Bu çalışma, Amerikan Kalp Derneği GRA Bahar 09 Doktora Sonrası Bursu (09POST2250268) ve NIH R01 CA097465 tarafından kısmen desteklenmektedir. Biz çok tümör implantasyonu üzerine ona yardım için Dr Wen Li ve Dr Vikas Gulani MRG protokolü test ve kurulum için, ve Bayan Yvonne Parker teşekkür ederiz.

Protokol Bir önceki çalışmada 11 uyarlanmıştır. 1.. CLT1 Peptide Gd-DOTA Konjügasyon <ol><li> Standart katı-faz peptid sentezi kullanılarak, bir 2-klorotritil klorür reçinesi (1.0 mmol) üzerine, Fmoc-korumalı amino asitlerden CLT1 peptid (CGLIIQKNEC) sentezler. </li><li> Nihai amino asit ilave edildikten sonra, 2 saat boyunca 0 ° C&#39;de DMF içerisinde talyum (III) trifloroasetat (1.09 g, 2.0 mmol, 2 eşdeğer) ile reçine lineer peptid (20 mi) dönüşür. </li><li> Daha sonra, PEG konjugatı ve Fmoc-NH-PEG-COOH (3.0 mmol), Fmoc-Lys (Fmoc)-OH (3.0 mmol) ile arka arkaya reaksiyona sokulması ile reçine üzerinde CLT1 peptid (1.0 mmol) of N-terminaline lisin sekans, ve Fmoc-Lys (Fmoc)-OH, (6 mmol) ihtiva eden bir ikinci parti. </li><li> Piperidin ile Fmoc çıkarın. DOTA-tris (t-Bu) (4.58 g, her bir amino grubuna 8.0 mmol, 2 eşdeğer), 2 saat için reçine üzerindeki lizin dendrimer üzerindeki dört serbest aminler ile reaksiyona ekle. </li><li> Son olarak, ayrılması ve ürünün korumasını kaldırmaktrifloroasetik asit, su, ve oda sıcaklığında 8 saat boyunca triisobutylsilane (TBS) (20 mi, 95/2.5/2.5) bir kokteyli ile muamele etmek suretiyle reçineden. Trifluoroasetik asit (TFA) ile yıkanarak, ardından süzme ile reçine çıkarın. Birleştirilen süzüntüler soğuk etil eter (200 mi), damla damla ekleme ile santrifüj ve etil eter ile 4x yıkayın. Renksiz bir katı (1.99 g,% 61 verim) vermek üzere vakum altında kurutun. </li><li> 10 dakika boyunca Çözücü A içinde 20 dakika ve% 40-90 çözücü B için% 0-40 çözücü B bir gradyan çözücü içinde asetonitril (% 0.1 TFA) (% 0.1 TFA sulu solüsyon) kullanılarak hazırlayıcı HPLC ile ham ürün saflaştırarak . </li><li> CLT1-dL-(DOTA) DI su içinde 4 (250 mg, 0.077 mmol) (15 mi) çözülür ve 1 M NaOH ile pH&#39;ı 6&#39;ya ayarlayın. Gd (OAc) 3 .4 H 2 O ekleyin (472 mg, 0.462 mmol, DOTA monoamid 1.5 eşdeğer) çözeltisine, kısımlar halinde, 1 M NaOH ile pH 6 olarak muhafaza edilirken. 48 saat için oda sıcaklığında reaksiyon çözeltisi ilave edin. </li><li> Kompleks e kalıntı Gd (III)thylenediaminetetraacetic asit (EDTA) (90 mg, 0.31 mmol) eklendi ve nihai ürüne CLT1-dL-(Gd-DOTA) 4 (169 mg,% 57) vermek üzere bir ebat eksklüzyon kolonu ile ham ürünü temizler. </li></ol> 2. Kontrast Ajanları karakterizasyonu <ol><li> Indüktif eşleşmiş plazma optik emisyon spektroskopisi ile Gd (III) içeriği ölçün. </li><li> Bir matris olarak 2,5-dihidroksibenzoik asit (2,5-DHB) ile doğrusal modda matris destekli lazer desorpsiyon / iyonizasyon time-of-flight (MALDI-TOF) kütle spektrumları elde edin. </li><li> Bir relaxometer ile CLT1-dL-(Gd-DOTA) 4&#39;ün kontrast ajanları ve nontargeted kontrol maddesi 60 MHz (1.5 T) farklı konsantrasyonları ile sCLT1-dL-(Gd-DOTA) 4 sulu çözeltinin gevşeme süreleri ölçmek 37 ° C&#39;de T 2 ölçmek için 500 yankıları ile T 1 ve Carr-Purcell-Meiboom-Gill dizisi