The authors would like to thank Drs. Helene Salmon and Miriam Merad from Icahn School of Medicine at Mount Sinai for providing the B16-F10-YFP cells and for their expert advice on melanoma mouse models. The authors further thank the Animal Imaging Core Facility, the Radiochemistry and Molecular Imaging Probes Core Facility, and the Molecular Cytology Core Facility at Memorial Sloan Kettering Cancer Center (MSK) for their support. This work was supported by National Institutes of Health grants NIH 1 R01 HL125703 (W.J.M.M.), R01CA155432 (W.J.M.M.), K25 EB016673 (T.R.) and P30 CA008748 (MSK Center Grant). The authors also thank the Center for Molecular Imaging and Nanotechnology (CMINT) at MSK for their financial support (T.R.).

Prosedür nanopartiküllerin ikili radyoaktif ve floresan etiketleme, in vivo PET-BT görüntüleme, ex vivo biyolojik dağılım ölçümleri ve ex vivo immün oluşur ve analiz akım sitometri. Tüm hayvan deneyleri Memorial Sloan Kettering Kanser Merkezi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. Çift etiketli lipozomların hazırlanması 1. Not: singenik B16 melanoma tümörleri% 2 -isoflurane içeren oksijen teneffüs anestezi altında C57BL / 6 farelerinin arka yanları halinde 300.000 B16-F10 hücreleri enjekte edilerek uyarılabilir. Ameliyat sırasında steril bir çalışma alanında steril reaktifler ve araçları kullanın. Ameliyattan sonra, dikkatlice anesteziden kendi kurtarma kadar hayvanları gözlemlemek. onlar tam bilinç ve hareketlilik yeniden sadece geri hayvanları kafeslerinde koyun. ksenogreflenmiş tümörü olan hayvanlar için steril odalarda onlara ev. boyutta tümörler300 mm 3 bağlı nanopartikül birikimini artırmak bunların belirgin gelişmiş geçirgenlik ve tutma etkileri (EPR) &#39;e nanopartikül karakterizasyonu için idealdir. Ayrıntılı protokoller aşağıda verilmiştir. <ol><li> Yuvarlak tabanlı bir şişe içinde içeren kloroform, 1,2-dipalmitoyl- sn -glisero-3-phophocholine (DPPC), kolesterol, 1,2-disearoyl- sn -glycero- 2 mL lipidlerin 20 mg karışımı çözülür 3-fosfoetanolamin-poli (etilen glikol) 2000 (DSPE-PEG2000) DSPE-deferoksamin (DSPE-DFO) 8 ve 1,1-diododecyl-3,3,3,3-tetramethylindodicarbocyanine-5,5-disülfonik asit ( diIC12 [5], sırasıyla% 61.3,% 33.4, 5,% 0.2 ve% 0.1, molar oranlarda) -ds. NOT: Bu lipit kompozisyonu, şu anda klinik 2&#39;de kullanılan doksorubisin lipozom formülasyonuna benzemektedir. Bizim prosedüründen elde edilen lipozomlar, yakın klinik nanomedic in vivo performansını taklit edecekine. </li><li> organik çözücünün çıkması ve oda sıcaklığında ince bir lipid film oluşturmak için bir döner buharlaştırıcı kullanarak. PBS ile 20 ml ilave edilir ve 30 dakika boyunca ses dalgalarına maruz buz üzerinde yeterli bir soğutma ile, bir 3.8 mm sonikasyon ucu ve 30 W arasında bir genliğe kullanarak lipozomal nano-tanecikleri oluşturmak için. </li><li> Santrifüj agrega kaldırmak ve yıkama PBS santrifüj filtrasyon (molekül ağırlıklı bir kesim (MWCO kullanarak nano-tanecikleri, 10 dakika boyunca 4000 x g&#39;de lipozom solüsyonu: ki olacak, lipozomlar bilgilerini lipidler ve çökelen maddelerin giderilmesi için 100 kDa) konsantre edin. üst bölmede kalır ve yeni bir tüpe aktarın. lipozomlar için sonraki basamaklardan önce 1 hafta kadar PBS içinde buzdolabında saklanır. </li><li> Kalite kontrolü için, dinamik ışık saçılımı (DLS) lipozomlar karakterize etmektedir. Özellikle, PBS 950 uL