The authors would like to thank the MSKCC Tissue Procurement Service Team (TPS), specifically, Maria Corazon Mariano, Priscilla McNeil, Anas Idelbi, Daniel Navarrete and Katrina Allen, in all of their efforts in the successful pursuit of this project and funding from the following sources: 5 R21 CA158609-02 and the Conquer Cancer Foundation and the Breast Cancer Research Foundation. In addition, the authors would like to thank Eric Cottington PhD, Vice President of the Office of Research and Project Administration, the Office of Technology Development, Research Outreach and Compliance and RTM Information Systems Support, in the support of the submission of this manuscript.

NOT: Hasta doku temini kurumsal inceleme kurulu (KİK) ile yetki verildi Memorial Sloan Kettering Kanser Merkezi&#39;nde Biospecimen ve Klinik Protokolleri (Protokolü numaralarını sırasıyla 09-121 ve 11-041) -onaylı. 1. Doku İhale <ol><li> Hasta primer tümör / metastaz Alımı NOT: Bugüne kadar, bu protokol ameliyatla çıkarıldı pankreas, mide ve meme tümör türleri, yanı sıra, lenfoma metastaz üzerinde yapılmıştır. <ol><li> Steril sıvı dökülmelerine karşı dayanıklı bir kap içinde sıkıca kapatılmış ve steril sızdırmaz plastik numune torbaya Patoloji bölümünde kurye veya pnömatik tüp sistemi ile numune teslim Cerrahi ekibi yönlendirin. Hiçbir formalin veya sabitleştirici ile taze halde numune taşıma Cerrahi ekibi yönlendirin. </li><li> Hangi inc, steril tekniği kullanarak taze örnek hasat patolog veya patolog yardımcısı yönlendirinLaminer akış kaputun altında steril eldiven ve araçların idarecisi kullanımı. </li><li> Cerrahi prosedürün tamamlanmasından 30 dakika altında titizlikle tutulur tedarik numunenin, hasat zamanını kaydedin. Soğuk iskemi dikkate etkileri de dikkate alınarak,&gt; 30 dakika 18 soğuk iskemi süresi ile rezeke numune yapmayız. </li><li> Steril bir ortam sağlamak için, bir laminar akış başlığı içinde 1,0 cm3 yaklaşık 0,5 cm3 primer tümör örnek kaldırmak için, Patoloji Bölümü personelin doğrudan. Mümkün olduğunda, olası Frank santral nekroz (hücre ölümü) önlemek için indeks lezyonun çevresinden tümör dokusu seçin. NOT: nekrotik doku aşağıdaki kriterlerden herhangi biri tarafından fena halde tanınabilir olabilir: renk veya doku solukluk kaybı; nekrotik doku yumuşak ve kırılgan olduğu gücü kaybı; nekrotik ve canlı doku arasında belirgin bir sınır. </li><li> Pathologi Doğrudanst veya patolog yardımcısı tanısal değerlendirme için tümörün hemen örnekleme sonra aşırı (cerrahi artıklar) ise periferik doku sağlayabilir. Yaklaşık 5 mi,% 1 antibiyotik (penisilin ve streptomisin) ihtiva eden en az temel ortam (MEM) içeren 15 ml&#39;lik steril bir konik santrifüj tüpü içinde örnek yerleştirin. </li><li> Mevcut (örneğin, mastektomi numune aksiller kuyruk lenf nodu), aynı boyutta (0.5 cm 3 cm 3 1.0) fena halde pozitif ilişkili lenf nodu örneği temin ve primer tümörün ilaç yanıtı ile karşılaştırırsanız. Primer tümör numunesinde gibi,% 1 antibiyotik (penisilin ve streptomisin) içeren yaklaşık 5 mi minimum temel ortam (MEM) içeren bir 15 ml&#39;lik steril bir konik santrifüj tüpüne lenf