Ohse Grant Vakfı Bu eser finanse.

1. hücre uzantısı özellikleri
<ol><li>SW-13, SW-Neo ve CO2 ' Dulbecco'nın modifiye kartal orta % 10 fetal sığır serum ve 10.000 U/mL penisilin ve streptomisin ('büyüme aracı olarak' belirlenmiş) ile desteklenmiş bir standart oksijen kuluçka 37,0 ° C'de ve %5 SW-DKK3 hücreleri korumak.<ol>
<li>Bir hemasitometre kullanarak hücreleri saymak ve iyi SW-13, SW-Neo ve SW-DKK3 hat başına 5000 hücre ayrı 6-şey kalıplara plaka ve gecede büyüme aracı olarak steril cam coverslips üzerinde büyür.</li>
	</ol>
</li>
	<li>Her şey için gecede büyüme Aspire edin sonra büyüme orta, hücreleri 1 mL sıcak fosfat tamponlu tuz (PBS) ile yıkayın ve hücreleri % 3.7 formaldehit 10 dk 1 mL ile düzeltin.</li>
	<li>Her şey ile 1 mL % 0.05 kristal Menekşe 30 dk. yıkama aşırı aspiratı formaldehit ve leke hücrelerinde uzak üç yıkama 1 mL deiyonize su kullanarak boya. Not: boya alımı düzeyine bağlı olarak, birkaç ek yıkar hücre görselleştirme tanıtmak için boyama arka plan çıkarmak için gerekli olabilir.<ol>
<li>İnce uçlu forseps kullanarak, coverslips almak ve yüzü aşağı bir damla temiz montaj reaktif etiketli cam Slayttaki yer onları.</li>
	</ol>
</li>
	<li>Işık mikroskobunda rastgele her coverslip hücre uzantısı tahlil için hücrelerin üst üste 20 sayısı seçin.<ol>
<li>Photomicrographs her görüş alanı 400 x büyütme oranında al. Doğrudan uzantısı türlerinin her biri 20 temsilcisi hücreleri için saymak. Not: Bu nedenle, güvenilir sonuçlar alabilmek sınanan her koşul için 20 izole hücreler incelenmesi gerekir. Filopodia 5 mikron veya daha az bir üs ve tek bir tepe ile hücresel uzantıları tarafından tanımlanan. Lobopodia bir üs 5 ila 20 mikron, birden çok apices uzunluğu içeren uzantıları ile hücresel uzantıları tarafından tanımlanan. Lamellipodia 20 mikron uzunluğunda olan veya olmayan apices daha büyük hücre uzantıları tarafından tanımlanan. Test edilmiş hücre türüne bağlı olarak hücre uzantıları boyutları farklı olabilir ve parametreleri tanımlama arıtma gerektirebilir.</li>
	</ol>
</li>
	<li>Her hücre türü (Yani, SW-13, SW-Neo, SW-DDK) her hücre uzantısında muayene 6 kuyular arasında belirlemek.</li>
	<li>Bir tek yönlü Varyans analizi (ANOVA) istatistiksel test kullanarak, hücre zarının uzantıları farklı test hücre tipleri arasında ortalama yüzdesini farklılıkları karşılaştırın.</li>
</ol>2. yapışma tahlil
<ol><li>SW-13, SW-Neo ve SW-DKK3 hücreleri 37,0 ° C ve % 5 CO2 büyüme orta'de standart bir oksijen kuluçka korumak.<ol>
<li>Bir hemasitometre kullanarak, say ve iyi başına 100.000 hücre SW-13, SW-Neo ve SW-DKK3 ayrı 6-şey kalıplara plaka ve gecede büyüme ortamında büyümek.</li>
		<li>Her biri aşağıda test 6 belirlenen zaman puan için bir tabak kullanarak tohum 6-şey plakaları her hücre türü için test. Not: Zaman Puan farklı hücre tipleri için farklı olabilir.</li>
	</ol>
</li>
	<li>Gecede kuluçka sonra sıcak PBS bir kez yıkama hücrelerle sonra her şey için 0.5 mL 1 x enzimatik olmayan hücre ayrılma çözümü ekleyin.<ol>
<li>Hücre ayrılma çözümü yaymak için plaka girdap. Tanımlanan zaman noktaları (Yani, 1, 2, 3, 5, 10 ve 15 dk) belirlenen plaka Aspire edin ve sonra üç kez 1 mL sıcak PBS solüsyon ile yıkayın.</li>
		<li>Yıkama arasında hafifçe gevşek bağlı hücreler ayırmak plakalara dokunun.</li>
	</ol>
</li>
	<li>Kalan PBS ve kalan hücreleri ile 1 mL % 3.7 formaldehit 10 min için plaka bağlı düzeltme Aspire edin.</li>
	<li>1 mL % 0.05 kristal Menekşe 30 dk için hücrelerle leke sonra aşırı boya uzak 1 mL deiyonize su ile yıkayın. Hücre görselleştirme tanıtmak için yıkama titre. Not: boya alımı düzeyine bağlı olarak, birkaç ek yıkar hücre görselleştirme tanıtmak için gerekli olabilir.</li>
	<li>100 x büyütme hafif bir mikroskop kullanarak, her şey her tabak içinde kalan ekli hücreleri saymak. Not: Saydam cetvelleri veya ince kalem kılavuzları plaka alt tarafında işaretleme kullanımı tekrarlanan aynı hücreleri sayma önlemek için yardımcı olacaktır.</li>
	<li>Her plaka için iyi başına bağlı hücre ortalama sayısını belirleyin.</li>
	<li>SW-13, SW-Neo ve SW-DDK3 hücreleri arasında hücre dekolmanı kullanarak iki yönlü ANOVA istatistiksel test belirli bir dönem içinde oranı karşılaştırın.</li>
</ol>

