Alman Kanser Merkezi'ndeki (DKFZ) Tek Hücreli Açık Laboratuvar'a (scOpenLab) yararlı tartışmalar için teşekkür ederiz. Bu araştırma Alman Kanser Yardımı, Max-Eder Programı hibe numarası 70111964 (S.T.) tarafından desteklendi.

Almanya'nın Heidelberg kentindeki Ulusal Tümör Hastalıkları Merkezi (NCT)- doku bankasından taze dondurulmuş glioma örnekleri alındı. Hasta materyalinin kullanımı Heidelberg Tıp Fakültesi Kurumsal İnceleme Kurulu tarafından onaylandı ve çalışmaya dahil edilen tüm hastalardan bilgilendirilmiş onam alındı.
1. Deneysel hazırlık
<ol><li>Tüm adımları buz üzerinde veya 4 °C'de gerçekleştirin.</li>
	<li>4 °C'ye kadar soğuk öncesi tüpler, bulaşıklar, jiletler, Douncers ve pestles.</li>
	<li>Tüm arabellekleri önceden hazırlayın. Bu tamponlar oda sıcaklığında stabildir. Steril filtrasyon, özellikle sakkaroz için önerilir. Stok hazırlıkları Corces ve ark.19'dan değiştirilmiştir. Bkz. Tablolar 1-7.</li>
	<li>Sıvı nitrojen veya -80 °C dondurucu deposundan numuneleri çıkarın ve Adım 2.1'e kadar kuru buzda tutun.</li>
</ol>2. Doku diseksiyonu ve ayrışması
<ol><li>Taze dondurulmuş doku örneğini (10-60 mg) önceden soğutulmuş bir Petri kabına aktarın. Taze donmuş dokuyu jiletle buz üzerinde küçük parçalara ayırın/doğrayın.</li>
	<li>Önceden soğutulmuş 1,5 mL'lik bir tüpe 500 μL soğutulmuş çekirdek liziz tamponu ekleyin. Doku parçalarını çekirdek liziz tamponu içeren 1,5 mL tüpe yerleştirin ve bir Douncer'a(Malzeme Masası) aktarın. NOT: İki Dounce homojenizatör pestlesi vardır: ilk numune azaltma için "gevşek" veya "A" pestle ve tam numune homojenizasyonu için "sıkı" veya "B" pestle.</li>
	<li>Sürtünme azaltılana kadar doku parçalarını yaklaşık 20 vuruş boyunca "gevşek" pestle ile yapın. Döküntü varsa, numune 100 mm'lik bir süzgeçle filtrasyon ile önceden temizlenebilir.</li>
	<li>Tam doku homojenizasyonu elde etmek için 20 inme için "sıkı" pestle ile yapın.</li>
	<li>Homojenatı (yaklaşık 500 μL) önceden soğutulmuş 2 mL'lik bir tüpe aktarın. 1 mL soğutulmuş lizis tamponu ekleyin. Hafifçe karıştırın ve 5 dakika boyunca buzda kuluçkaya yatırın. Geniş delikli pipet ucu ile hafifçe karıştırın ve kuluçka sırasında 1-2 kez tekrarlayın.</li>
	<li>30 μm süzgeç ağı kullanarak tüm homojenatı filtreleyin, 15 mL Falcon tüpüne toplayın ve önceden soğutulmuş yeni bir 2 mL tüpe geri aktarın. Tek bir süzgeç tipik olarak tüm homojenat için yeterlidir.</li>
	<li>Büyük kalıntıların giderilmesini ve nükleer zarın sağlamlığını doğrulamak için hafif bir mikroskop altında kontrol edin. Çekirdeklerin yuvarlak olması ve nükleer zarın bozulmaması gerekir. Enkaz varsa, çekirdekler yeniden filtrelenebilir.</li>
	<li>Çekirdekleri 500 x g'da 4 °C'de 5 dakika boyunca bir tezgah üstü santrifüj üzerinde santrifüj. Çekirdeği içeren pelet ile ~50 μL geride bırakarak süpernatant çıkarın. Peleti 1 mL'lik başka bir çekirdek liziz tamponunda hafifçe yeniden dürt ve buzda 5 dakika kuluçkaya yatır.</li>