ölçmek için bir inversiyon-kurtarma darbe dizisi kullanın. T 1 ve T inci 2 relaksivitelerine hesaplayınGd konsantrasyonlara karşılık 1 / T 1 ve 1 / T 2 araziler yamaçlarından e ajanlar. </li></ol> 3. Ortotopik PC3 prostat tümörü ile Hayvan Hücre Kültürü Modeli Geliştirme <ol><li> / Ul PBS 2.5 x 10 4 hücre yoğunluğunda,% 5 fetal sığır serumu ve penisilin / streptomisin / fungizon, tripsinizasyon ile hasat ve tekrar süspansiyon ile desteklenmiş RPMI ortamı içinde, yeşil floresan proteini (GFP) yapısal ifadesi ile kültür PC3 prostat kanseri hücreleri. </li><li> CWRU Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından onaylanmış bir hayvan protokole göre Atimik Hayvan Core Tesisleri&#39;nde, Case Western Reserve Üniversitesi (CWRU) at (4-5 haftalık) erkek NIH atımık çıplak fareler koruyun. </li><li> Cerrahi yüzeyler% 2 klorheksidin çözeltisi püskürtüldü ve daha sonra emici perdeler ile örtülmüştür. Steril cerrahi eldiven işlem sırasında giyilirdi. Hayvan Ameliyattan önce, ip anesthetiz için Avertin (250 mg / kg) enjektefareler e. Seçenek olarak ise, ketamin / ksilazin / asepromazin 50/5/1 mg / kg &#39;lik bir konsantrasyonda kullanılabilir. Oftalmik merhem anestezi sırasında yeterli nemi korumak için fareye uygulanmıştır. </li><li> Steril bir bisturi bıçağını kullanarak, yaklaşık 1 cm&#39;lik bir uzunluğa da alt orta hat boyunca deri ve periton içinden küçük bir insizyon keserek ameliyat başlayın. Bir steril cerrahi örtü hayatta kalma ameliyatları sırasında insizyon etrafında kaplıydı. </li><li> Yavaşça exteriorize ve prostat maruz prostat dorsal lobları stabilize. Bir 30 gauge iğne ile prostatın içine PBS (20 ul) in-GFP PC3 hücrelerinin süspansiyon enjekte edilir. Nihayet 12 AutoClip yara ile kesi kapatın. </li><li> Post-prosedür izlenmesi için, 1 hafta boyunca günde iki kez hayvanları izlemek. Hastalığın belirtileri yeme eksikliği, üretra çevresindeki koyu idrar toplama ve zımba içinde mesane enfeksiyonu veya tıkanıklığına işaret olabilir kararsız yürüyüş içerir. </li><li> 10 gün sonra, t kaldırmako zımba zımba sökücü kullanıyor. Tümör büyüklüğünü ve bu kilo kaybı, davranış sapmaları, ve motor kusurlar gibi ağrı herhangi bir kanıt değerlendirmek için günlük hayvan izleyin. Tümör büyüme sırasında ağrı etik izin verilen sınırı veya delil aşan bir tümör büyüklüğünü gösterdi hayvanları Euthanize. </li></ol> 4. Floresan Görüntüleme ve Histoloji ile peptidlerin tümör bağlanma özelliğinin teyidi <ol><li> Tümör hücrelerinin aşılanmasından sonra tümör büyümesinin yaklaşık 4 hafta görüntüleme ile başlamadan önce izin verir. Önceki peptid sondaların enjeksiyon, tümör varlığını doğrulamak için bir floresan kamera üzerinde canlı farelerde ile GFP floresan görüntü kazanır. </li><li> IV Texas Red 10 nmol / fare &#39;lik bir dozda taşıyan farelerin tümör etiketli peptidleri enjekte edilir. </li><li> Servikal dislokasyon ile daha sonra fareler 2 saat Kurban, tümör ve önemli organları toplamak ve bir floresan kamera üzerinde hemen görüntü. GFP (uyarma için yeşil ışık filtreleri kullanın: 444-490 nm; emisyon: 515 nm uzun-pass filtre, satın alma ayarları: 500-720 10 nm adım) ve