<ol>
	<li>Libe, R., Fratticci, A., Bertherat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenocortical+cancer:+pathophysiology+and+clinical+management.">Adrenocortical cancer: pathophysiology and clinical management.</a> Endocrine-related cancer. 14 (1), 13-28 (2007).</li><li>Veeck, J., Dahl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+Wnt+pathway+in+cancer:+the+emerging+role+of+Dickkopf-3.">Targeting the Wnt pathway in cancer: the emerging role of Dickkopf-3.</a> Biochimica et biophysica acta. 1825 (1), 18-28 (2012).</li><li>Friedl, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+in+morphogenesis,+regeneration+and+cancer.">Collective cell migration in morphogenesis, regeneration and cancer.</a> Nat Rev Mol Cell Biol. 10 (7), 445-457 (2009).</li><li>Jacquemet, G., 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=L-type+calcium+channels+regulate+filopodia+stability+and+cancer+cell+invasion+downstream+of+integrin+signalling.">L-type calcium channels regulate filopodia stability and cancer cell invasion downstream of integrin signalling.</a> Nat Commun. 7, 13297 (2016).</li><li>Machesky, L. M., Li, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fascin:+Invasive+filopodia+promoting+metastasis.">Fascin: Invasive filopodia promoting metastasis.</a> Commun Integr Biol. 3 (3), 263-270 (2010).</li><li>Petrie, R. J., Harlin, H. M., Korsak, L. I., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activating+the+nuclear+piston+mechanism+of+3D+migration+in+tumor+cells.">Activating the nuclear piston mechanism of 3D migration in tumor cells.</a> J Cell Biol. 216 (1), 93-100 (2017).</li><li>Korah, R., 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=Epigenetic+silencing+of+RASSF1A+deregulates+cytoskeleton+and+promotes+malignant+behavior+of+adrenocortical+carcinoma.">Epigenetic silencing of RASSF1A deregulates cytoskeleton and promotes malignant behavior of adrenocortical carcinoma.</a> Molecular cancer. 12, 87 (2013).</li><li>Cheng, J. 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=A+novel+FOXO1-mediated+dedifferentiation+blocking+role+for+DKK3+in+adrenocortical+carcinogenesis.">A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis.</a> BMC cancer. 17 (1), 164 (2017).</li><li>Barry, D. J., Durkin, C. H., Abella, J. V., Way, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+software+for+quantification+of+cell+migration,+protrusions,+and+fluorescence+intensities.">Open source software for quantification of cell migration, protrusions, and fluorescence intensities.</a> J Cell Biol. 209 (1), 163-180 (2015).</li><li>Cliffe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+3D+analysis+of+complex+single+border+cell+behaviors+in+coordinated+collective+cell+migration.">Quantitative 3D analysis of complex single border cell behaviors in coordinated collective cell migration.</a> Nat Commun. 8, 14905 (2017).</li></ol>

Yazarlar ifşa gerek yok.