ile lipozomlar 50 mcL karıştırın ve ardından boyutlandırma küvet içine karışımı aktarın. analizör küvet koyun ve parçacık ölçmekboyutları ve boyut dağılımı. sırasıyla, 0.2, - 120 nm ve 0.1 - parçacık boyutları ve boyut dağılımı (polidispersite indeksi (PDI)) 90 olması beklenmektedir. </li><li> Radyoaktif, (1.5 ml bir tüp içinde, 2 saat boyunca 37 ° C &#39;de 1 mCi 89, Zr-oksalat ile 6.9 ve 7.1 arasında bir pH değerinde toplam hacim PBS lipidlerin 2 mg eşdeğer saflaştırılmış lipozomlar, karışımı: ~ 100 uL). 1.3 adıma benzer santrifüj filtrasyon (MWCO = 100 kDa), serbest, reaksiyona girmemiş 89 Zr çıkarın. steril PBS ile bakiyenin yıkayın ve istenilen hacme seyreltin. % 95 - radyokimyasal verim 80 olmalıdır. DİKKAT! Radyoaktif maddeler ile çalışılırken yüksek düzenlenir. Kurumlar belgeli ve gerekli tesisleri, personel eğitimi ve radyoaktivite kullanımı sıkı izleme sağlıyor olması gerekir. Araştırmacılar yeterli eğitimi almış ve radyoaktivite işlerken kişisel koruyucu donanım ve dozimetre halkaları / rozetleri giymek gerekir. </li><li&gt; Tipik% 98 üzerinde 10 olmalıdır boyut dışlama radyo-HPLC ile radyo etiketli lipozom radyokimyasal saflığı belirleyin. % 95 daha düşük Radyokimyasal saflık adım 1.5 yetersiz yıkama göstergesidir. </li><li> radyo-etiketlenmesi sonra, dinamik ışık saçılımı ile nanopartiküller boyutunu belirler. Büyüklüğü ve PDI 1.4 adım 10&#39;da ölçümleri ile aynı aralıkta olması beklenir. Not: Radyoaktif örnekleri akış sitometresi veya floresan mikroskobu izin verilmez ise, radyoaktif olmayan Alan kulüp Zr-oksalat adım 1.5 lipozomlar etiketlemek için kullanılabilir. Bu radyoaktif olmayan lipozomlar, radyoaktif analoglanna özdeş özelliklere sahiptir. Bu prosedürün bir tasviri, Şekil 2&#39;de sağlanmıştır. </li></ol> Çift etiketli lipozomlar Vivo PET-BT Görüntüleme ve biodistribution 2. <ol><li> W bir ikili-etiketli lipozomların, yaklaşık 100 ul enjektei ölçülü melanom farelere radyoaktivitenin yaklaşık 300 uCi - yaklaşık 10 mCi 89 Zr / kg vücut ağırlığı kendi kuyruk damarlar yoluyla (C57BL / 6 kadın, 10 16 haftalık). </li><li> yarılanma ömrü belirlemek için, 28-G insülin şırınga kullanarak% 2 izofluran içeren oksijen altında anestezi hayvanlardan kuyruk damarından kan çizin. 2 dakika, 15 dakika, 1 saat, 4 saat, 8 saat, 24 saat, ve enjeksiyondan sonra 48 saat daha önceden tartılmış tüplerde - (20 uL 10) kan toplamak. , Farkla kan tartılır, bir gama sayacı kullanılarak radyoaktivite içeriğini belirlemek ve başına enjekte edilen dozun (% ID / g) yüzdesi olarak radyoaktivite konsantrasyonunu hesaplamak. NOT: Bir anestezi sistemi, kurtarma kafesleri, ısıtma ve biyolojik tehlike kaput ile donatılmış bir odada yordamı gerçekleştirin. hayvanlar tamamen iyileşir ve kafesleri tutan kurtarma kafeslerden aktarmadan önce işlem sonrası normal bir hareketlilik kazandırmak emin olun. Ev uygulandığı hayvanlar için belirlenmiş oda tümör taşıyan hayvanlarradyoaktif maddeler. </li><li> 24 veya 48 saat sonra, enjeksiyon Küçük hayvan MicroPET-BT tarayıcısında 10 lipozomlar, görüntü fareler. karnı yatay anestezi hayvan koyun ve anestezi tutmak. Bir burun konisi ile% 2 izofluran-oksijen verin. kurumasını önlemek için taramadan önce gözlerinin oftalmik merhem sürün. </li><li> 15 dakika yaklaşık bir süresi olan bir tüm vücut PET statik tarama kayıt en az 50 milyon tesadüf olaylar gerçekleştirin. sırasıyla 350-700 keV ve 6 ns enerji ve tesadüf zamanlama aralığı ayarlayın. Daha sonra, 5 dk bilgisayarlı tomografi (BT) taraması gerçekleştirin. 