Name: lung tumor cell recruitment assay
Uploaded: 2019-02-26T12:30:00
Description: Watch this Scientific Journal Video about Lung Tumor Cell Recruitment Assay at JoVE.com

Fare ile gerçekleştirilen tüm yordamları hayvan Araştırma Komitesi, Tokyo kadın Tıp Üniversitesi tarafından onaylanmıştır.
1. kuyruk ven enjeksiyon Lewis akciğer Karsinomu (LLC) hücre
<ol><li>Bir oksijen % %5 10 FCS ile takıma DMEM LLC hücreleri korumak CO2 kuluçka 37 ° C'de Hücreleri bir floresan protein ifade sistemi (Örneğin, GFP) içermelidir.</li>
	<li>Hücre kültür yemek bir enzimatik olmayan hücre ayrılma reaktif kullanarak kaldırın. Ür...

<ol>
	<li>Valastyan, S., 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=Tumor+metastasis:+molecular+insights+and+evolving+paradigms.">Tumor metastasis: molecular insights and evolving paradigms.</a> Cell. 147 (2), 275-292 (2011).</li><li>Kaplan, R. 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=VEGFR1-positive+haematopoietic+bone+marrow+progenitors+initiate+the+pre-metastatic+niche.">VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.</a> Nature. 438 (7069), 820-827 (2005).</li><li>Hiratsuka, S., Watanabe, A., Aburatani, H., Maru, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-mediated+upregulation+of+chemoattractants+and+recruitment+of+myeloid+cells+predetermines+lung+metastasis.">Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis.</a> Nature Cell Biology. 8 (12), 1369-1375 (2006).</li><li>Hiratsuka, S., 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=The+S100A8-serum+amyloid+A3-TLR4+paracrine+cascade+establishes+a+pre-metastatic+phase.">The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase.</a> Nature Cell Biology. 10 (11), 1349-1355 (2008).</li><li>Tomita, T., Sakurai, Y., Ishibashi, S., Maru, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imbalance+of+Clara+cell-mediated+homeostatic+inflammation+is+involved+in+lung+metastasis.">Imbalance of Clara cell-mediated homeostatic inflammation is involved in lung metastasis.</a> Oncogene. 30 (31), 3429-3439 (2011).</li><li>Deguchi, 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=Serum+amyloid+A3+binds+MD-2+to+activate+p38+and+NF-&#954;B+pathways+in+a+MyD88-dependent+manner.">Serum amyloid A3 binds MD-2 to activate p38 and NF-&#954;B pathways in a MyD88-dependent manner.</a> Journal of Immunology. 191 (4), 1856-1864 (2013).</li><li>Erler, J. 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=Hypoxia-induced+lysyl+oxidase+is+a+critical+mediator+of+bone+marrow+cell+recruitment+to+form+the+premetastatic+niche.">Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche.</a> Cancer Cell. 15, 35-44 (2009).</li><li>Huang, Y., 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=Pulmonary+vascular+destabilization+in+the+premetastatic+phase+facilitates+lung+metastasis.">Pulmonary vascular destabilization in the premetastatic phase facilitates lung metastasis.</a> Cancer Research. 69 (19), 7292-7237 (2009).</li><li>Sceneay, J., Smyth, M. J., M&#246;ller, 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+pre-metastatic+niche:+finding+common+ground.">The pre-metastatic niche: finding common ground.</a> Cancer Metastasis Reviews. 32 (3-4), 449-464 (2013).</li><li>Castle, J., Shaker, H., Morris, K., Tugwood, J. D., Kirwan, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+circulating+tumor+cells+in+breast+cancer:+A+review.">The significance of circulating tumor cells in breast cancer: A review.</a> The Breast. 23, 552-560 (2014).</li><li>Wolf, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+CCR2+signaling+induced+by+colon+carcinoma+cells+enables+extravasation+via+the+JAK2-Stat5+and+p38MAPK+pathway.">Endothelial CCR2 signaling induced by colon carcinoma cells enables extravasation via the JAK2-Stat5 and p38MAPK pathway.</a> Cancer Cell. 22 (1), 91-105 (2012).</li><li>Hiratsuka, S., 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=Primary+tumours+modulate+innate+immune+signalling+to+create+pre-metastatic+vascular+hyperpermeability+foci.">Primary tumours modulate innate immune signalling to create pre-metastatic vascular hyperpermeability foci.</a> Nature Communications. 4, 1853 (2013).</li><li>Deguchi, 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=Eritoran+inhibits+S100A8-mediated+TLR4/MD-2+activation+and+tumor+growth+by+changing+the+immune+microenvironment.">Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment.</a> Oncogene. 35 (19), 1445-1456 (2016).</li><li>Ieguchi, 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=ADAM12-cleaved+ephrin-A1+contributes+to+lung+metastasis.">ADAM12-cleaved ephrin-A1 contributes to lung metastasis.</a> Oncogene. 33 (17), 2179-2190 (2014).</li></ol>

