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 border="0" cellpadding="0" cellspacing="0" width="598">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:96px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vibratome Leica VT1000s</td>
			<td style="width:96px;">Leica</td>
			<td style="width:119px;">14047235613</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">UltraPure agarose</td>
			<td style="width:96px;">Invitrogen</td>
			<td style="width:119px;">16500500</td>
			<td style="width:167px;">Prepare 4% and 6% before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Injector blade</td>
			<td style="width:96px;">Ted Pella</td>
			<td style="width:119px;">121-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">MEM with Penicillin&#160; + Streptomycin</td>
			<td style="width:96px;">Media Core Facilities (MSKCC)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">The media is prepared at Memorial Sloan Kettering Cancer Center&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scalpel no. 10</td>
			<td style="width:96px;">Thermo Scientific</td>
			<td style="width:119px;">31-200-32</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable forceps</td>
			<td style="width:96px;">Cole-Parmer</td>
			<td style="width:119px;">84011182</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Embedding mold</td>
			<td style="width:96px;">Electron Microscopy Science</td>
			<td style="width:119px;">70181</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS (heat inactivated)</td>
			<td style="width:96px;">Gemini</td>
			<td style="width:119px;">100106</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24 well plates</td>
			<td style="width:96px;">Corning</td>
			<td style="width:119px;">3524</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Formalin (10%)</td>
			<td style="width:96px;">Sigma Diagnostics</td>
			<td style="width:119px;">SDHT501128&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">16% Formaldehyde solution</td>
			<td style="width:96px;">Thermo Scientific</td>
			<td style="width:119px;">28908</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Embedding microsettes</td>
			<td style="width:96px;">Simport</td>
			<td style="width:119px;">M503-2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol (70%)</td>
			<td style="width:96px;">Fisher Scientific</td>
			<td style="width:119px;">A405P-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Waterbath</td>
			<td style="width:96px;">Fisher Scientific</td>
			<td style="width:119px;">15-462-2SQ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microwave</td>
			<td style="width:96px;">General Electric</td>
			<td style="width:119px;">ModelJES2051DNBB</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Adhesive (Ethyl Cyanoacrylate)</td>
			<td style="width:96px;">Sigma-Aldrich</td>
			<td style="width:119px;">E1505-5G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10mm dishes</td>
			<td style="width:96px;">BD Falcon</td>
			<td style="width:119px;">353003</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">15ml tubes</td>
			<td style="width:96px;">BD Falcon</td>
			<td style="width:119px;">352096</td>
			<td style="width:167px;"></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

Research

JoVE Journal

Medicine

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

14.6K 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

Thermal Ablation for the Treatment of Abdominal Tumors

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ablation procedure, a thin applicator is guided into the target tumor under imaging guidance. Energy is then applied to the tissue until temperatures rise to cytotoxic levels (50-60 &deg;C). Various energy sources are available to heat biological tissues, including radiofrequency (RF) electrical current, microwaves, laser light and ultrasonic waves. Of these, RF and microwave ablation are most commonly used worldwide.
During RF ablation, alternating electrical current (~500 kHz) produces resistive heating around the interstitial electrode. Skin surface electrodes (ground pads) are used to complete the electrical circuit. RF ablation has been in use for nearly 20 years, with good results for local tumor control, extended survival and low complication rates1,2. Recent studies suggest RF ablation may be a first-line treatment option for small hepatocellular carcinoma and renal-cell carcinoma3-5. However, RF heating is hampered by local blood flow and high electrical impedance tissues (eg, lung, bone, desiccated or charred tissue)6,7. Microwaves may alleviate some of these problems by producing faster, volumetric heating8-10. To create larger or conformal ablations, multiple microwave antennas can be used simultaneously while RF electrodes require sequential operation, which limits their efficiency. Early experiences with microwave systems suggest efficacy and safety similar to, or better than RF devices11-13.
Alternatively, cryoablation freezes the target tissues to lethal levels (-20 to -40 &deg;C). Percutaneous cryoablation has been shown to be effective against RCC and many metastatic tumors, particularly colorectal cancer, in the liver14-16. Cryoablation may also be associated with less post-procedure pain and faster recovery for some indications17. Cryoablation is often contraindicated for primary liver cancer due to underlying coagulopathy and associated bleeding risks frequently seen in cirrhotic patients. In addition, sudden release of tumor cellular contents when the frozen tissue thaws can lead to a potentially serious condition known as cryoshock 16.
Thermal tumor ablation can be performed at open surgery, laparoscopy or using a percutaneous approach. When performed percutaneously, the ablation procedure relies on imaging for diagnosis, planning, applicator guidance, treatment monitoring and follow-up. Ultrasound is the most popular modality for guidance and treatment monitoring worldwide, but computed tomography (CT) and magnetic resonance imaging (MRI) are commonly used as well. Contrast-enhanced CT or MRI are typically employed for diagnosis and follow-up imaging.