düğümü örnek yerleştirin. </li><li> Eğer örneğin, cerrahi örnek sağlar (örneğin, mastektomi numune), (primer tümörden uzak) çıkarmak, normal doku örneği (büyüklüğü,yaklaşık 5 ml transferi amaçlıdır% 1 antibiyotik içeren minimum temel ortam (MEM) içeren 15 ml steril konik santrifüj tüpü normal yoğun lifli / meme parankim) ve yer. Laboratuar tesislerine transferi takiben, bir de kriyotüpe, &#39;ek&#39; donma ve mağaza &#39;normal&#39; örneği aktarmak -80 ° C derin dondurucuda gelecekteki moleküler analizler için. </li><li> Islak buz üzerinde tüm örneklerini yerleştirin ve hemen ex vivo taze doku kesit için laboratuvar uzaya taşıyacak. BÖLÜM Normal numune yapmak değil, gelecekteki moleküler analizler için ° C derin dondurucuda bir -80 saklayın. Gelecekteki moleküler analizler için -80 ° C derin dondurucuda kriyotüpe, &#39;ek&#39; donma ve mağaza için bir primer tümörün bölümünü ve ilişkili metastaz aktarın. </li></ol></li><li> Klinik çalışmalarda doku İhale (örneğin, opsiyonel iğne biyopsisi (CNB)) NOT: Biz hastaların tümör dokuları eldeTedavinin başlamasından önce erişilebilir bir tümör metastaz CNB geçmesi onların rıza klinik protokolleri üzerine alındı. CNBs, bugüne kadar meme, marjinal zon lenfoma, mantle hücreli lenfoma, metastatik paragangliomalardır, serviks (skuamöz hücreli) ve yumurtalık kanserleri olan hastalarda yapılmıştır. <ol><li> Girişimsel Radyoloji veya ilgili bölümünden klinik personeli içeren steril konik santrifüj tüpü tedavi öncesi iğne biyopsisi yapmak ve bir 15 ml numune yerleştirin sahip yaklaşık 5 ml% 1 antibiyotik (penisilin ve streptomisin) içeren minimum temel ortam (MEM). </li><li> Islak buz üzerinde biyopsi örneği yerleştirin ve hemen ex vivo taze doku kesit için laboratuvar uzaya taşıyacak. </li></ol></li></ol> Kesit ve Vibratome Ayarları için Malzemelerinin 2. Hazırlık <ol><li> Kesit için Malzemelerinin Hazırlanması <ol><li> Aga 4 g eklenerek,% 4 agaroz solüsyonu yapmakPBS, 100 ml yükseldi. Otoklav (devir ayarı: sıvı, 121 ° C, 30 dak, inç kare başına £ 15), ilk kullanımdan önce çözelti. Daha sonraki kullanımlar için, agaroz oda sıcaklığında katılaşması dikkatli her kullanımdan önce (30 saniye aralıklarla için yüksek ısı) sıvılaştırılması ve uygun bir şekilde ~ 25 ° C (yani, sıcak bir dokunma) soğumaya bir mikrodalga içinde% 4 agaroz yerleştirin. </li><li> Kalıcı olmayan bir doku gömme ortamı olarak% 4 agaroz kullanın. % 4 agaroz yeterli miktarda soğutulur ve steril bir forseps gömme kalıp doku örneğin yerleştirilmesi ve% 4 agaroz akma, sıvı halde hala demektir. </li><li> Gömme kalıp kesme yüzeyi doğrudan duvara yakın bir doku örneği (~ 2 mm mesafe), fakat yerleştirin. % 4 agaroz, hızlı katılaşmaya başlar, bu nedenle hızlı ve uygun şekilde doku örneği yer alacak. Kesitlere ayırma esnasında çözelti içinde tutmak için bir 55 ° C su banyosu içinde,% 4 agaroz tutun. </li><li> Bölümü analiz için 24 plaka için, 1 ml o eklemekHer bir oyuğa f tüm ortam (örneğin, MEM +% 10 cenin sığır serumu +% 1 antibiyotik). Kesit alma süresi boyunca, oda sıcaklığında (-25 ° C) &#39;de 24-yuvalı plaka tutun. </li></ol></li><li> Ayarlar Vibratome