Burada hücre uzantıları kolaylığı, birkaç çukur-falls ve çeşitli test koşulları uygulanabilir güvenilir tekrarlanabilirlik ile karakterize bir vitro nicel yöntemi açıklanmaktadır. Ayrıca, basit, ölçülebilir yapışma ve/veya hareketliliği deneyleri fonksiyonel önemini potansiyel: hücre zarı uzantılarında gözlenen değişiklikler ilişkilendirmek için aynı anda gerçekleştirilebilir.
Ancak, bu yöntemlerin olası bazı sınırlamalar takdim edebilir miyim...

Dysregulated WNT sinyal Adrenokortikal maligniteler1' de kritik bir rol oynamaktadır. Bu çalışmada kullanılan yöntemleri DKK3 susturmak, WNT sinyal, negatif bir regülatör adrenal korteks dedifferentiation olayını temsil eder ve bağlamında tümör oluşumu hücre-uzantı repertuar değişir teşvik olup olmadığını araştırmak. DKK3 38 kDa ile bir N-terminal sinyal peptid glikoprotein salgılanan ve önceki çalışmalar zorunlu ifadesini hücre döngüsü tutuklama sonuçlandı, saldırgan kötü huylu davranış inhibe ve Mezenkimal epitel ters göstermiştir olduğunu geçiş2.
Adrenokortikal Karsinom (ACC) ve diğer kanser Malign davranışını tümör hücreleri çevreleyen yüzeyler ile arayüz yeteneği etkisinde bölümünde sırayla tümör hücre işgali kolaylaştırır hücre dışı matriks dahil olmak üzere ve geçiş3. Öncelikle filopodia oluşumu ile çeşitli bağlamlarda belirli hücre zarı kanseri ilerleme uzantılarında rolünün giderek gösterilen. Örneğin, overexpression L tipi kalsiyum kanal filopodia oluşumu teşvik ve tümör hücre işgali4teşvik bulundu. Benzer şekilde, Fascin, en az bağlayıcı protein bağlıdır bir aktin dokusu normal ifade de kanser hücrelerinin filopodia oluşumu5ile birlikte overexpressed mevcuttur. Lobopodia oluşumu sağlar etkili ekstra hücresel matris ile geçirmek non-Malign fibroblastlar, Fibrosarkom hücreleri metalloproteinaz etkinliği hücre geçişi kolaylaştırmak için lobopodia yerine itimat gösterilmiştir ancak, ve istila6. Biz o tümörü göstermiştir bastırıcılarının Ras Derneği etki alanı aile 1 izoformu (RASSF1A) de dahil olmak üzere ve DKK3, hücre iskeleti öğeleri değiştirebilir ve lamellipodia oluşumu teşvik ve invaziv özellikleri7,8taş koymak için işlev görebilir.
Bu nedenle, bu genlerin karsinojenezis ve hücre zarının uzantısı değişiklikler, özellikle filopodia, lobopodia ve lamellipodia oluşumu test koşullarında değerlendirilmesi ile ilişkilerini katılan etkileri karakterize etmek için önemlidir. Geçerli state-of-art teknikleri veri toplama ve yorumu giderek karmaşık mikroskobu yöntemleri, floresan etiketleme ve/veya karmaşık bilgisayar algoritmaları kullanımını içerir. Bu yöntemler yeni ve güçlü analitik araçlar sağlarken, kendi karmaşıklığı onların yaygın kullanımı ve adaptasyon hücre biyolojisi deneylerde sınırlar. Ayrıca, kesin miktar ve hücre uzantısı Morfoloji değişimler gözlenmesi değil genellikle ölçülen9,10. Buna ek olarak, biz doğru bir şekilde standart mikroskobik teknikler ve kolayca adapte vitro yöntemleri kullanarak hücre uzantısı değişiklikler quantifies bir teknik tanıtmak. Bu yöntemler aynı zamanda her hücre uzantı türü aynı anda her hücre analiz için sayısal ve hücre zarının uzantısı repertuar genel değişiklikleri belirlemek. Biz de nasıl bu değişiklikler hücre adezyon özellikleri için ilgili olabilir gösterir.
Deneysel örnek olarak, önceden oluşturulmuş hücre kültürünü SW-13, SW-DDK3, stabil pCMV6-giriş/DKK3 plazmid vektörleri ile transfected ve yapısal DKK3, böbrek üstü bezi bir farklılaştırıcı faktör overexpresses belirlenmiş kullanacağız. Sigara transfected SW-13 hücreleri ve stabil SW-Neo, belirlenmiş boş ile vektör (pCMV6-giriş), transfected SW-13 hücreleri deneysel denetimler olarak görev yapacak.