Ön arıtma farelerin antikorlar, uyuşturucu, yüksek yağlı diyet ya da tümör şartına orta önce tümör hücre enjeksiyon ile mümkündür. En zor bu tahlil kuyruk ven enjeksiyon adımdır. Eksik enjeksiyon sonuçsuz verilerde sonuçlanır. Kuyruk ven C57BL/6 başarısız enjeksiyonları sonucu tespit etmek, özellikle zordur. Enjeksiyon daha kolay olur, böylece bir Isıtma yastığı (37 ° C) fareyi yerleştirerek yardımcı olur kuyruk ven genişletmek.
Bu tahlil çok hasta kan iç...

Tümör metastazı kanserle ilgili hastalıklarda yüksek ölüm oranı hesaplar. Moleküler seviyede araştırmalar bakış açısıyla, metastaz birden çok adımı ayrılabilir: tümör başlangıcında birincil tümör sitesi, birincil tümör büyüme ve çevreleyen dokular, intravasation, kan dolaşımı kan damarları aracılığıyla içine işgali , uzak organlara ve tümör yeniden büyüme ekstravazasyonu. Her adım molekülleri/sinyalleri1farklı grupları içerir.
Çalışmalar bugüne tümör hücreleri olarak da bilinen önceden metastatik faz2,3,4,5olan kanda dolaşan başlamadan önce uzak organlarda meydana gelen olaylarla ilgili, 6. Fare modeli çalışmalarımız önceden metastatik faz akciğerlerde uzun ömürlü ve düşük seviyeli inflamatuar yanıt oluşturarak akciğer metastazı kolaylaştırır saptandı. Önceden metastatik faz ile karakterize themicrovasculature hyperpermeability, kemik iliği türetilmiş hücre (BMDC) alımı ve upregulation3,4 S100A8 ve SAA3 de dahil olmak üzere sitokin/Kemokin-gibi moleküllerin tarafından . Bu o lysyl oksidaz BMDCs7askere akciğerlerde hücre dışı matriks değiştirir bildirilmiştir. Başka bir rapor önceden metastatik faz8' Angpt2, MMP3 ve MMP10 önemini ortaya koymuştur. Başka bir deyişle, birincil tümörler, kemik iliği ve uzak organlara arasındaki karmaşık etkileşimler anlaşılır olması ve düzgün (örneğin, proteinler bu etkileşimlerde yer hedef uyuşturucu kullanarak) modülasyonlu gerek gelecekteki metastaz9 önlemek için . Önceden metastatik faz düzenlemek için olmalı ilk görüntülenir ve tanı veya tedavi müdahaleler için değerlendirilir. Burada, önceden metastatik akciğer aşamasında çalışma sağlar bir akciğer tümör hücre işe alım tahlil raporu.
Tümör hücreleri doğrudan fareyi damar enjekte olabilir her ne kadar kültürlü hücreleri arasındaki farklar tümör hücreleri kanser hastalarında dolaşan ve tümör hücreleri dolaşan benzer. Dolaşım birincil tümöründen girdiğinizde tümör hücreleri dolaşan kendilerini bazı karakteristik değişiklikleri meydana. Yine de, onlar kuyruk damarda enjekte edildiğinde kültürlü hücreleri immünyetmezligi farelerde tümör oluşabilir. Ayrıca, fare modeli sistemi tümör hücreleri etiketli Floresans izleme bu hücreler vücuttaki izin kullanılabilir. Onlar kanser hastaları10nihai sonuçları belirlemek gibi kritik tümör hücreleri dolaşan davranıştır. Kan tümör hücreleri2hayatta kalmak için iyi bir yer değil. Ana bilgisayar bağışıklık sistemi tarafından saldırısından kaçıp apoptosis fiziksel destek eksikliği nedeniyle önlemek için anti-apoptotik sinyal gerekir ihtiyaçları var. Tümör hücrelerinin tümör başlama metastatik sitelerde daha yüksek yetenekleri ile daha fazla tümör nodüller oluşturur. Tümör hücreleri belirli organ tropism gösterirsen, metastaz belirli organları dikkat edilmelidir. Bu tahlil, metastatik cascade (birincil tümör büyüme ve işgali) ilk adımlar dahil değildir de odak dolaşan tümör hücreleri ve stromal hücreler (endotel hücreleri, epitel hücreleri ve kan hücreleri) arasındaki etkileşimi öyle. Geleneksel tümör hücre enjeksiyon deneyleri araştırmacılar tümör nodüller metastaz algılamak için somut bir boyuta büyümek kadar beklemek zorunda. Bu durumda, tümör hücreleri kan damarı içinde tam sayısını sayısal olamaz. Ayrıca bu işlem tümör hücreleri diğer faktörler sonucu etkileyebilir gösteren bir tümör büyütme aşama içerir.
Sayma tümör hücreleri floresan stereomicroscope altında etiketli basit bir işlemdir. Böylece bütün akciğer taranabilir stereomicroscope geniş görüş alanı sağlar. Akış Sitometresi da tümör hücreleri doku doğru sayıda anında veren yararlı bir araçtır. Floresan confocal mikroskop altında doku hücrelerinde sayma olanağı da sağlar ve metastatik tümör hücreleri en doğru resimlerini görüntüler, ama zaman alıcı ve seçili bir alanın sayılır Eğer sadece önyargılı sonuçlar verebilir. Bu tahlil, nihai hedefi ne kadar kolay Akciğerlerde tümör hücreleri birikir gözlemlemektir. Akciğerleri inflamatuar birincil tümöründen sinyal nedeniyle önceden metastatik aşamasında ise3,4, daha önce bildirildiği gibi enjekte tümör hücreleri böylece daha yüksek akciğer metastazı olasılığı ima akciğerlerde biriken yatkındır. Diğer koşullar (romatoid artrit, astım, vb) ve anti-enflamatuar ajanlar (gibi anti-TNFα) ile tedavi akciğer metastazı azaltmak. Böylece, bu tahlil akciğer metastazı olasılığı değerlendirmek için güçlü bir araç olduğu sonucuna varıldı.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C6885</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>DN-25</td>
			<td>trace amount</td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>BD</td>
			<td>352340</td>
			<td>40 micrometer mesh</td>
		</tr>
		<tr>
			<td>Fluorescence labeling kit</td>
			<td>Sigma</td>
			<td>MIN26-KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m syringe filter</td>
			<td>sartorius</td>
			<td>17597K</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde Phosphate Buffer Solution</td>
			<td>Wako</td>
			<td>163-20145</td>
			<td></td>
		</tr>
	</tbody>
</table>