80 kV ve 500 uA bir akımının bir voltaj X-ışını tüpü ayarlayın; CT taraması çerçeve 10 için yaklaşık 145 ms, 220 derecelik bir toplam 120 dönme adımları kullanır. </li><li> PET-BT taramadan sonra, hemen Karbondioksit boğulma fareler kurban ve Pinchi tarafından ölümleri onaylamakHayvanların pençeleri ng. teyit sonra, servikal çıkıkları gerçekleştirin. </li><li> Önce, sol ventrikül içine 20 ml PBS enjekte sağ atriyum, açık kesme ve fare dokuları içinde kan çıkarın ve daha sonra, tümör, karaciğer, dalak, akciğer, böbrek, kas ve diğer 16 alakalı dokuların toplar. </li><li> , Doku tartılır 8, 9 sayma gama bunların radyoaktivite içerikleri, ölçebilir ve gramı başına enjekte edilen doz (% ID / g) bir yüzdesi olarak, doku radyoaktivite birikimini hesaplayabilir. </li></ol> 3. Ex Vivo Sitometrisi ve immün Not: vücut ağırlığının her bir kg&#39;ı başına boya, 0.5 mg bir dozda kuyruk damarı yoluyla farelere melanom flüoresan lipozomların, yaklaşık 100 ul enjekte edilir. Radyoaktif örnekleri akış sitometresi ve flöresanlı mikroskop izin verilmez ise, radyoaktif olmayan lipozomlar, kullanılmalıdır. <ol><li> Aşama 2.5 gibi, fareler 24 saat sonra enjeksiyon Kurban ve soğuk PBS depolanmalıdır tümörleri, toplar. </li><li> Flow sitometri için aşağıdaki adımları 17 izleyin: <ol><li> % 0.5 BSA ve 1 (PBS tampon akış sitometrisi saflaştırıldı kolajenaz I ve II (4 U / ml), hiyaluronidaz (60 U / ml) ve DNase I (60 U / mL) içindeki bir karışımından oluşan bir enzim kokteyli elde mM EDTA). </li><li> 1.5 ml bir tüp içinde, enzim kokteyli tamponu 200 uL tümör dokusu, 100 mg karıştırılır ve ince makasla küçük parçalar için doku zar. tüp içine enzim kokteyli bir 1.3 ml ilave edilir ve 6 oyuklu plaka içeriğini aktarın. </li><li> 60 rpm&#39;de, 60 dakika boyunca 37 ° C fırın içine yerleştirilmiştir yatay çalkalayıcı üzerinde 6 oyuklu plaka çalkalayın. </li><li> tek bir hücre süspansiyonu elde etmek için akış sitometrisi tampon ile 3 kez doku Homojenat yıkayın. </li><li> Belirli CD45, CD11b, LY6C, CD11c dahil biyomarkerların antikorların bir kokteyl ile hücreleri leke,CD64, CD3, MHC II ve CD31 (1: Tüm antikorlar için 200 seyreltme, klon bilgi materyalleri elektronik tablodaki sağlanır). İlgili hücre tiplerini belirlemek ve (5) analiz yazılımı sitometri uygun bir akış ile her bir hücre popülasyonunda -ds diIC12 bunların ortalama floresan yoğunluğu (MFI) belirler. </li></ol></li><li> immün için, şu adımları izleyin: <ol><li> Optimal kesme ortamı (OCT) ile dolu bir kaset içine bir tümör yerleştirin ve kuru buz üzerinde dondurma. </li><li> Tümör dokusu 6 mikron dondurulmuş bölümleri yapmak ve histoloji cam slaytlar üzerine koyun; Bölümler doku üzerinde en iyi temsil eden bilgi tümörün en kesitlere kapsamalıdır. </li><li> Biyobelirteçlerini Leke (örneğin, endotel hücreleri için CD31) karşılık gelen antikorların (örneğin anti-CD31, klon MEC13.3) ile ilgi. paraformaldehid ile bölümleri düzeltmek; Serum sekonder antikor türlerinin kökeni eşleşen onları engellemek; inkubasyon4 ° C&#39;de 12 saat süre ile primer antikorlar ile yedi ve daha sonra 2 saat boyunca sekonder antikor ile; ve nihayet, PBS ile yıkayın ve kapak slipleri ile kaplayın. Görüntü Leica dik konfokal SP5 mikroskop 13, 16 ile boyanmış slaytlar. </li></ol></li></ol>