<table width="653">
	<tbody>
		<tr height="63">
			<td height="63" style="width:216px;height:63px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:115px;">ThermoFisher</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:203px;">Supplemented with fetal bovine serum and penicllin and streptomycin</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Deulbeco&#39;s Phosphate Buffered Saline</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td>1X solution, used directly</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">3.7% formaldehyde&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">F8775</td>
			<td>Dilute stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">0.05% crystal violet&#160;</td>
			<td style="width:115px;">Harleco</td>
			<td style="width:119px;">192-12</td>
			<td>Dilute stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">De-ionized water</td>
			<td style="width:115px;">NA</td>
			<td style="width:119px;">NA</td>
			<td>NA</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Microscope with 400X magnification&#160;</td>
			<td style="width:115px;">NA</td>
			<td style="width:119px;">NA</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td>NA</td>
		</tr>
		<tr height="43">
			<td height="43" style="width:216px;height:43px;">Cell dissociation solution non-enzymatic 1X</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">C5914&#160;</td>
			<td>1X solution, used directly</td>
		</tr>
	</tbody>
</table>

characterization of cell membrane extensions and studying their roles in cancer cell adhesion dynamics

Hücre zarı'nın uzantısı repertuar onların ya da göçmen potansiyelleri de dahil olmak üzere kanser hücrelerinin çeşitli kötü huylu davranışlar modüle. Nasıl hücre sinyallemesi olayların etkisi kanser hücre davranış ve saldırganlık belirlemek için kritik doğru şekilde sınıflandırmak ve hücre uzantıları ölçmek ve bir hücrenin yapışkanlı kapasitesi üzerindeki etkisini ölçmek için yeteneğidir. Burada, vitro tasarım ve bir hücre uzantısı quantification yöntemiyle birlikte kurulan vitro modelinde bir yapışma kapasite tahlil Adrenokortikal Karsinom (ACC) için kullanımını açıklar. Özellikle, DKK3, sözde Tümör baskılayıcı ve bir yanlısı farklılaşma faktörü üzerine etkileri membran uzantısı fenotip ACC hücre satırının SW-13 test ediyoruz. Biz nispeten basit, güvenilir sağlamak için bu deneyleri teklif ve kolay yorumlanabilir ölçümleri bu özellikleri çeşitli deneysel koşullarda ölçer.

Yukarıdaki deneyleri kullanarak, hücre uzantısı morfoloji ve hücre adezyon özellikleri DKK3 overexpression etkileri kurulan ACC hücre hattında SW-13, vitrotest edildi. DKK3 overexpressing hücreleri stabil Myc-DDK etiketli pCMV6-giriş/DKK3 plazmid Vektörler ve SW-DKK3 belirlenmiş hücrelerle SW-13 transfecting tarafından üretildi. Benzer şekilde, stabil boş vektör pCMV6-giriş ile transfected hücreleri transfection ve passaging denetimi olarak oluşturulan ve SW-...