A thermal tumor ablation procedure is described. The entire procedure is detailed, including pretreatment planning and imaging studies, anesthesia, adjuvant techniques to facilitate a percutaneous approach, imaging guidance of the ablation device to the tumor, thermal treatment, post-treatment care and follow-up.

Karın Tümörler Tedavi için Termal Ablasyon

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ...

Tıp

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Biyoparlaklık ve Micro CT Görüntüleme Tümör Metastazlari ve osteolitik Lezyonlar İzleme

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

Yeni Bir Yüksek çözünürlüklü<em&gt; In vivo</emTümör Büyüme ve Tedavi için İntrakranial Yapıların Dinamik Tepki Eğitim&gt; Görüntüleme Tekniği

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Klinik ve Klinik Öncesi Araştırma Uygulamaları Tümör Hücre (CTC) Tahliller Sirkülasyon semiautomated adaptasyon

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Karaciğer metastazları Klinik öncesi fare modeli

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Uveal Melanom Karaciğer Metastazlı için bir tedavi olarak izole Karaciğer Perfüzyon

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

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 ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&amp;#946; and Tau Neuropathology

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Amiloid-β ve Tau Nöropatolojisinin Transgenik Fare Modellerinde Spontan Dalgalanmanın Değerlendirilmesi, Yeni Nesne Tanıma ve Limb Klapması

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Treatment of Liver Metastases Using an Internal Target Volume Method for Stereotactic Body Radiotherapy

The prognosis of patients with metastatic cancers has improved in the past decades due to effective chemotherapy and oligometastatic surgery. For inoperable patients, local ablation therapies, such as stereotactic body radiotherapy (SBRT), can provide effective local tumor control with minimal toxicity. Because of its high precision and accuracy, SBRT delivers a higher radiation dose per fraction, is more effective, and targets smaller irradiation volumes than does conventional radiotherapy. In addition, steep dose gradients from target lesions to surrounding normal tissues are achieved using SBRT; thus, SBRT provides more effective tumor control and exhibits fewer side effects than conventional radiotherapy. The use of SBRT is prevalent for treating intracranial lesions (known as stereotactic radiosurgery); however, it is now also used for treating spinal and adrenal metastases. Because of advancements in image-guided assistance and respiratory motion management, several studies have investigated the use of SBRT for treating lung or liver tumors, which move as a patient breathes. The results of these studies have suggested that SBRT favorably controls tumors in the case of moving lesions.
Four-dimensional computed tomography (4D-CT) with an abdominal compressor (AC) is clinically convenient for effective respiratory motion management. Because this method is noninvasive and allows free breathing, its use reduces complications. Furthermore, patients consider this method convenient. Moreover, it is considered more efficient than other methods of respiratory motion management by physicians and therapists. The use of 4D-CT with an AC for treating pulmonary lesions has also been widely investigated, and the technique is gaining acceptance for treating hepatic lesions. However, the protocols for using 4D-CT with an AC for treating hepatic lesions are different from those used for treating pulmonary lesions. In this article, we describe a new protocol for SBRT with 4D-CT and an AC for treating liver metastases.

Stereotactic body radiotherapy (SBRT) requires rigorous accuracy and precision for delivering high radiation doses per fraction to small treatment volumes to improve tumor control and simultaneously reduce toxicity. Herein, we present a noninvasive and clinically convenient respiratory motion management protocol for SBRT for liver metastases.

Stereotaksik vücut radyoterapi için bir iç hedef birim yöntemini kullanarak karaciğer metastaz tedavisi

The prognosis of patients with metastatic cancers has improved in the past decades due to effective chemotherapy and oligometastatic surgery. For ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

384-şey plakaları ısı kontrollü bir Ca duyarlı floresan boya ile ölçülen insan IPSC türetilmiş Cardiomyocyte Syncytia kasılmalar

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...