasyonu PBS önce seyreltme tedaviler; Şekil 1B). Daha kolay işlemek için polimerizasyon önce Matrigel yapmak için, tüm plasticware (örneğin, ipuçları, tüpler, vb.) Buz olmalıdır. </li><li> Kaplanmamış birçok 8 mikron gözenek 6.5 mm çapında 24 iyi doku kültürü plakası kuyuların içine gerekli Transwells olarak yerleştirin, sonra dikkatli bir şekilde kuyuları seyreltilmiş Matrigel 100 ul pipet ve 37 ~ 30 dakika inkübe bırakın ° C katılaşmaya (Şekil 1C). </li><li> Bu süre zarfında hazırlamak, bir veya daha fazla floresan etiketli kendi normal büyüme her tedavi öncesi durum (e. g siRNA, ilaç tedavisi) hücre hattı bağlı olarak 4-1 ml başına x 10 5 hücre, hücre süspansiyonları orta (Şekil 1D). </li><li> Matrigel filtre (Şekil 1E) yukarı bakacak şekilde alt ve hücre süspansiyonu pipetle 100 ul üzerine Transwells invert katılaşmış vardır. </li><li> 24 iyi doku tabanı ile dikkatlice Transwells kapağıkültür plakası, hücre süspansiyonu (Şekil 1F) her damla ile temas. </li><li> Hücre eki (Şekil 1G) izin vermek için, 4 saat süreyle ters devlet plaka inkübe edin. </li><li> Bu süre sonrasında, plakalar sağ taraftaki yukarıya döner ve ardışık 2 x 1 ml serum serbest orta (Şekil 2A) içine batırarak her transwell yıkayın. </li><li> (Şekil 2B) gerekli olan herhangi bir ilaç ya da tedavilerin de içeren bir üçüncü Transwells bırakın. </li><li> Katılaşmış Matrigel / PBS karışımı üstüne transwell hafifçe (25 ng / ml &#39;e. g AGF) FBS / DMEM artı kemoatraktan% 10 ul 100 pipeti az 3-5 gün, 37 kapak ve inkübe yerine ° C,% 5 CO 2 (Şekil 2C). </li></ol> 3. Boyama ve görselleştirme <ol><li> Hücreler floresan proteinleri ifade konfokal mikroskobi önce aşağıdaki adımları atlayabilirsiniz </li><li> Taze 24 iyi yemekler ve 4 mcM pipet 1 ml Matrigel, yer Transwells işgalci görüntü floresan olmayan hücreler içinCalcein taraf dökülme ve alt ve üst leke sağlayan, her Matrigel fiş üstünde leke çözüm AM. Calcein AM (Calcein acetoxymethyl ester) bu lekeler tüm hücrelerin canlı bir hücre boya yeşil ve hiçbir fiksasyon gerektirir. </li><li> 1 saat 37 ° ° C&#39;de% 5 CO noktası hücreleri tamamen boyanmış ve konfokal mikroskobi tarafından görüntülendi hazır olan 2 nemlendirilmiş atmosfer. </li><li> Alternatif olarak, floresan olmayan hücrelerin Matrigel işgalci sabit ve 3,9-3,5 aşağıda açıklandığı gibi boyanmış olabilir. </li><li> Yeni bir 24 plaka her transwell aktarın. Yerleşimi 1% 4 para-formaldehyde/0.2% Triton-X 100 ml. </li><li> 0.5 saat boyunca oda sıcaklığında inkübe edin. </li><li> Fiksatif çıkarın ve 1 ml PBS ile 3 kere yıkayın. </li><li> 100 mcg / ml RNaz kullanarak 30 dakika RNaz tedavi ile sitoplazmik RNA çıkarın. PBS ile 2 kez yıkayın. </li><li> Görselleştirme için PBS içinde sulandırılmış 0.01 mg / ml Propidium İyodür (PI) ve oda sıcaklığında da terk0.5 saat rk. PBS ile 3 kez yıkayın. Bu aşamada, PI transwell karanlık oda sıcaklığında en az 1 ay süreyle tutulabilir boyandı. </li><li> Konfokal mikroskobu hassas görüntüleme yöntemleri kullanılabilir kaynakları bağlıdır. Olmayan daldırma 20 X objektif ve optik bölümleri matrigel fişi alttan her 10-15 mikron yakalamak üzerinde artık PBS büyük lamel üzerine küçük bir miktar (hava kabarcığı sağlamak) ile ters bir konfokal mikroskop, yer transwell kullanma. Bireysel optik dilim işgali ölçüde (Şekil 3A) ölçmek veya Volocity olarak uygun yazılım (Şekil 3B) kullanılarak 3 boyutlu nesneler oluşturmak için kullanılabilir. </li></ol> 4. Temsilcisi Sonuçlar Şekil 3A optik dilim Z-serisi bir örnek gösterilmiştir. Bu durumda, hücreler Calcein AM ile boyandı ve mesafe ile filtre kadar işgalci hücrelerinin sayısını azaltmak görülebilir. De, ölçümüvasion oranı Calcein, AM olumlu olumsuz her aralıkta, ya da yukarıda ayrıntılı olarak tespiti / boyama yöntemi kullanarak ve her pozisyonda PI pozitif çekirdek sayma piksel piksel analiz ederek yapılabilir. Calcein bir avantajı. Boyama PM hücre işgalinin bu 3-boyutlu rekonstrüksiyonlar işgali modu (Şekil 3B) görsel bir tasviri vererek, böyle bir Volocity olarak yazılımı kullanarak monte edilebilir. Hücreleri floresan proteinlerle ifade etiketli iseniz, o zaman her renk hücre konumları ya tarafında (Şekil 3C) veya yeniden yapılanma (Şekil 3D) aracılığıyla dilimleri yaparak görüntülenebilir 3 boyutlu rekonstrüksiyonlar görüntülenmiştir olabilir. <img alt="Şekil 1" src="/files/ftp_upload/3525/3525fig1.jpg" /> Şekil 1 şematik ters işgali tahlil kurma alan adımları. A) Matrigel ECM buz üzerinde çözülmüş. B) Matrigel iki kez final konsantrasyon herhangi bir ilaç tedavileri içeren PBS ile 1:1 seyreltilir. C)<html> Transwell ekler multiwell plakaları yerleştirilir ve Matrigel her pipetlenir. D) Hücre süspansiyonlar istenilen konsantrasyonda yaptı. E) Matrigel sonra, plaka ters ve kaldırılır hücreleri transwell ekler alt filtre üzerine kaplanmıştır. F) ters pozisyonda, multiwall plaka dikkatle hücre süspansiyonu ile temas eden, transwell ekler üzerine yerleştirilir. G) Hücreler, 4 saat için filtre uymak izin verilir. <img alt="Şekil 2" src="/files/ftp_upload/3525/3525fig2.jpg" /> Şekil 2 ters işgali tayini için şematik diyagramı devamı. Hücreleri yapıştırılır A) Bir kez, iki kez gevşek hücreleri çıkarmak için serum ücretsiz medya her transwell batırın. B) Yer gerektiği gibi de içeren nihai bir medya artı tedaviler transwell yıkanır. C) Medya, gerektiği gibi tedavileri ile kemoatraktan (örneğin% 10 fetal sığır serumu) içeren dikkatle Matrigel üzerine katmanlı. ent &quot;&gt; <img alt="Şekil 3" src="/files/ftp_upload/3525/3525fig3.jpg" /> Şekil 3. Ters işgali testinin sonuçları Temsilcisi görüntüleri. A) Calcein ile boyanmış hücreler Optik bölümler konfokal mikroskobu ile 15 mikron aralıklarla alınan Matrigel işgalci PM. B) İmar, yandan bakıldığında konfokal Z-serisi görüntüleri, bir yığın hücre işgali 3 boyutlu rekonstrüksiyon. Referans, 10 yayımlanmaktadır. C) Üç boyutlu yan bakıldığında Matrigel işgalci GFP ve RFP etiketli hücrelerinin yeniden inşası. D) Optik, GFP ve RFP etiketli hücreleri 3 boyutlu rekonstrüksiyon ile dilim. Referans, 10 yayımlanmaktadır.