	<li>Çekirdeği 500 x g'da 4°C'de 5 dakika santrifüj edin. Peletin rahatsız etmeden süpernatantını çıkarın, 500 mL 1x homojenizasyon tamponu (HB)(Tablo 4)ekleyin ve yeniden canlandırmadan 5 dakika kuluçkaya yatırın. Ardından, çekirdeği 1x HB'nin 1,0 mL'lik başka bir yerine yeniden kullanın.</li>
	<li>Çekirdeği 500 x g'da 4 °C'de 5 dakika santrifüj edin. Süpernatant çıkarın ve çekirdeği 200 μL 1x HB'de yeni bir 2.0 mL tüpe hafifçe yeniden kullanın.</li>
</ol>3. Degrade santrifüjleme
<ol><li>% 25 iyodixanol son konsantrasyon vermek için 200 μL%50iyodixanol çözeltisi ( Tablo 5 ) ekleyin. 300 mL'ye ayarlanmış pipetle 10 kez iyice karıştırın.</li>
	<li>% 25 karışımın altına 300 μL% 29 iyodiksanol çözeltisi (Tablo 6) ekleyin. Katmanların karışımını önlemek için P1000 ince uç kullanın.</li>
	<li>% 29 karışımın altına 300 μl% 35 Iyoksisanol çözeltisi (Tablo 7) ekleyin. Katmanların karışımını önlemek için P1000 ince uç kullanın. Dikkat: Bu adım, aşırı hacimli yer değiştirmeyi önlemek için pipet ucunun pipet ucunun pipet ucunun kademeli olarak kaldırılmasını gerektirir.</li>
	<li>Numuneleri sallanan bir kova santrifüjüne yerleştirin, fren kapalıyken 4 °C'de 3.500 x g'da 20 dakika döndürün.</li>
	<li>Numuneleri sallamadan hafifçe çıkarın ve ışık altında gözlemleyin. İkinci ve üçüncü katman arasında % 95 saf çekirdekten oluşan net bir beyaz bant görünmelidir (Şekil 1).</li>
</ol>4. Çekirdeklerin izolasyonu
<ol><li>%29-%35 ara fazda beyaz çekirdek bandına kadar üst katmanları aspire edin.</li>
	<li>Çekirdek bandını 200 mL hacimde toplayın, taze bir tüpe aktarın ve 20 μm filtreli filtre(Malzeme Masası). NOT: Çekirdeğin filtrasyondan önce yeniden dürtülmesi gerekmez.</li>
	<li>Büyük kalıntıların giderilmesini ve nükleer zarın sağlamlığını doğrulamak için hafif bir mikroskop altında kontrol edin. Çekirdeklerin yuvarlak olması ve nükleer zarın bozulmaması gerekir.</li>
	<li>Bir hemositometrede Trypan mavi boyama ve snRNA-seq / snATAC-seq için aliquot çekirdekleri kullanarak çekirdekleri sayın.</li>
</ol>5. Tek çekirdek RNA ve ATAC seq
<ol><li>Çekirdekleri tek hücre gen ekspresyon ve tek hücreli ATAC reaktif kitlerini ( Malzeme Tablosu ) kullanarak hemenişleyin. NOT: 10x Genomik sistemi için çekirdek örnek konsantrasyonları snRNA-seq için μL başına 1500-3000 çekirdek ve snATAC-seq için m μL başına 3500-7000 çekirdektir. Çekirdek 1x PBS kullanılarak seyreltilebilir.</li>
	<li>Genomik Çekirdek Tesisi'nde elde eden kitaplıkları sıralayın.</li>
	<li>Verilerin kalite kontrol analizini gerçekleştirin. Çekirdekler, Benzersiz Moleküler Tanımlayıcı (UMI) >1000, gen sayısı >500 ve mitokondriyal genlerin yüzde <5'i içeriyorsa ve ÜM'lerin ve genlerin ortalama + üç standart sapması içindeyse daha fazla analiz için dahil edilir.</li>
</ol>