lung tumor cell recruitment assay

Tümör metastazı yöneten moleküler mekanizmaları araştırmak için çeşitli deneyleri fareyi kullanarak bir modeli hayvan olarak önerilmiştir. Burada, tümör hücre ekstravazasyonu veya micrometastasis değerlendirmek için basit bir tahlil göstermektedir. Bu tahlil, tümör hücreleri kuyruk ven enjekte edildi ve kısa bir süre sonra akciğerleri disseke ve birikmiş etiketli tümör hücreleri saymak için sindirilmiş. Bu tahlil birincil tümör işgalinin ilk adımı damar atlar ve tümör metastazı oluştuğu uzak organ etkinlikte çalışma kolaylaştırır. Damar enjekte hücre sayısı Metastazlari sınırlı sayıda gözlemlemek için optimize edilmiş olması. Stromal hücreler uzak organ metastazı için katkıda rapor edildi. Böylece, bu tahlil potansiyel terapötik uyuşturucu ya da tümör metastazı önlenmesi için aygıt keşfetmek için yararlı bir araç olabilir.

Akciğerler birçok GFP-LLC hücreleri 2 h sonra enjeksiyon (şekil 1A) mevcut. Bu da kırmızı filtrede tespit floresan noktalar dan hücre sayı say silinmemesi gerektiğini belirtmek gerekir. LLC hücreler büyük çoğunluğu akciğerlerden 24 h enjeksiyon (şekil 1 c) sonra kaybolur.
Akciğerlerde GFP LLC sayısını doğrulamak için bir LOB akış sitometrik ana...

Watch this Scientific Journal Video about Lung Tumor Cell Recruitment Assay at JoVE.com

Lung Tumor Cell Recruitment Assay

Akciğer tümör hücre işe alım tahlil

Bu el yazması tümör hücresi birikimi Akciğerlerde tümör metastazı, bir hayvan modeli ölçmek için bir yöntem açıklanır.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