<ol>
	<li>Olson, M. F., Sahai, 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+actin+cytoskeleton+in+cancer+cell+motility.">The actin cytoskeleton in cancer cell motility.</a> Clin. Exp. Metastasis. 26, 273-287 (2008).</li><li>Hoelzinger, D. B., Demuth, T., Berens, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+factors+that+sustain+glioma+invasion+and+paracrine+biology+in+the+brain+microenvironment.">Autocrine factors that sustain glioma invasion and paracrine biology in the brain microenvironment.</a> J. Natl. Cancer. Inst. 99, 1583-1593 (2007).</li><li>Chaffer, C. L., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+cancer+cell+metastasis.">A perspective on cancer cell metastasis.</a> Science. 331, 1559-1564 (2011).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hallmarks+of+Cancer.">The Hallmarks of Cancer.</a> Cell. 100, 57-70 (2000).</li><li>Francia, G., Cruz-Munoz, W., Man, S., Xu, P., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics.">Mouse models of advanced spontaneous metastasis for experimental therapeutics.</a> Nat. Rev. Cancer. 11, 135-141 (2011).</li><li>Hooper, S., Marshall, J. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+migration+in+three+dimensions.">Tumor cell migration in three dimensions.</a> Methods. Enzymol. 406, 625-643 (2006).</li><li>Croft, D. R., Olson, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+the+conversion+between+rounded+and+elongated+modes+of+cancer+cell+movement.">Regulating the conversion between rounded and elongated modes of cancer cell movement.</a> Cancer Cell. 14, 349-351 (2008).</li><li>Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-step+pericellular+proteolysis+controls+the+transition+from+individual+to+collective+cancer+cell+invasion.">Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion.</a> Nat. Cell. Biol. 9, 893-904 (2007).</li><li>Hennigan, R. F., Hawker, K. L., Ozanne, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fos-transformation+activates+genes+associated+with+invasion.">Fos-transformation activates genes associated with invasion.</a> Oncogene. 9, 3591-3600 (1994).</li><li>Scott, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIM+kinases+are+required+for+invasive+path+generation+by+tumor+and+tumor-associated+stromal+cells.">LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells.</a> J. Cell. Biol. 191, 169-185 (2010).</li></ol>

Yazarlar ifşa etmek başka bir şey var.

Matrigel işgali testleri geleneksel ve alt kısmında bir filtre aracılığıyla doğru kemoatraktan indüklenen motilite ile ekstraselüler matriks proteini bir tabaka üzerine yerleştirilen hücreleri ile kurulmuştur. Invazivlik filtrenin alt kısmında kaç hücre olabilir sayılır bir fonksiyonu olarak gol oldu. Çok az bir fark ile, yukarıda açıklanan &quot;ters&quot; işgal tahlil pratik olmasına rağmen, Matrigel ile yukarı taşımak ve işgalci hücreleri görselleştirme tarafından tespit edilebilir ...

<table border="1"><tbody><tr><th> Reaktifi Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> DMEM (Dulbecco&#39;nun Modifiye Kartal Orta) </td><td> Gibco </td><td> 21969 </td></tr><tr><td> Fetal sığır serumu </td><td> PAA </td><td> A15-101 </td></tr><tr><td> Penisilin Streptomisin </td><td> Gibco </td><td> 15140 </td></tr><tr><td> 200 mM L-Glutamine (100x) </td><td> Gibco </td><td> 25050-032 </td></tr><tr><td> Puromycin </td><td> Sigma-Aldrich </td><td> P8833 </td></tr><tr><td> % 0.05 tripsin EDTA </td><td> Gibco </td><td> 25300 </td></tr><tr><td> Polybrene </td><td> Sigma-Aldrich </td><td> AL-118 </td></tr><tr><td> Lipofectamine 2000 Reaktif </td><td> Invitrogen </td><td> 11668019 </td></tr><tr><td> 6.5mm Transwells, 8.0 mikron gözenek boyutu </td><td> Corning </td><td> 3422 </td></tr><tr><td> Komple Matrigel </td><td> BD Biosciences </td><td> 354234 </td></tr> &lt;tr&gt; <td> Calcein AM </td><td> Invitrogen </td><td> C1430 </td></tr><tr><td> RNaz </td><td> Qiagen </td><td> 19101 </td></tr><tr><td> Propidium İyodür </td><td> Sigma-Aldrich </td><td> P4864 </td></tr><tr><td> Konfokal microcope </td><td> Leica </td><td> SP2MP </td></tr></tbody></table>