hazırlanması <ol><li> Titreşimli mikrotomu kullanılarak, 10 bıçak örneği yaklaşır hızını [10 (2,5 mm / sn) 0 (0.00 mm / sn)] ve bıçak [0 (0 Hz) frekansını ayarlamak (100 Hz) ]. Doku (örneğin yoğunluk / sertlik) bir kıvama göre ayarları yapın. </li><li> Invaziv duktal karsinom, primer meme tümörü için, sırasıyla 6 ve 3, hız ve frekansa ayarlar. Az yoğun / firma tümör dokusu için hızını azaltmak ve buna göre sıklığını arttırırlar. </li><li> (- 999 mikron 1) enstrüman sınırları içinde kesit kalınlığı ayarlayın. Bu analizler için, 200mm&#39;lik bir kesit kalınlığı ayarlayın. </li><li> Örnek disk tha sıvı daha ince bir yapışkan tabaka kullanılarak Agaroz yerleştirilen örneği montet ıslak buz kaplı olan medya bir rezervuar batırıldıktan. </li></ol></li></ol> 3. Doku Kesit, Tedavi ve Analizleri <ol><li> Hemen tam ortam 1 ml ile bir çok-çukurlu bir tabla içindeki steri forseps ve yer kullanarak kesit üzerine hassas dilimlenmiş bölümleri çıkarın. Yeterli bölümleri tepki ve bağlaşık analizler için yakalandığı zaman, standart doku kültür koşulları altında ayarlanmış bir doku kültürü kuluçka makinesi içinde tedaviye yanıt için çok çukurlu plaka yerleştirilir. </li><li> Kültürlü eserler karşı doza bağlı bir cevap teyit etmek için bir çaba muamele edilmiş bölümleri bağlayıcının birden fazla kontrol bölümleri al. (- 4 sa ~ 2) 18 de soğuk iskemi zaman zararlı etkilerin içinde, - (40 dk ~ 30) doku hızla kesit gerçekleştirin. Sadece 1 saat alışma dönemi aşağıdaki bölümlerden tedavisini yapın. </li><li> 1 saat alışma süresinden sonra, doz (terapötik ilgili artan ekleyin; <em.&gt;, Örneğin 0 - tedavi süresi için standart kültür koşulları altında doku bölümleri ve ev İlacın konsantrasyonu 10 uM). Bu çalışma için, 24 ve / veya 48 saat değerlendirme dönemleri kullanın. Ve 96 saat değerlendirme dönemleri dahil olmak üzere dönemler için, bu çalışmada test Ancak, kontrol (yalnızca araç) doku bölümleri, canlı kalacaktır. </li><li> Tedaviyi takip ederek, RT O&#39;da% 10 tamponlu formalin ve sırayı içeren bir tüpe doku bölüm yerleştirerek, steril koşullar ve formalin ile düzeltme altında bölümleri çıkarmak / N veya% 4 paraformaldehid içeren bir tüpe (bir şişesinden,% 16 paraformaldehit seyreltik 2 saat boyunca oda sıcaklığında bir nihai konsantrasyonu% 4) ve bir yer için PBS ile yıkanmıştır. <ol><li> Daha büyük doku bölümleri saptamak (örneğin,., ~ 6 mm x 4 mm),% 10 tamponlu formalin O 200 mikron kalınlığında / oda sıcaklığında K. Daha küçük bölümleri Fix (örneğin,., 1 mm x 1 mm), oda sıcaklığında 2 saat süresince% 4 paraformaldehit içinde 200 mikron kalınlığındadır. NOT: fiksatif f hacmi verimli penetrasyon içinixative doku hacmi 20x olmalıdır. Verilen bu doğru tespiti kullanımı tüm doku boyutları için fiksatif 1 ml güvencesi için son derece küçük doku boyutları (örneğin, 6 mm 3 doku = 6 ul hacmi), bu protokol sonuçları. </li><li> Tespitin ardından doku örnekleri Patoloji Bölümü personeli tarafından gömülü parafine var. Tahsil slaytlar üzerine parafin bloklar örnek Bölüm hematoksilen ve eozin lekeli ve değerlendirilir immunohistokimyasal (örneğin, apoptoz) olmak. </li></ol></li></ol>

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No conflicts of interest disclosed.

Etkili tedavi stratejileri geliştirmek için çalışırken Kanser biyologlar önemli zorluklarla karşı karşıya. Kurulan kanseri hücre çizgileri üzerindeki geliştirme ilaçlar in vivo test doğru yanıt olarak ve PDX modellerinde in vivo deneyler, emek yoğun ve çok pahalıdır yansıtmıyor olabilir. Yukarıda verilen, primer hastanın ex vivo tekniklerin uygulanması 15 hemen kurulmuş olan kanser hücre çizgileri ve hasta türetilmiş ksenograftları (PDX) mol...

Kanser terapötik olarak etkili geliştirilmesi son derece zor olduğu kanıtlanmıştır. Kanser hücre hatları ve tümör eksplant - hem de ksenograftları üzerinde yarım asır 1,2,3 kanser araştırmalarında kullanılmıştır. Bugüne kadar, ilaç hassasiyeti ve mukavemet hem de kurulmuş olan kanser hücre çizgileri ve hasta türetilmiş ksenograftlarının moleküler analizi (PDX) vazgeçilmezdir. Bununla birlikte, sabitlenen kanser hücresi hatlarında bileşiklerin testleri çoğu zaman in vivo etkinliğe işaret eden değildir ve özellikle de PDX modellerinde, hayvanlarda in vivo çalışmalarda da karşılık gelen çok pahalı ve zaman alıcı bir işlemdir. Bu model sistemlerde, tümör ilerlemesi ve tedavi stratejileri yanıt yerli mikroçevresinin etkisi hakkında bilgilendirmek için yani yetersizlik sınırlamalar, bu analizleri iltifat için ek yöntemler geliştirmek araştırma alanını açmıştır. Yakın zamandaki, artan dikkat hasta tum ex vivo analizi doğru ödeniyorKanser terapötik cevap, kanser hücrelerinin temel molekül bileşime özel değildir ama daha çok büyük, geleneksel yöntemler ile kültürleme değinmeyecek edilemez tümör hücresi mikro-6, 7 özelliği etkilenir olmasından dolayı daha iyi anlaşılması için eksplantlar ya da 4, 5 / veya PDX. yukarıdaki bağlamda ex vivo analizi (yani., bitişik çevreleyen tümör hücre mikro-etkisi) daha çok hücre izolatları 8, 9 ex vivo analizine göre, uygun bir primer tümör / metastaz bölme değerlendirilmesini gerektirir. Biz de hasta primer tümörlerin (hassas dilimlenmiş taze doku bölümleri yani.) Ve ilişkili metastazlar (yani, lenf düğümleri) sadakatle yanıt üzerine bilgilendirir (IC50), hedef dışı etkileri bir ex vivo tekniği burada rapor ve moleküler sağlar direnci ve geri besleme mekanizmalarının analizi. Fototerapi ek olarak, bir analiz bağıntılıbiyomarkör ve gen ekspresyon profilinin karşı utic duyarlılık / direnç (yani., yüksek ilaç tepkisi belirli bir biyolojik profil ile hastayı maçları) ilgi deneysel ilaca cevap daha muhtemel hastaları belirlemek için bir çaba yapılabilir. Bir çok parametre moda ex vivo tekniği ve değerlendirme uygulama hasta seçimi ve klinik sonuçların genel iyileştirilmesine yönelik bir harekettir. Ex vivo tedavi yanıtı analizi kanser tedavileri klinik öncesi ve klinik geliştirme standart bir araç haline gelebilir ve tedavi kalkınma stratejilerinde kişiselleştirilmiş tıp yaklaşımına doğru bir adım olarak tasavvur edilir.

<table><tbody><tr><td>Vibratome Leica VT1000s</td><td>Leica</td><td>14047235613</td><td></td></tr><tr><td>UltraPure agarose</td><td>Invitrogen</td><td>16500500</td><td>Prepare 4% and 6% before use</td></tr><tr><td>Injector blade</td><td>Ted Pella</td><td>121-4</td><td></td></tr><tr><td>MEM with Penicillin&amp;nbsp; + Streptomycin</td><td>Media Core Facilities (MSKCC)</td><td></td><td>The media is prepared at Memorial Sloan Kettering Cancer Center&amp;nbsp;</td></tr><tr><td>Scalpel no. 10</td><td>Thermo Scientific</td><td>31-200-32</td><td></td></tr><tr><td>Disposable forceps</td><td>Cole-Parmer</td><td>84011182</td><td></td></tr><tr><td>Embedding mold</td><td>Electron Microscopy Science</td><td>70181</td><td></td></tr><tr><td>FBS (heat inactivated)</td><td>Gemini</td><td>100106</td><td></td></tr><tr><td>24 well plates</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>Formalin (10%)</td><td>Sigma Diagnostics</td><td>SDHT501128&amp;nbsp;</td><td></td></tr><tr><td>16% Formaldehyde solution</td><td>Thermo Scientific</td><td>28908</td><td></td></tr><tr><td>Embedding microsettes</td><td>Simport</td><td>M503-2</td><td></td></tr><tr><td>Ethanol (70%)</td><td>Fisher Scientific</td><td>A405P-4</td><td></td></tr><tr><td>Waterbath</td><td>Fisher Scientific</td><td>15-462-2SQ</td><td></td></tr><tr><td>Microwave</td><td>General Electric</td><td>ModelJES2051DNBB</td><td></td></tr><tr><td>Adhesive (Ethyl Cyanoacrylate)</td><td>Sigma-Aldrich</td><td>E1505-5G</td><td></td></tr><tr><td>10 mm dishes</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>15 ml tubes</td><td>BD Falcon</td><td>352096</td><td></td></tr></tbody></table>

ex vivo treatment response of primary tumors and/or associated metastases for preclinical and clinical development of therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Bu çalışma için, ex vivo olarak teknik, bir ısı şoku proteini 90 inhibitörleri (Hsp90i) terapötik hassaslık / duyarlılık bir bağıntılı analizinde kullanıldı. Bu Hsp90i, meme kanseri primer tümörün, bir ER + invaziv duktal karsinom (IDC) ve ilişkili lenf nodu metastazı klinik öncesi değerlendirmede tedaviye yanıt ex vivo olarak analiz edildi &#160; (Şekil 1). Birden fazla 200 um seri bölümleri Hsp90i bölgesinin sadece araç ve artan dozlard...

Watch this Scientific Journal Video about Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics at JoVE.com

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Kurulmuş kanser hücre hatları ve ksenogratflardır son birkaç yıldır kanser araştırma dayanak noktası olmuştur. Bununla birlikte, son bulgular terapötik tepki özellikle, tümör hücre mikro-ortamı tarafından etkilendiğini göstermektedir. Bu nedenle, ilaç geliştirme amaçları için primer tümör numunelerinin bir ex vivo analiz geliştirdik.

İlköğretim Tümörleri ve / veya klinik öncesi için İlişkili Metastazlarının ve Tedavi Klinik Geliştirme Ex Vivo Tedavisi Tepki

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

ex-vivo-treatment-response-primary-tumors-andor-associated-metastases

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Medicine

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

14.7K Görüntüleme.  Memorial Sloan Kettering Cancer Center.  Kurulmuş kanser hücre hatları ve ksenogratflardır son birkaç yıldır kanser araştırma dayanak noktası olmuştur. Bununla birlikte, son bulgular terapötik tepki özellikle, tümör hücre mikro-ortamı tarafından etkilendiğini göstermektedir. Bu nedenle, ilaç geliştirme amaçları için primer tümör numunelerinin bir ex vivo analiz geliştirdik. 

Video: Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

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 ...

Tıp

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 ...

Kanser Araştırmaları

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

Yapısal DNA Alterasyon Analizi ve Hasta Kaynaklı Ksenogreftler Kullanılarak Kanserde Hedefli Tedavilerin Test Edilmesi

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Kuyruk Ven Metastaz Tahlilleri ve Gerçek Zamanlı Vivo Görüntüleme Kombine Kullanımı Meme Kanseri Metastatik Kolonizasyon ve Yük Akciğerlerde Quantify için

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Organotipik Tümör Doku Dilimlerinde Gerçek Zamanlı İlaç Yanıtının Ölçülmesi

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

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. ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

Hasta Kaynaklı Tümör, Hastalarda İlaç Direncini Tahmin Etmek İçin "Canlı" Preklinik Bir Platform Olarak Eksplantlar

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

EGFR-Mutant NSCLC Hasta Kaynaklı Organoidlerde Hedefe Yönelik Tedavilere Duyarlılığın Profillenmesi

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Toplu ve Tek-Organoid Analizi için Geniş Alan Canlı Hücre Görüntüleme Kullanılarak Multiparametrik Tümör Organoid İlaç Taraması

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Kanser İlaç Duyarlılık Etkinliğinin Değerlendirilmesi<em&gt; İn Vitro</em&gt; Ve<em&gt; İn Vivo</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

İlköğretim Tümörleri ve / veya klinik öncesi için İlişkili Metastazlarının ve Tedavi Klinik Geliştirme Ex Vivo Tedavisi Tepki

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