Bu yöntem, tümör biyolojisi alanında metastazın moleküler mekanizmaları ile ilgili anahtar soruların cevaplatını yardımcı olabilir. Bu tekniğin en büyük avantajı, uzak bir organ içinde tümör hücrelerinin işe quantitate olabilir. Floresan protein ifade Lewis akciğer karsinomu kaldırmak için bir enzimatik olmayan hücre dissosiasyon reaktifi kullanarak başlayın, veya LLC, standart protokollere göre kendi kültür konteyner hücreleri.Ortaya çıkan hücre çözeltisini santrifüjden önce 15 mililitrelik konik bir tüpe aktarın ve santrifüj peletleri yıkama başına beş mililitre PBS'de iki kez yıkayın. İkinci yıkamadan sonra hücreleri 40 mikrometrelik gözenek hücresi süzgecinden geçirin ve süspansiyonu mililitre konsantrasyonu başına altı hücreye kadar bir kez 10'a kadar seyreltin. Sonra hücreleri 29 kalibrelik bir iğneyle donatılmış bir mililitrelik şırıngaya yükleyin ve her sekiz ila 10 haftalık C57-Black-6 erkek farenin kuyruk damarına 100 mikrolitre hücre enjekte edin.Uygun deneysel uç noktada, her farenin ventral yüzeyinden deri kaldırmak için makas kullanın ve göğüs boşluğu ortaya çıkarmak için kaburga ve diyafram kesti. Sağ ventrikül aracılığıyla PBS 15 mililitre floş ve kalp ve timus kaldırmak için 25-gauge kanatlı infüzyon iğne ve tüp kullanın. Nefes borusunu forceplarla tutarak, yukarı çekin, trakeanın etrafındaki bağ dokularını parçalayarak akciğerleri hasat edin.Sonra PBS akciğerleri yıkayın, ve floresan mikroskop altında yerinde floresan hücrelerin görselleştirilmesi için bir lob ayırmak. Akciğer dokusunu sindirmek için, önce makasla kaudal loba zar atve akciğer parçalarını 10 mililitrelik şırıngaya yerleştirin. Şırıngayı pistonla kapatın ve beş mililitre sindirim çözeltisini şırınganın içine aspire edin, akciğer dokusu çözelti boyunca yayılana kadar şırınga şaftına girip çıkar.Akciğer bulamacını 50 mililitrelik bir tüpe dağıtın ve 37 santigrat derece ve 150 rpm'de 15 dakika boyunca karşılıklı bir çalkalayıcıda dokuyu sindirin. Kuluçka sonunda, akciğer hücreleri tamamen dağılına kadar kalan doku triturate, ve ek bir 30 dakika için shaker tüp dönmek. Daha sonra hücreleri 40 mikrometrelik gözenek süzgecinden geçirin ve santrifüj ile hücreleri toplayın.Akış sitometri ile izole akciğer hücrelerini analiz etmek için, oda sıcaklığında bir dakika için kırmızı kan hücre lisis tampon iki mililitre pelet resuspend, ve santrifüj ile hücreleri toplamak. Sonra taze PBS beş mililitre pelet iki kez santrifüj yıkama başına% 1 sığır serum albumin ile takviye. İkinci santrifüjden sonra, taze PBS artı BSA bir mililitre pelet resuspend, ve akış sitometri ile tümör hücrelerinin sayısallaştırılması için yeni bir 40 mikrometre gözenek süzgeci ile hücreleri filtre.Akciğerler enjeksiyondan iki saat sonra birçok GFP-LLC hücresi sunar, hücrelerin büyük çoğunluğu 24 saat içinde akciğerlerden kaybolur. Kırmızı filtre içinde algılanan floresan lekelerin hücre sayısından çıkarılması gerektiğini unutmayın. Akciğerlerdeki GFP-LLC sayısını doğrulamak için loblardan biri gösterildiği gibi akış sitometrik analizi için kullanılabilir.Bu prosedürü çalışırken, dondurulmuş bir kesit prosedürü tümör hücrelerinin tam yerini gözlemlemek için daha doğru bir yöntem olduğunu hatırlamak önemlidir.

Research

JoVE Journal

Cancer Research

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

Monosit/makrofajlar tümör Microenvironment için işe alım çalışmaya kanser hücre hatlarında gen ifadesinin koşullu nakavt

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Kanser Araştırmaları

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Tümör hücre görselleştirme akciğer dokusu içinde dolaşan bir akciğer kolonizasyon tahlil kurulması

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

Tümör metastazı çalışmaya acellular ve hücresel akciğer modeli

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Canlı hücre görüntüleme deneyleri için çalışma Glioblastoma Beyin tümör kök hücre göç ve işgali

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

Dijital polimeraz zincir reaksiyonu tahlil için tüp içinde çip biçimini kullanarak bir sporadik ailesel Adenomatous poliposis hastada genetik varyasyon

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Mezenkimal kök hücre izolasyon hamuru doku ve kanser hücreleri ile ortak kültür ilişkileri incelemek için

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Gerçek zamanlı hafiye-in Vitro tümör hücre Apoptosis içinde CD8 tarafından indüklenen+ T hücreleri Myeloid hücrelerin tümör infiltre bağışıklık baskılayıcı işlevlerini çalışmaya

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

İmmünfluoresans Ile Küçük Hücreli Olmayan Akciğer Kanseri Hastalarından Dolaşımdaki Tümör Hücrelerinin Yarı Otomatik PD-L1 Karakterizasyonu ve Numaralandırılması

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

In vitro assay tümör çalışma-macrophage etkileşim

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

Ultrason Görüntüleme ile Farelerde Akciğer Tümörü İlerlemesinin Saptanması

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...