Watch this Scientific Journal Video about Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics at JoVE.com

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

Bu çalışmada yardımcı programı ve kolaylığı nicel hücre zarı uzantısı ölçüm ve hücre yapışkanlı kapasite için onun korelasyon gösterir. Temsil edici bir örnek olarak, biz burada Dickkopf ilgili biraz protein 3 (DKK3) artan lobopodia oluşumu teşvik ve vitroAdrenokortikal Karsinom hücre adhesiveness hücreleri göstermek.

Hücre zarının uzantıları ve rolleri kanser hücre adezyon dinamikleri eğitim karakterizasyonu

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

characterization-cell-membrane-extensions-studying-their-roles-cancer

Research

JoVE Journal

Cancer Research

7.4K Görüntüleme.  Yale University School of Medicine. Bu çalışmada yardımcı programı ve kolaylığı nicel hücre zarı uzantısı ölçüm ve hücre yapışkanlı kapasite için onun korelasyon gösterir. Temsil edici bir örnek olarak, biz burada Dickkopf ilgili biraz protein 3 (DKK3) artan lobopodia oluşumu teşvik ve vitroAdrenokortikal Karsinom hücre adhesiveness hücreleri göstermek.

Video: Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

Diffüz İçsel Pontine Glioma Hızlı post-mortem Hücre Kültürü için Protokol (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

Kanser Araştırmaları

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

Mitokondri zar potansiyeli CLIC4 nakavt kaynaklı HN4 hücre Apoptosis Vitro çalışma tespiti

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Hedef hücre ön zenginleştirme ve tüm genom amplifikasyon tek hücre aşağı akım karakterizasyonu için

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

3-b hücre kültür sistemi istila eğitim ve Mesane kanserinde tedavi değerlendirmek için

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

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 ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

Mide kanseri hasta-türetilen xenograft modelleri ve primer hücre hatları kurulması

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

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 ...

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide clues to overcoming radioresistance. Voltage-gated calcium channel &#945;2&#948;1 subunit isoform 5 has been reported as a marker for radioresistant CSCs in non-small cell lung cancer (NSCLC) cell lines. Using calcium channel &#945;2&#948;1 subunit as an example of a CSC marker, methods to study the radiosensitivity of CSCs in NSCLC cell lines are presented. CSCs are sorted with putative markers by flow cytometry, and the self-renewal capacity of sorted cells is evaluated by sphere formation assay. Colony formation assay, which determines how many cells lose the ability to generate descendants forming the colony after a certain dose of radiation, is then performed to assess the radiosensitivity of sorted cells. This manuscript provides initial steps for studying the radiosensitivity of CSCs, which establishes the basis for further understanding of the underlying mechanisms.

The presence of cancer stem cells have been associated with relapse or poor outcomes after radiotherapy. This manuscript describes the methods to study the radiosensitivity of cancer stem cells in lung cancer cell lines.

Akciğer Kanseri Hücre Hatlarında Kanser Kök Hücrelerinin Radyosensitivite

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

Fare Böbreği ve Tavuk Koryoallantoik Membranda Kurulan Metastatik Berrak Hücreli Renal Hücreli Karsinom Modelinin Karşılaştırılması

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence of disseminated tumor cells in patients with different types of cancer. The detection of these LN-residing tumor cells is an important biomarker associated with poor prognosis and adjuvant therapy decisions. Recent mouse models have indicated that LN-residing tumor cells could be a substantial source of malignant cells for distant metastases. The ability to quantify the adhesivity of tumor cells to LN parenchyma is a critical gauge in experimental research that focuses on the identification of genes or signaling pathways relevant for lymphatic/metastatic dissemination. Because LNs are complex 3D structures with a variety of appearances and compositions in tissue sections depending on the plane of section, their matrices are difficult to replicate experimentally in vitro in a fully controlled way. Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to LN cryosections. Using serial sections of the same LN, we adapt the classic method developed by Brodt to use nonradioactive labels and directly count the number of adhering tumor cells per LN surface area. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the cell-binding affinity to LN parenchyma, which is suggestive evidence of molecular alterations in the affinity binding of integrins to their correlate LN-ligands.

Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to lymph node (LN) cryosections. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the tumor cell-binding affinity to LN parenchyma.

Lenf Nodu Kriyosiyonlarında Tümör Hücre Adezyonunun Ölçülmesi

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence ...