Yazarlar Dr Dr teşekkür ederim. Fahad Bafakih ve Jim Mandell, hasta numunelerinin edinilmesi ve tanımlanması için Garrett F. Beeghly, Biyorepository ve Doku Araştırma Tesisi, Kardiyovasküler Araştırma Merkezi Histoloji Çekirdeği ve Virginia Üniversitesindeki Biyomoleküler Analiz Merkezi ile yardımda bulunmak için yardım ettiler. Örnek alma, immünohistokimya ve görüntüleme.

Bu protokol, bloke edilmiş klinik hasta örnekleri için normal olduğu gibi, formalin-sabit parafin gömülü (FFPE) numunelerdeki hücresel bileşenleri tanımlar. Parafin gömülmesi, hücresel ve doku morfolojisinin en iyi şekilde korunmasına ve bölümlerin daha uzun ömürlü olmasına olanak tanır. Bu analiz için kullanılan örneklere Virginia Üniversitesi Biyorepository ve Doku Araştırma Tesisi aracılığıyla erişilmiştir. Hasta numuneleri, 2010 ve 2013 yılları arasında Virginia Üniversitesi&#39;nde tümör rezeksiyonlarını tamamlayan ve glioblastoma (astrositom, DSÖ grade IV) kesin bir teşhisine dayanan bir nöropatolog tarafından seçildi ve bu analizden önce tanımlanmadı 11 . 1. FFPE Örneği Deparafinizasyon ve Rehidrasyon NOT: Protokolün bu kısmı FFPE örneklerine özeldir. Parafin gömülü numuneler, bu analiz için hücresel ve t korunmasından dolayı daha yararlı olabilirkenMorfoloji yayınlarken, bu analiz donmuş bölümlerle de yapılabilir. Dondurulmuş kesitler kullanılıyorsa bu bölüm atlanabilir ve direkt olarak kromojenik immünohistokimyaya geçebilir. <ol><li> Ksinilen, ksilen,% 100 etanol,% 100 etanol,% 95 etanol,% 95 etanol,% 70 etanol, Deiyonize su, Deiyonize su </li></ol> 2. Antijen Geri Alma NOT: Protokolün bu kısmı, FFPE numunelerinin formalin fiksasyonu sırasında oluşan metilen köprülerini kırmak ve bağlamak için antikorlar için antijen alanlarını ortaya çıkarmak için gereklidir. <ol><li> Damıtılmış suda üreticinin tavsiyesi üzerine Tris bazlı yüksek pH antijen maskesini çözündürünüz. </li><li> Bir mikrodalga kullanarak ısı aracılı antijen alımını gerçekleştirin. Isı aracılı geri alımın diğer biçimleri (basınçlı ocak, sebze buharı veya kaynar su gibi) de yeterli olacaktır. <ol><li> Seyreltilmiş maskelenmeyen çözeltiyi, mühürlenmemiş bir mikrodalgaya tabi tutulabilirliğe ilave edindamar. Kayıkları tekneye yerleştirin. Slaytları mikrodalga fırına yerleştirin. </li><li> Yüksek güçlü, 20 dk kaynatın. Buharlaşma için sıvı seviyelerini izleyin ve gerekirse damıtılmış su ile doldurun. </li></ol></li><li> Numunelerin, çözeltide oda sıcaklığında 1 saat bekletilmesine izin verin. </li></ol> 3. Kromojenik İmmünohistokimya <ol><li> Örneği örtmek için gereken reaktif hacmini en aza indirgemek için hidrofobik bir kalem ile doku örneğini ana hatlarıyla çizin. NOT: Doku numunelerini sulu tutun ve lekelenme etkinliğini etkileyeceği için kurumasına izin