modeling and imaging 3-dimensional collective cell invasion

Kanser malignite tanımlayan ayırt edici özelliği, invazyon ve metastaz 1 . Bazı kanser türleri (E. G glioma 2), çevredeki sağlıklı doku içine lokal invazyon, hastalık ve ölüm kök nedenidir . Diğer kanserler için (e. g meme, akciğer, vb.), Tümör hücrelerinin primer tümör kitlesi hareket distal siteleri kolonize ve sonuçta organ yetmezliğine katkıda metastaz süreci, sonunda morbidite ve ölüm 3. Invazyon ve metastaz, kanser ölümlerinin 4% 90 oranında sorumlu olduğu tahmin edilmektedir . Sonuç olarak, gelişen tanı ve tedavi 5 amaçları için moleküler süreçler ve invazyon ve metastaz kritik protein arabulucular tespit yoğun ilgi olmuştur. Kanser bilim adamları için bir meydan okuma in vivo durumu yeterince benzer işgali testleri geliştirmek içindoğru hastalığı modelleme 6 etkinleştirin. İki boyutlu hücre motilite testleri işgali yalnızca bilgilendirme yaklaşık bir yönü vardır ve aynı zamanda önemli bir unsurudur hesap ekstraselüler matriks (ECM) protein remodeling yapmayız. Son zamanlarda, tümör hücresi invazyonu anlayışımız araştırma rafine ve tek tek hücreler, uzatılmış veya yuvarlak modları 7 hareket etmiş olabileceğini göstermiştir . Buna ek olarak, özellikle epitel özellikleri korumak son derece farklılaşmış tümörler kanser 8 yayılmasını hücreleri lifler, çarşaf ve kümeler halinde işgal toplu işgali katkısı büyük takdir,, olmuştur. Biz kolektif işgali 10 katkıları aday proteinlerin incelenmesi için zarif bir yöntem 9 sunuyoruz. Özellikle, farklı floresan proteinleri ifade hücreleri mühendislik ayrı havuzları, moleküler faaliyetler ve protei incelemek mümkünns aşağıdaki hücreleri gerekli olan karşı önde gelen hücreleri gereklidir. RNAi kullanımı deneysel yanı sıra toplu işgalinin farklı pozisyonlarda bireysel hücre invazyonu ilgili işlemleri sökmeye moleküler bir araç sağlar. Bu prosedürde, floresan etiketli hücrelerinin karışımları Matrigel ECM protein ile dolu bir transwell eklemek altındaki kaplama, daha sonra, filtre ve Matrigel içine &quot;yukarı&quot; işgal izin. Konfokal görüntüleme ile elde edilen üç boyutlu gösterimleri içine z serisi görüntü yığınlar, İmar lideri konumunu karşı aşağıdaki floresan etiketli hücrelerinin temsil toplu işgalci iplikçikleri ve analiz görselleştirme sağlar.

Watch this Scientific Journal Video about Modeling and Imaging 3-Dimensional Collective Cell Invasion at JoVE.com

Modeling and Imaging 3-Dimensional Collective Cell Invasion

Üç boyutlu hücre dışı matriks içine tümör hücresi invazyonu modelleri daha iyi yansıtacak<em&gt; In vivo</em&gt; Iki boyutlu motilite testleri daha durum. Floresan etiketli hücreleri, işgali modları ve önde gelen karşı aşağıdaki hücreleri farklı katkıları hakkında detaylı bilgi konfokal görüntüleme ile birlikte matris işgali testleri kullanılarak elde edilebilir.

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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16.6K Görüntüleme.  University of Strathclyde. Üç boyutlu hücre dışı matriks içine tümör hücresi invazyonu modelleri daha iyi yansıtacak In vivo Iki boyutlu motilite testleri daha durum. Floresan etiketli hücreleri, işgali modları ve önde gelen karşı aşağıdaki hücreleri farklı katkıları hakkında detaylı bilgi konfokal görüntüleme ile birlikte matris işgali testleri kullanılarak elde edilebilir.

Video: Modeling and Imaging 3-Dimensional Collective Cell Invasion

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Oral Skuamöz Hücre Karsinomu Ortotopik Fare Modeli Tümör Hücre Invasion Multi-foton Görüntüleme

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

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

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

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

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

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 ...

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

3-Boyutlu Reçine Döküm ve Fare Portal Ven veya intrahepatik safra Sisteminin Görüntüleme

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Nakledilen Muscle Progenitör Hücreler Hedefleyecek Moleküler Görüntüleme

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

Üretim ve Farelerde arteriyel Lezyonlarının 3-Boyutlu kantitasyonu Optik Projeksiyon Tomografi ile

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Moleküler Kolorektal Kanser Hücrelerinin bir 3-Boyutlu Modeli İnvaziv tümör mikro Profili ve<em&gt; Ex vivo</em&gt; Fibroblastlar

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

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 ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

Farelerde spontan metastatik renal hücreli karsinom (MRCC) modelleme ardından nefrektomi

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Hastalık Modelleme için kas biyopsisi Hasta türetilmiş Hücre Hatları İzolasyonu ve Ölümsüzleştirme

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

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 ...