Texas Red (uyarma için kırmızı ışık filtreleri: 576-621 nm; emisyon: 635 nm uzun geçiş filtresi; edinimi ayarları: 630-800 10 nm adım). GFP ve Texas Red için 150 msn 10 msn maruz kalma süresi. </li><li> Hemen bütün doku floresan ölçümünden sonra, tümör dokuları toplamak formalin ile düzeltmek ve cryosection 5 mikron dilimler halinde. </li><li> PBS ile yıkandıktan sonra, hemen konfokal lazer tarama mikroskobu 4 içeren bir montaj orta 1 damla &#39;,6-diamidino-2-fenilindol (DAPI), görüntü ile monte edilir. </li></ol> 5. Manyetik Rezonans Görüntüleme (MRI) <ol><li> Yaklaşık 4 hafta, tümör hücresi aşılamasından sonra tümörler çapı 0,3-0,6 cm kadar büyümeye olanak sağlar. MRG çalışma için bir hacim radyo frekansı (RF) bobini ile 7 T MR tarayıcı kullanın. </li><li> Izofluran indüksiyon odası içinde bir% 2 izofluran-oksijen karışımı ile fare anestezisi. Ophthalmic merhem anestezi sırasında yeterli nemi korumak için fare uygulanmıştır. </li><li> Heparinize tuzlu su ile dolu bir 1.0 m uzunluğunda bir boru içine bir 30 gog iğne takın. Fare kuyruk damarı içinde kendi kendine yapılan kateter yerleştirin. </li><li> Mıknatıs içine fare koyun ve bir burun konisi üzerinden% 1.5 izofluran-oksijen inhalasyon anestezi altında tutmak. </li><li> Hızı ve solunum derinliği izlemek için karın üzerinde bir izleme sistemine bağlı bir solunum sensörü yerleştirin. Bir geri besleme kontrol sistemi vasıtasıyla mıknatıs içine sıcak havanın üflenmesi ile 37 ° C &#39;de vücut ısısını korumak. </li><li> Tümör konumunu (TR / TE = 200/3.7 msn, FOV = tanımlamak için bir lokalizasyon dizisi kullanarak sagital kesit görüntüleri ile Başlayan 3.0 cm, kesit kalınlığı = 2.2 mm, dilim sayısı = 16, ortalama = 2, çevirme açısı = 45 °, matriks = 128 x 128). </li><li> CE-MR (TR / TE = 151.2/1.9 msn, FOV = 3.0 cm x 3.0 cm, 2D eksenel görüntüler elde etmek için bir 2D T1-ağırlıklı gradient yağ baskılama sırasını kullanabilirsiniz sbitleri kalınlığı = 1.2 mm, dilim sayısı = 12, ortalama = 1 flip açısı = 80 °, matriks = 128 x 128). </li><li> Enjeksiyon öncesi taban çizgisi MR Görüntü alındıktan sonra, 200 ul serum fizyolojik ile yıkanarak 0.03 mmol Gd / kg &#39;lik bir dozda hedef madde veya kontrol maddesi enjekte başlar. </li><li> Kadar 30 dakika boyunca farklı zaman noktalarında CE-MRI görüntüleri elde etmek için devam edin. </li></ol> 6. Görüntü İşleme ve Analizi <ol><li> Görüntü analizi için görüntüleme yazılımı kullanın. </li><li> Iki boyutlu görüntüleme düzlemde bütün tümör üzerinde faiz (ROI) ve bölgeleri ve böbrekler çizin ve ortalama sinyal yoğunluğunu ölçmek. </li><li> (S 0 - ΔSNR = (S t / σ t): CE-MRG verileri ölçmek için, aşağıdaki denklem kullanılarak kontrast öncesi SNR aşırı kontrast sonrası SNR&#39;de artışın hesaplanmasında ile tümör veya böbrek kontrast (ΔSNR) ölçmek / σ 0), S 0 ve S t varolmadığı iddiası sinyalini göstermek neredeya da ya da böbrek önce ve kontrastı ve σ 0 ve σ t sonra kontrast önce ve sonra arka plan havadan ölçülen gürültü standart sapmadır. </li><li> P &lt;0.05 istatistiksel anlamlılık varsayarak, öğrencinin iki yönlü t-testi kullanılarak p değerlerini hesaplayın. </li></ol>

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+evaluation+of+a+peptide+targeted+small+molecular+Gd-DOTA+monoamide+conjugate+for+MR+molecular+imaging+of+prostate+cancer.">Synthesis and evaluation of a peptide targeted small molecular Gd-DOTA monoamide conjugate for MR molecular imaging of prostate cancer.</a> Bioconjugate Chem. 23, 1548-1556 (2012).</li><li>Burden-Gulley, S. M., Gates, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+molecular+diagnostic+of+glioblastomas:+detection+of+an+extracellular+fragment+of+protein+tyrosine+phosphatase+mu.">A novel molecular diagnostic of glioblastomas: detection of an extracellular fragment of protein tyrosine phosphatase mu.</a> Neoplasia. 12, 305-316 (2010).</li><li>McBride, J. 	. D., Birgit, H., Leatherbarrow, R. 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Çıkar çatışması ilan etti.

Kritik Adımlar Uygun Biyomarker ve küçük peptid Hedefleme Seçimi Başarılı küçük boyutu ile bir hedef kontrast madde geliştirmek için, iki önemli noktaları dikkate alınması gerekir. İlk olarak, normal dokularda çok az varlığı ile hastalıklı dokuda bol miktarda mevcut olan, uygun moleküler belirteçler seçmek önemlidir. Bizim seçilmiş kanserle ilgili belirteçler, pıhtılaşmış plazma proteinleri, bu gereksinimi karşı...

Kanseri ile ilgili moleküler hedeflerin etkili görüntüleme erken kanser teşhis ve tanıyı doğruluğunu artırmak için büyük önem taşıyor. Manyetik rezonans görüntüleme (MRG) yüksek uzaysal çözünürlük ve hiçbir iyonizasyon radyasyon 1 ile güçlü bir klinik görüntüleme yöntemidir. Ancak, hedeflenen kontrast ajan klinik MR kanser moleküler görüntüleme için kullanılabilir. Hedeflenen MRG kontrast maddelerin Yenilikçi tasarımı ve gelişimi büyük ölçüde MR kanser moleküler görüntüleme uygulamasını ilerlemek olacaktır. Önemli çabalar, kanser hücrelerinin yüzeyi üzerinde ifade edilen biyolojik MR görüntüleme için hedeflenmiş kontrast maddelerin geliştirilmesi için girişimlerde bulunulmuştur. Nedeniyle MRG ve bu biyolojik düşük konsantrasyonda nispeten düşük duyarlılığı, küçük moleküler hedefli kontrast maddeleri 2,3 kullanarak etkili MR moleküler görüntüleme için yeterli kontrast oluşturmak için bir meydan okumadır. Yeterli donanım elde edilmesi amacıyla, çeşitli iletim sistemleri sucparamanyetik Gd (III) şelatları yüksek bir yük ile, lipozomlar, nano partiküller ve polimer birleşiklerinin olarak h, hedef merkezler 4,5 kontrast maddelerin lokal konsantrasyonunu artırmak için hazırlanmıştır. Bu iletim sistemleri, hayvan modellerinde önemli tümör geliştirme elde etmek mümkün olmasına rağmen, bunların büyük boyutlarda ciddi bir güvenlik kaygıları 6 neden olabilir toksik Gd (III) iyonları, uzun süreli birikimi ile sonuçlanan, vücuttan yavaş ve eksik ortadan kaldırılması ile sonuçlanmıştır. Son zamanlarda, bazı çalışmalar, moleküler görüntüleme için MR sınırlamaları lezyonlarında yüksek lokal bir ifade ile, uygun belirteçler molekül seçilerek ve kolayca 7,8 atılır olabilir küçük moleküler maddeler kullanılarak aşılabilir göstermiştir. Bu ajanların en önemli özelliği normal dokularda çok az varlığı ile hastalıklı dokuda bol miktarda moleküler işaretçileri hedef olmasıdır. Kontrast maddelerin yüksek bir konsantrasyonu ile sonuçlanır yeterliyken, bu hedeflere bağlanabiliretkili MR moleküler görüntüleme için cient kontrast. Boyutları renal filtrasyon eşik değerinden daha küçük olduğu için, bağlanmamış kontrast maddeleri hali hazırda düşük arka plan gürültüsü ile vücuttan atılır. Biz bol tümör stroma var evrensel kanserle ilgili biyogöstergesini, pıhtılaşmış plazma proteinleri, seçilmiş ve normal dokularda 9 nadiren mevcuttur var. Biz güçlü PC3 prostat tümörü modeli 10 bağlanabilen spesifik ve dört Gd-DOTA monoamid çelatlar gösteren küçük bir hedef peptit CGLIIQKNEC (CLT1), ihtiva eden bir hedef kontrast ajanı sentezlendi. Burada, farelerde tümörleri tespit etmek için MR Kanser, moleküler görüntüleme için bir yöntem sağlar.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>Fmoc protected amino acids</td>
			<td>EMD Chemicals Inc</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>DOTA-tris(t-Bu)</td>
			<td>TCI America</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>PyBOP, HOBt, HBTU</td>
			<td>Nova Biochem</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>DIPEA, Thallium(III) trifluoroacetate, TIS</td>
			<td>Sigma-Aldrich Corp.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Texas Red, succinimidyl ester, single isomer</td>
			<td>Invitrogen</td>
			<td>T20175</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENTS</td>
		 </tr>
		 <tr>
			<td>Agilent 1100 HPLC system</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>ZORBAX 300SB-C18 PrepHT column</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>ICP-OES Optima 3100XL</td>
			<td>Perkin-Elmer</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>MALDI-TOF mass spectrometer</td>
			<td>Bruker</td>
			<td>AutoflexTM Speed</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Maestro FLEX In Vivo Imaging System</td>
			<td>Cambridge Research &amp; Instrumentation, Inc.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Biospec 7T MRI scanner</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