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Yazarların açıklayacak bir şeyi yok.

Tümör içi heterojenlik alanı heyecan verici bir aşamadadır ve mevcut bilgiye meydan okumak ve genişletmek için yeni tahliller ve platformlar geliştirilmektedir. Tümör içi heterojenlik gliomalarda hastalığın ilerlemesine ve mevcut tedavi yöntemlerine direnç gösteren önemli bir faktördür28. Beyin tümörleri üzerinde yapılan son çalışmalar, aynı tümör içindeki hücresel heterojenliği daha iyi karakterize etmek için tek hücreli transkriptomik ve epigenomik tahliller k...

Yetişkinlerde en sık görülen primer beyin tümörü olan dağınık alt derece gliomalar (LGG), sıklıkla serebral yarımkürede ortaya çıkan infiltratif neoplazmlardır. LGG'ler sadece tümör popülasyonu tarafından değil, aynı zamanda tümör gelişimi ve ilerlemesinde karmaşık bir şekilde yer alan kötü huylu olmayan hücreler tarafından da yönlendirilen tümör içi heterojenliği hem inter-hem de intra-tümör heterojenliği sergiler 1,2,3,4,5.
Son on yılda gliomalar alanında toplanan bir dizi genomik veri vardır. Bu veriler esas olarak toplu tümör dizileme çalışmalarından gelir ve moleküler karakterizasyona ve beyin tümörlerinin mevcut sınıflandırılmasına son derece katkıda bulunmuştur5,6, 7,8,9,10,11. Bununla birlikte, bu çalışmalar gliomalarla ilişkili geniş moleküler manzarayı ortaya ortaya sürse de, terapötik müdahale konusunda hala hayal kırıklığı yaratan bir ilerleme eksikliği vardır. Beyin tümörlerinde tedavi direncinin önündeki engellerden biri de tümör içi heterojenliktir. Bu sorunu çözmek için, çeşitli çalışmalar tek hücreli düzeyde bir tümörde bulunan genomik, transkriptomik, proteomik ve epigenetik heterojeniteye odaklanmıştır12,13 ,14,15,16,17.
Son birkaç yıldır tek hücre alanında dikkat çekici teknolojik gelişmeler olmasına rağmen, en önemli sınırlayıcı faktörlerden biri, hücreleri izole etmek ve bu deneyleri gerçekleştirmek için gereken taze örneklerin mevcudiyetidir. Bu sınırlamanın üstesinden gelmek için,18 , 19hücreleri yerine çekirdek kullanarak donmuş dokulardan snRNA-seq ve snATAC-seq gibi tahliller yapmak için birkaç başarılı girişimolmuştur. Bu yöntemlerin çoğu floresan ile etkinleştirilen hücre sıralama (FACS) veya filtrasyon stratejilerine dayanır. Hem tek hücreli hem de tek çekirdekli yaklaşımların güçlü ve dezavantajları vardır. Tek hücreli yaklaşımlar mitokondriyal transkriptleri korur, bu da bilgilendirici olsa da, yüksek bollukları nedeniyle transkriptome kapsamını azaltabilir. Tek çekirdek izolasyon yaklaşımları mitokondriyal fraksiyonun yüksek bir yüzdesini ortadan kaldırır, böylece nükleer transkriptlerin daha derinlemesine kapsanmasına izin verir20.
RNA-seq ve ATAC-seq dahil olmak üzere tek hücreli genomik verileri test etmek için son yıllarda kullanılan çeşitli ticari platformlar vardır. En belirgin platformlardan biri, tek hücreli gen ekspresyumu ve tek hücreli ATAC profilleme için 10x Genomik Krom platformudur. Platform mikroakışkan odaların yardımıyla çalıştığından, herhangi bir döküntü veya agrega sistemi tıkayabilir ve bu da veri, reaktif ve değerli klinik örneklerin kaybına yol açabilir. Bu nedenle, tek hücreli çalışmaların başarısı büyük ölçüde tek hücrelerin/ çekirdeklerin doğru izolasyonuna bağlıdır.
Burada göstereceğimiz protokol, DroNc-seq ve Omni-ATAC-seq protokollerinin biraz değiştirilmiş bir kombinasyonudur ve insan beynindeki nörolojik bozuklukları ve nöronal hücre tiplerini anlamak için snRNA-seq kullanan son çalışmalara benzer bir yaklaşım kullanır18,19,21,22,23,24. Protokol, donmuş numunelerin enzipmatik/mekanik ayrışmasının ve ardından filtrasyon ve gradyan santrifüjlemenin bir kombinasyonunu kullanır ve tek çekirdeğin taze dondurulmuş glioma dokularından hızlı ve doğru bir şekilde izole edilmesini sağlar. Bu protokolü, beyin tümörü örneklerinden aynı çekirdek hazırlığından snRNA-seq ve snATAC-seq verileri oluşturmak için başarıyla kullandık.

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>21115-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce Homogenizer</td>
			<td>Active motif</td>
			<td>40401</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Thermo Scientific</td>
			<td>R1021</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 5 mL Round Bottom Polystyrene Test Tube</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodixanol (aka Optiprep)</td>
			<td>Stem cell technologies</td>
			<td>07820</td>
			<td></td>
		</tr>
		<tr>
			<td>MACs Smart Strainers (30 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-458</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS SmartStrainers (100 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-463</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg(Ac)2</td>
			<td>Sigma</td>
			<td>63052-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Abcam</td>
			<td>ab142227</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclei Isolation Kit: Nuclei EZ Prep</td>
			<td>Sigma</td>
			<td>NUC101-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-Separation Filters (20 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-101-812</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe lock tubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td>0030120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe lock tubes 2.0 mL</td>
			<td>Eppendorf</td>
			<td>0030120094</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Cell ATAC</td>
			<td>10x Genomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Cell Gene Expression</td>
			<td>10x Genomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Bore pipette tips (1000 &#181;L)</td>
			<td>Themo Fisher Scientific</td>
			<td>2079GPK</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Bore pipette tips (200 &#181;L)</td>
			<td>Themo Fisher Scientific</td>
			<td>2069GPK</td>
			<td></td>
		</tr>
	</tbody>
</table>

nuclei isolation from fresh frozen brain tumors for single-nucleus rna-seq and atac-seq

Erişkin dağınık gliomalar tümörler arası ve tümör içi heterojenlik sergiler. Yakın zamana kadar, büyük ölçekli moleküler profilleme çabalarının çoğu, beyin tümörlerinin moleküler sınıflandırılmasına yol açan toplu yaklaşımlara odaklanmıştır. Son beş yılda, tek hücreli dizileme yaklaşımları gliomaların birkaç önemli özelliğine vurgu yaptı. Bu çalışmaların çoğunluğu, akış sitometrisi veya antikor bazlı ayırma yöntemlerini kullanarak tek hücreleri izole etmek için taze beyin tümörü örneklerinden yararlanmıştır. İleride, biyobankalardan taze dondurulmuş doku örneklerinin kullanılması tek hücre uygulamalarına daha fazla esneklik sağlayacaktır. Ayrıca, tek hücreli alan ilerledikçe, bir sonraki zorluk tümör karmaşıklığını daha iyi çözmek için tek bir hücreden veya aynı örnek hazırlığından çoklu omik verileri oluşturmak olacaktır. Bu nedenle, tek çekirdekli RNA dizilimi (snRNA-seq) ve transposase-Accessible Chromatin için tek çekirdekli Tahlis gibi çeşitli yöntemler için veri üretimine izin veren basit ve esnek protokoller alan için önemli olacaktır.
10x genomik platformu gibi erişilebilir mikroakışkan aletlerle birleştiğinde tek hücre alanındaki son gelişmeler, araştırma laboratuvarlarında tek hücre uygulamalarını kolaylaştırmaktadır. Beyin tümörü heterojenliğini incelemek için, tek çekirdeğin taze donmuş gliomalardan izolesi için geliştirilmiş bir protokol geliştirdik. Bu protokol varolan tek hücre protokollerini birleştirir ve bir homojenleştirme adımini ve ardından filtrasyon ve arabellek aracılı gradyan santrifüjünü birleştirir. Elde edilen örnekler, aynı çekirdek preparatından tek çekirdek gen ekspresykülü ve kromatin erişilebilirlik verileri oluşturmak için kullanılabilecek saf tek çekirdek süspansiyonlarıdır.

Tek çekirdek genomik sınırlı veri ve protokollere sahip gelişen bir alandır. Tek çekirdek tahlillerinin sonucunu etkileyen kritik bir faktör saf ve sağlam çekirdeklerin izolasyonudur. Yayınlanan iki protokolü (DroNc-seq ve Omni-ATAC-seq protokolleri) birleştirerek taze donmuş glioma doku bloklarından yüksek kaliteli ve saf çekirdekleri nispeten kısa sürede izole ettik ve böylece transkriptlerin stabilitesini koruduk (Şekil 1).
İyodixanol/sakk...

Watch this Scientific Journal Video about Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq at JoVE.com

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Tümör içi heterojenlik gliomalar da dahil olmak üzere tümörlerin doğal bir özelliğidir. Tek çekirdek RNA ve ATAC dizileme çalışmaları için tek çekirdekleri taze donmuş glioma dokularından izole etmek için tamponlar ve gradyan santrifüjleme kombinasyonunu kullanan basit ve verimli bir protokol geliştirdik.

Tek Çekirdekli RNA-seq ve ATAC-seq için Taze Donmuş Beyin Tümörlerinden Çekirdek İzolasyonu

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused ...

nuclei-isolation-from-fresh-frozen-brain-tumors-for-single-nucleus

Research

JoVE Journal

Cancer Research

11.9K Görüntüleme.  University Hospital Heidelberg. Tümör içi heterojenlik gliomalar da dahil olmak üzere tümörlerin doğal bir özelliğidir. Tek çekirdek RNA ve ATAC dizileme çalışmaları için tek çekirdekleri taze donmuş glioma dokularından izole etmek için tamponlar ve gradyan santrifüjleme kombinasyonunu kullanan basit ve verimli bir protokol geliştirdik.

Video: Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

Katı ve Sıvı Tümörlerinde harekete geçirici Mutasyonlar Tespiti için Yeni Nesil Dizi

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

Kanser Araştırmaları

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

Zebra balığı Pediatrik Beyin Tümörlerinin Tümör Hücre Davranışının ve İlaç Yanıtının Uzun Vadeli İncelenmesi için Bağışıklık Yetenekli Ev Sahibi Olarak Transplantasyonu

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

Osteoclastogenesis periferik kan monosit kültürlü meme kanseri hücre hatları ile birlikte gelen bir insan preklinik modelinin geliştirilmesi

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

Bir CDH1 nadir transkript değişken mide kanseri taze donmuş dokularda çip tabanlı dijital PCR tarafından algılanması

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Jinekolojik ve Meme Kanseri Tümörlerinden Kanser Kök Hücre Kürelerinin Alınması

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

Ortotopik Pankreas Tümörlerinin Üretimi ve Tümör Efiltrasyonu T Hücre Sitotoksisitesinin Ex vivo Karakterizasyonu

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Meme Kanseri için Preklinik Modeller Olarak Meme Tümörlerinin Ortopedik Transplantasyonu

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

Hasta Kaynaklı Glioma Kök Hücreleri Kullanarak GBM için Potansiyel Kombinasyon Tedavisini Belirlemek için Hızlı Tarama İş Akışı

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

ATAC-Seq Optimization for Cancer Epigenetics Research

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the hyperactive Tn5 transposase. Tn5 cuts and ligates adapters for high-throughput sequencing within accessible chromatin regions. In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins, which acts as a physical barrier to the transcriptional machinery. In response to extrinsic signals, transcription factors recruit chromatin remodeling complexes to enable access to the transcriptional machinery for gene activation. Therefore, identifying open chromatin regions is useful when monitoring enhancer and gene promoter activities during biological events such as cancer progression. Since this protocol is easy to use and has a low cell input requirement, ATAC-seq has been widely adopted to define open chromatin regions in various cell types, including cancer cells. For successful data acquisition, several parameters need to be considered when preparing ATAC-seq libraries. Among them, the choice of cell lysis buffer, the titration of the Tn5 enzyme, and the starting volume of cells are crucial for ATAC-seq library preparation in cancer cells. Optimization is essential for generating high-quality data. Here, we provide a detailed description of the ATAC-seq optimization methods for epithelial cell types.

ATAC-seq is a DNA sequencing method that uses the hyperactive mutant transposase, Tn5, to map changes in chromatin accessibility mediated by transcription factors. ATAC-seq enables the discovery of the molecular mechanisms underlying phenotypic alterations in cancer cells. This protocol outlines optimization procedures for ATAC-seq in epithelial cell types, including cancer cells.

Kanser Epigenetiği Araştırmaları için ATAC-Seq Optimizasyonu

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the ...

Pankreas Kanseri Kaynaklı Tümör Organoidleri ve Taze Dokudan Fibroblastlar

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Yazar Spotlight: Pankreas Kanseri Kaynaklı Tümör Organoidlerinin ve Taze Dokudan Fibroblastların Kurulması  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Taze dokudan pankreas kanseri kaynaklı tümör organoidlerinin ve fibroblastların oluşturulması

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...