vermeyin. </li><li> Doku örneğini örtmek için yeterli permeabilizasyon solüsyonu (Tris-tamponlu salin (TBS) +% 0.01 Triton-X) pipetleyin (tipik olarak yaklaşık 100 - 200 μL). </li><li> Çözümü çıkarıp atın ve adım 3.2&#39;yi tekrarlayın. </li><li> Örnekleri engelleme çözeltisi (% 2.5 at serum + permeabilization çözüm) oda sıcaklığında inkübe edin. </li><li> Seyreltilmiş primer antikorlarla gece boyunca 4 ºC&#39;de örnekleri inkübe edinEngelleme çözümünde. NOT: Burada kullanılan tüm birincil antikorlar 1: 200&#39;de seyreltilir, ancak en iyi seyreltiler imalatçının tavsiyesi ile başlayan seri seyreltmeler kullanılarak belirlenebilir. Bu protokolde kullanılan antikorlar hakkında ayrıntılı bilgi 11 yayınlanmıştır. </li><li> Birincil antikorları, birincil antikor konakçı hayvana karşılık gelen bir yaban turbu peroksidaz polimer reaktifi kullanarak, üretici protokolünü izleyerek tespit edin. </li><li> Doku numunesini örtmek için 5 dakika yeterli inkübasyon solüsyonu pipetleyin ve inkübe edin. </li><li> Çözümü çıkarın ve atın ve Adım 3.7&#39;yi tekrarlayın. </li><li> 15 dakika boyunca 1 x TBS,% 0.3 H2O 2 slaytlar inkübe edin. </li><li> Bir peroksidaz diaminobenzidin (DAB) substrat ile istenilen leke yoğunluğuna ulaşılıncaya kadar 2-10 dakika örnekler geliştirin. </li><li> Hematoksilen gibi hücre çekirdeğini üreticinin protokolünü izleyerek tanımlamak için leke örnekleri hazırlayın. </li><li> Örnekleri% 100 oranında kurutunEtanol ve ksilen. </li><li> Örnekleri montaj malzemeleri ile kalıcı olarak monte edin. </li></ol> 4. İlgi Alanı Tanımlama Bölgeleri <ol><li> Yüksek çözünürlüklü görüntülere sahip parlak alan mikroskobu altında görüntü slaytları. NOT: Görüntüleme hücresel bileşenlerini en az 20x çözünürlükte kullanın. </li><li> Fotoğraf makinesini doku örnekleri boyunca belirli bölgelere taşıyın. </li><li> Niceleme için resimleri TIFF dosyaları olarak kaydedin. </li></ol> 5. Görüntü Analizi <ol><li> Yüzde kapsama alanının miktarını belirlemek için ImageJ&#39;de resim açın. </li><li> Hematoksilenle boyalı çekirdeğin mor rengini çıkarmak için Eşik Renk eklentisini kullanın. </li><li> Görüntüyü 8 bit&#39;e dönüştürün. </li><li> Karanlık arka plan olmadan eşik ekleyin. </li><li> 17 önceden yüklenmiş ImageJ eşik filtrelerinden ( örn. MaxEntropy) birini kullanarak işlem görüntüsü, böylece yalnızca DAB lekeli kısımları eşiğe dahil edilir. NOT: Minimize eden en iyi ön yüklemeli eşik filtresini seçinArka plan boyamanın dahil edilmesi. Bu, lekelenme kalitesine ve antikorun spesifitesine bağlı olabilir. Her hasta örneğindeki tüm teknik çoğaltmalar için aynı eşiği kullanın. </li><li> Eşik uygula. </li><li> Eşikli görüntünün yüzde alanını ölçün. </li><li> Her örnek içindeki birden çok bölge için yüzde yüzdelik kapsam. </li></ol>

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Burada önerilen yöntemimiz, geleneksel kromojenik immünhistokimyayla boyanan histolojik örneklerin analizinde niceliksel bir yaklaşımdır. Bu tür analizler için güncel metodoloji, bağımsız patologlar tarafından derecelendirilen benzer boyama protokollerini içerir. Bu yöntem güvenilir olmuştur, ancak birçok uygulama için, tümör ile ilişkili heterojenliği daha iyi anlamak ve in vitro çalışmalar için tümörlerin doğru yeniden özetlenmesi gibi, hücresel makyajın daha kesin bir şekilde anlaş...

En yaygın ve malign beyin kanseri olan Glioblastoma (GBM), birincil tümör hacminden çevreleyen sağlıklı beyin parankimine 1 , 2 oranlarında dağılmış istila ile karakterizedir. Bu dağınık istila, tümörün tamamen kesilmesini zorlaştırır ve tedaviden sonra kalan istilacı kanser hücreleri, kaçınılmaz nüksetmenin en yaygın nedenidir 2 , 3 , 4 . Daha önce, diffüz glioma hücre invazyonunu inhibe ederek terapötik açıdan faydalı 5 bulduk, ancak GBM istilasına katkıda bulunan kompleks mekanizmalar hakkında çok az şey biliniyordu. Tümör mikro ortamı veya kanseri çevreleyen doku, çoklu kanserlerde tümörlerin ilerlemesinde rol oynadı 6 , 7 . Glioblastoma tümör mikro ortamı, pEkstraselüler matriks, çözünür faktörler ve biyofiziksel faktörlerin yanı sıra, astrositler, mikroglia ve oligodendrositler gibi çok sayıda glial hücrenin oluşturduğu benzersiz bir şekilde karmaşıktır. Deneysel olarak, astrositler ve mikrogliaların glioma progresyonunu ve istilasını 8 , 9 , 10 arttırdığı gösterilmiştir, ancak doğal insan beyninin mikro ortamındaki tüm glial hücrelerin kompozisyonu bilinmemektedir. Daha önce, mikro çevre bileşenleri, glioblastoma mikro ortamının hücresel bileşenlerini nicel olarak analiz ederek ve analizlerimizi orantısal bir tehlike modeli 11 içine dahil ederek, hastanın hayatta kalmasını öngörebildiğini gösterdi. Burada, glioblastoma hastasından tümör rezeksiyonlarının tümör hacmi ve komşu bölgelerindeki glial hücrelerin popülasyonlarının tanımlanması için nicel analiz metodunu açıklıyoruzs. Glial hücre popülasyonlarını tanımlamak için kromojenik immünohistokimya ve her glial popülasyon için boyamanın yüzde kapsama oranını analiz etmek için ImageJ kullanılmıştır. Kapsam yüzdesinin değerlendirilmesi, özellikle kanser hücreleriyle etkileşimlerden etkilenen hücrelerin morfolojik farklılıklarını belirlemek için basit bir ölçüm oluşturur. Histopatolojik boyamanın nicelleştirilmesi için yapılan önceki çalışmalar, hematoksilin ve eozin 12 veya Masson&#39;s trikrom 13 gibi standart boyayı antikor esaslı immünhistokimyasal boyanmanın özgüllüğünden yararlanmayan boyayı kullanır. Metodumuz, kompleks glioblastoma mikro ortamını aydınlatmak için kullanmayı düşündüğümüz, glioblastoma hasta tümör rezeksiyonlarında glial popülasyonları doğrudan ölçmek için geliştirilmiştir.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Xylene</td>
			<td style="width:71px;">Fisher Chemical</td>
			<td style="width:119px;">X3P</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">High pH antigen unmasking solution</td>
			<td style="width:71px;">Vector Labs</td>
			<td>H-3301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TBS</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton-X</td>
			<td style="width:71px;">Amresco</td>
			<td style="width:119px;">9002-93-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Horse serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-ALDH1L1&#160;</td>
			<td style="width:71px;">abcam&#160;</td>
			<td style="width:119px;">ab56777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Iba1&#160;</td>
			<td style="width:71px;">abcam&#160;</td>
			<td style="width:119px;">ab5076</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-Oligodendrocyte Specific Protein1&#160;</td>
			<td style="width:71px;">abcam&#160;</td>
			<td style="width:119px;">ab53041</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ImmPRESS anti-goat</td>
			<td style="width:71px;">Vector Labs</td>
			<td style="width:119px;">MP-7405</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ImmPRESS Universal (anti-mouse/rabbit)</td>
			<td style="width:71px;">Vector Labs</td>
			<td style="width:119px;">MP-7500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydrogen peroxide</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">216763</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ImmPACT DAB substrate</td>
			<td style="width:71px;">Vector Labs</td>
			<td style="width:119px;">SK-4105</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hematoxylin counterstain</td>
			<td style="width:71px;">ThermoScientific</td>
			<td style="width:119px;">72404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Histochoice Mounting Media</td>
			<td style="width:71px;">Amresco</td>
			<td style="width:119px;">H157-475</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Aperio Scanscope</td>
			<td style="width:71px;">Leica Biosystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Image Scanscope</td>
			<td style="width:71px;">Leica Biosystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Super HT PAP Pen</td>
			<td style="width:71px;">Research Products International</td>
			<td style="width:119px;">195506</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative immunohistochemistry of the cellular microenvironment in patient glioblastoma resections

Tümör mikro ortamındaki artan ilgiyle, ölümcül ve en invaziv beyin kanseri olan glioblastomun hasta örnekleri içindeki mikro çevre bileşenlerini spesifik olarak belirlemek için bir yöntem geliştirdik. Kantitatif yöntemler hastalıklı dokuları doğru bir biçimde tanımlamak için yararlı değildir, aynı zamanda potansiyel olarak daha doğru prognoz, teşhis ve doku mühendisliğindeki sistemlerin ve replasmanların geliştirilmesine katkıda bulunabilirler. Glioblastomada, mikroglia ve astrositler gibi glial hücreler, patoloğun derecelendirmesine dayalı zayıf prognoz ile bağımsız olarak ilişkilendirilmiştir. Bununla birlikte, bu hücrelerin ve diğer glial hücre bileşenlerinin durumu nicel olarak iyi tanımlanmamıştır. Bu, bu glial hücreleri işaretleyen büyük süreçler nedeniyle zor olabilir. Dahası, histolojik analizlerin çoğu, genel doku örneğine veya yalnızca tümör hacmine odaklanır; zira bölgeleri esas alan kantifikasyonları tasvir ederÇok heterojen dokuda. Burada, glioblastoma hastalarından tümör rezeksiyonlarının tümör hacmi ve komşu bölgelerindeki glial hücrelerin popülasyonlarının tanımlanması ve nicel olarak analiz edilmesi için bir yöntem açıklanmaktadır. Hastanın tümör rezeksiyonlarında glial hücre popülasyonlarını ve ImageJ&#39;de kromojenik immünohistokimya kullandık ve her glial popülasyon için boyama yüzdesini analiz ettik. Bu tekniklerle, glioma tümör mikro ortamının bölgeleri boyunca glial hücreleri daha iyi tarif edebiliyoruz.

Bu analiz için, tümör rezeksiyonlarımızda iki bölgenin (esas olarak diffüz istilalı kanser hücreli sağlıklı dokudan oluşan primer tümör hacmi ve komşu bölgeler), hematoksilin ve eozin boyalı hastada nöropatologların işbirliği yaptığı tespit edildi ( Şekil 1A , 1B ) örnekleri. Her hasta örneği içinde, astrositler ( Şekil 1C ), mikroglia ( Şekil 1D ) ve...

Watch this Scientific Journal Video about Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections at JoVE.com

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

Hasta Glioblastoma Rezeksiyonlarında Hücresel Mikro Çevrenin Kantitatif İmmünohistokimyası

Bu protokol, kromojenik immünhistokimya ve ImageJ kullanılarak glioblastoma hasta rezeksiyonlarında tümör mikro çevre bileşenlerini nicel olarak tanımlamak için geliştirildi.

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within ...

quantitative-immunohistochemistry-cellular-microenvironment-patient

Research

JoVE Journal

Medicine

9.6K Görüntüleme.  University of Virginia.  Bu protokol, kromojenik immünhistokimya ve ImageJ kullanılarak glioblastoma hasta rezeksiyonlarında tümör mikro çevre bileşenlerini nicel olarak tanımlamak için geliştirildi. 

Video: Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

Kantitatif Görselleştirme ve Dinamik Termal Görüntüleme Deri Kanseri Tespiti

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

Tıp

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Karboksi Flöresein sukkinimidil ester (KAKE) Kullanarak İnsan Glioblastoma Yavaş-bölme Hücreleri Tanımlama ve İzolasyonu

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Meme Kanseri Fare Modelleri Tümör Mikroçevre İlaç Yanıtlarının Görüntüleme Canlı

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

Hücresel Dielektrik Spektroskopisi kullanarak İdiopatik Skolyoz Prognostik Tahmininde Hücre-tabanlı Deney Protokolü

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

Tekrarlayan Glioblastoma&#39;nın Tedavisinde Bevacizumab ile Kombine Alan Terapi Tedavisi Tümör

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

Bir &quot;Hasta gibi&quot; Ortotopik singenik Fare Modeli Hepatoselüler karsinomu metastazı

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Tümör Hücre Göçü ve İnvaziv Olmayan İlaçların Hastaya Özel Etkilerinin İncelenmesi İçin Bir İnsan Glioblastoma Organotipik Dilim Kültürü Modeli

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Kinomic Profil Yöntemi ve İlaç Tepki Testi için 3D İnsan BIOGEL Kültür Sisteminin Kullanılması Microtumors ve Hasta kaynaklı Glioblastom Hücreleri Üretimi

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

Tümör mikroçevresinde Gerçek zamanlı Multisellüler Dynamics Genişletilmiş Time-lapse intravital Görüntüleme

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

Nicel [18F]-Facetogenic alçak sırt ağrısı olan bir hastada dinamik kemik cirosunun değerlendirilmesi Için NAF-Pet-MRI Analizi

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

Hasta Glioblastoma Rezeksiyonlarında Hücresel Mikro Çevrenin Kantitatif İmmünohistokimyası

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