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The authors have no disclosure to make.

Protokol çerçevesinde kritik Adımlar: Çift etiketli lipozomların, yüksek kaliteli, uzun bir süre boyunca tutarlı sonuçlar üretmek için bir anahtardır. Free floresan boyalar ya da 89 Zr iyonları tamamen farklı hedefleme desen oluşturabilir tamamen saflaştırma adımı sırasında uzaklaştırılmalıdır. bağışıklık sistemi önemli ölçüde deney kanseri Nanomedicine performansını etkiler, buna ek olarak, bağışıklık sistemi fare modelleri kullanılması, b...

Nanotıp kanser tedavisi geliştirme 1 paradigmasını değiştiriyor. Böyle lipozom ve albümin-temelli nanotherapies 2, 3 olarak önceki kanser nanomedicines, muazzam bir klinik etkisi esinlenerek, birçok yeni formülasyonlar geçtiğimiz on yıl içinde üretildi. Ancak, bu kanser nanomedicines klinik çeviri başarı son analizler bunlardan sadece bir kaç klinik kullanım 4, 5 için onaylanmış olduğunu göstermektedir. Yeni kanser nanomedicines klinik çeviri biri büyük engel ücretsiz terapötik bileşikler 6 doğrudan yönetimi ile karşılaştırıldığında terapötik indeksi sınırlı bir gelişmedir. Klinik öncesi hayvan modellerinde sistemik dokuda nanomedicines ve hücresel seviyelerinin in vivo performans gibi, doğru bir değerlendirme olarak kimliklendirmek esastıry gelecekteki klinik çeviri için optimal terapötik endeksleri olanlar. Nanomalzemeler tüm klinik görüntüleme yöntemleri 7 arasında mükemmel hassasiyet ve tekrarlanabilirlik vardır pozitron emisyon tomografisi (PET) görüntüleme ile yaşayan hayvanlar kantitatif karakterizasyonu için radyoaktif işaretli olabilir. Örneğin, 89 nanomedicines kanser 8, 9, 10, hem de diğer hastalık modellerinde 11 fare modellerinde karakterize edilmiştir uzun sirkülasyon Zr etiketli. Buna ek olarak, nanomedicines kan yarı ömrü ve biyolojik dağılım yoğun Bireysel dokular 8 ex vivo radyoaktivite ölçümü ile değerlendirilebilir. Bu nedenle, radyo-etiketlenmesi, sistemik ve doku düzeylerinde nanomedicines nicel değerlendirilmesi yapılır. Önemli olarak, Radyoizotopund nanomedicines nedeniyle genellikle radyoaktif sinyalin sınırlı uzaysal çözünürlük tek hücre ya da hücre içi düzeyde analiz edilemez. Bu nedenle, floresan etiketleme gibi flow sitometri ve floresan mikroskobu 12 optik görüntüleme teknikleri ile nanopartiküllerin değerlendirilmesi için tamamlayıcı bir yöntem olduğunu kanıtlamaktadır. Bu amaçla, radyoizotoplar floresan etiketler ile etiketlenmiş nanopartiküller kantitatif nükleer görüntüleme ile in vivo değerlendirilebilir ve ex vivo radyoaktivite sayımı ile ve aynı zamanda geniş bir optik görüntüleme hücresel düzeyde karakterize edilebilir. Daha önce, yüksek yoğunluklu lipoprotein (HDL), 11, lipozomlar 9, 10, polimerik nano partiküllerin, antikor fragmanları ve nanoemu dahil olmak üzere çeşitli nanopartiküller, radyoaktif ya da floresan etiketleri dahil modüler prosedürler geliştirdiklsions 10, 13. Bu etiketli nanopartiküller kendi özel uygulamalar için bu nanomateryallerin optimizasyonu güdümlü farklı düzeylerde ilgili hayvan modellerinde, kantitatif karakterizasyonu için izin vermiş. Bu çalışmada amaç, en köklü nanotıp platformu 14 çift etiketli nanoparçacık oluşturmak için kapsamlı prosedürleri göstermek için bir örnek gibi- ve iyice klasik singeneik melanom B16-F10 fare modelinde 15 bunu karakterize lipozomal-nanopartiküller kullanmaktır . sonuçlardan, bu nanopartikül karakterizasyonu yaklaşım ilgili fare modellerinde diğer kanser nanomedicines değerlendirmek için adapte edilebilir inanıyoruz.

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DPPC</td>
			<td style="width:136px;">Avantilipids</td>
			<td align="right" style="width:119px;">850355</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cholesterol</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">C8667</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DSPE-PEG2000</td>
			<td style="width:136px;">Avantilipids</td>
			<td style="width:119px;">880120P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DSPE-DFO</td>
			<td style="width:136px;">Home made</td>
			<td align="right" style="width:119px;">110634</td>
			<td>Perez-Medina et al, JNM, 2014</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DiIC12[5]-DS</td>
			<td style="width:136px;">AAT Bioquest</td>
			<td align="right" style="width:119px;">22051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifugal filter</td>
			<td style="width:136px;">Vivaproducts</td>
			<td style="width:119px;">VS2061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rotary evaporator</td>
			<td style="width:136px;">Buchi</td>
			<td style="width:119px;">R-100</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Radio-HPLC</td>
			<td style="width:136px;">Shimadzu HPLC with 2 LC-10AT pumps</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">89Zr-oxalate</td>
			<td style="width:136px;">MSKCC</td>
			<td style="width:119px;">Synthesized in house</td>
			<td>TR19/9 variable beam cyclotron (Ebco Industries Inc)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micro PET-CT</td>
			<td style="width:136px;">Siemens</td>
			<td style="width:119px;">Inveon Micro-PET/CT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gamma counter</td>
			<td style="width:136px;">PerkinElmer</td>
			<td style="width:119px;">2470-0150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flow cytometry</td>
			<td style="width:136px;">BD Biosciences</td>
			<td style="width:119px;">Fortessa</td>
			<td>Any multi-parametric flow cytometry analyzers would suffice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C57BL/6 mice</td>
			<td style="width:136px;">Jackson Laboratories</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B16-YFP melanoma cells</td>
			<td style="width:136px;">Home made</td>
			<td style="width:119px;">N/A</td>
			<td>Salmon et al, Immunity, 2016</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ly6C (clone HK1.4)--APC-Cy7</td>
			<td align="right" style="width:136px;">128025</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MHCII (M5/114/152)--APC</td>
			<td align="right" style="width:136px;">107613</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD45 (30-F11)--BV510</td>
			<td align="right" style="width:136px;">103137</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD64 (X54-5/7.1)--PE-Cy7</td>
			<td align="right" style="width:136px;">139313</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD11b (M1/70)--BV605</td>
			<td align="right" style="width:136px;">101237</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD3 (17A2)--BV711</td>
			<td align="right" style="width:136px;">100241</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD31 (13.3)--PE</td>
			<td align="right" style="width:136px;">561073</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD11c (M418)--PerCP-Cy5.5</td>
			<td align="right" style="width:136px;">117327</td>
			<td style="width:119px;">BD Biosciences</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD31 (13.3) no fluorophore</td>
			<td align="right" style="width:136px;">550274</td>
			<td style="width:119px;">BD Biosciences</td>
			<td></td>
		</tr>
	</tbody>
</table>

a comprehensive procedure to evaluate the in vivo performance of cancer nanomedicines