mr molecular imaging of prostate cancer with a small molecular clt1 peptide targeted contrast agent

Tümör hücre dışı matris Kanser, moleküler görüntüleme için biyolojik olarak kullanılabilir kanseri ile ilgili proteinlerin bol miktarda vardır. Bu çalışmada, tümör stromasında pıhtılaşmış plazma proteinlerine özel bir hedef MRI kontrast madde olarak Gd-DOTA monoamid kompleksi hedefleyen küçük bir molekül peptit ile etkili MR kanser moleküler görüntüleme gösterdi. Biz bir fare orthotopic PC-3 prostat kanseri modelinde MRG ile prostat tümör non-invaziv tespiti için ajanın etkinliğini değerlendirmek deney gerçekleştirdi. Hedeflenen kontrast maddesi Gd 0.03 mmol / kg &#39;lik düşük bir dozda anlamlı tümör kontrast üretmek için etkilidir. MRI kontrast ajanı hedeflenen peptit prostat tümörünün MR moleküler görüntüleme için umut vericidir.

Şekil 1, bu hedef kontrast madde CLT1-dL-(Gd-DOTA) 4 ve deney genel düzeni sentezini tasvir etmektedir. Klinik Gd-DOTA (Tablo 1) &#39;den CLT1-dL-(Gd-DOTA) Şekil 4, çok daha yüksek relaksivitesi. 1.5 T de, PBS içinde CLT1-dL-(Gd-DOTA) 4 (pH 7.4) gadolinyum başına T 1 relaksivitesi Gd-DOTA 10 daha yaklaşık 3 kat daha fazladır. Spesifik olmayan (sCLT1-TR) (Şekil 2) bağlama çok az bir tümör göstermektedir peptidi karı...

Watch this Scientific Journal Video about MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent at JoVE.com

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Fare prostat kanseri modelinde tümör stroma içinde pıhtılaşmış plazma proteinlerine özel bir MRT-kontrast maddesi hedefleyen küçük peptid ile MR kanser moleküler görüntüleme göstermek için.

Küçük Moleküler CLT1 Peptide Hedefli Kontrast Agent ile Prostat Kanseri MR Moleküler Görüntüleme

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

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Research

JoVE Journal

Medicine

11.1K Görüntüleme.  Case Western Reserve University.  Fare prostat kanseri modelinde tümör stroma içinde pıhtılaşmış plazma proteinlerine özel bir MRT-kontrast maddesi hedefleyen küçük peptid ile MR kanser moleküler görüntüleme göstermek için. 

Video: MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

İnflamatuar Plak Kızılötesi Floresans (NIRF) İntravasküler Moleküler Görüntüleme, Multimodal Yaklaşım civarında in vivo Ateroskleroz Görüntüleme

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

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Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

Kontrast Geliştirilmiş Gemi Görüntüleme MicroCT kullanarak

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Intravital Görüntüleme Gerçek Zamanlı tümörler Nanopartikül Alımı Değerlendirme

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Nakledilen Muscle Progenitör Hücreler Hedefleyecek Moleküler Görüntüleme

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

Septik Artrit Boyuna değerlendirilmesi için MR Hedefli Kontrast Ajan kullanarak in vivo Makrofaj Görüntüleme

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

İnsan Prostat Kanser metastaz Ortotopik fare modeli

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

İnsan Prostat Kanseri Örneklerinden Kanser Kök Hücreleri İzolasyon

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Ortotopik Pankreas Kanseri Fare Modeli Dinamik Kontrast Geliştirilmiş Manyetik Rezonans Görüntüleme

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Prostat Kanseri Hasta Üretimi Tümör Hücreleri Sirkülasyon gelen Xenograftlarında Modeller Türetilmiş

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

İlaç Tedavileri tümörün mikro incelenmesi ve Hedeflenen A Beyin Tümörü / Organotipik Dilim Co-kültür Sistemi

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...