Klinikte önceki kanser nanomedicines başarısı esinlenerek, araştırmacılar son on yıl içinde yeni formülasyonların çok sayıda yarattı. tıbbi gelişim altındadır nanomedicines çoğunluğu hayal kırıklığı yaratan sonuçlar vermiştir ise Ancak nanomedicines yalnızca az sayıda, klinik kullanım için onaylanmıştır. Yeni kanser nanomedicines başarılı klinik çeviri için bir büyük engel in vivo performansını doğru anlama olmaması. Bu makalede, pozitron emisyon tomografisi-bilgisayarlı tomografi (PET-BT), radyoaktivite ölçümü yöntemleri entegrasyonu yoluyla sistemik, doku, tek hücre ve hücre içi seviyede tümör taşıyan farelerdeki nanomedicines in vivo hareketinin karakterize edilmesi sıkı bir prosedürle , flow sitometri ve floresan mikroskobu. Bu yaklaşımı kullanarak, araştırmacılar doğru önemsizdir ilgili fare modellerinde yeni nano formülasyonlan değerlendirebilirr. Bu protokoller yüksek öteleme potansiyeli en umut verici kanser nanomedicines belirlemek ya da gelecekteki çeviri için kanser nanomedicines optimizasyonuna yardımcı becerisine sahip olabilir.

Şekil 1 prosedürün bir özetini göstermektedir. Şekil 2, 1. adımda 10&#39;da tarif edilen bir ikili-etiketli lipozomların şematik sentez prosedürü göstermektedir. Şekil aşama 2&#39;de tarif edildiği gibi 3 görüntüleyen Örnek PET BT görüntüsünde (Şekil 3a), PET görüntülemesi (Şekil 3b), kan yarı-ömür (Şekil 3C) ve rad...

Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines at JoVE.com

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Kapsamlı Usulü değerlendirin<em&gt; İn Vivo</em&gt; Kanser Nanomedicines performans

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

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Research

JoVE Journal

Cancer Research

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

8.8K Görüntüleme.  Memorial Sloan Kettering Cancer Center. The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Video: A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression. Traditional in vitro models of cancer cell invasion of endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio

This method utilizes zebrafish embryos to efficiently test the vascular invasive ability of cancer cells. Fluorescent cancer cells are injected into the precardiac sinus or yolk sac of developing embryos. Cancer cell vascular invasion and extravasation is assessed via fluorescence microscopy of the tail region 24 to 96 hr later.

Zebra balığı Kanser Hücrelerinin Vasküler İnvaziv Yetenek (Test<em&gt; Danio rerio</em&gt;)

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression.  Traditional in vitro models of cancer cell invasion of ...

Kanser Araştırmaları

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

Vivo EPR pH, pO2, Redox durumu ve fosfat konsantrasyonları ve glutatyon tümör Microenvironment olarak değerlendirilmesi

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

Sözde Hemangioblastoma tahlil çimlenme küresel kullanarak neovaskülarizasyon Vitro performansını değerlendirmek için kapsamlı bir yordam

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Prostat kanserinde Phosphoproteome araştırmak için etiket içermeyen nicel kütle spektrometresi ile birleştiğinde Phosphopeptide zenginleştirme

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

Farelerde Tümör Büyümesini Azaltmak için MikroRNA'nın Vivo İnhibisyonu

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

In vitro assay tümör çalışma-macrophage etkileşim

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Permeable Membran Destekleri ile Co-Culture kullanarak Tümör Salgılanan Parakrin Ligandların Makrofaj Aktivasyonu Üzerine Etkilerinin İnceleştirilmesi

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Tümör Hücrelerinin Lateral Kuyruk-Damar Enjeksiyonu Sonrasında Akciğer Metastazının Patolojik Analizi

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

İntrakraniyal Enjeksiyon ve Manyetik Rezonans Görüntüleme Ile Beyin Metastazlarının Modellenmesi

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

Polianhidrit IL-1α Nanopartiküllerinin In vivo Antitümör Aktivitesinin Değerlendirilmesi

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...