Yazarlar moleküler Sitoloji tesis ve hayvan tesisin Memorial Sloan Kettering Kanser Merkezi tarafından sağlanan teknik hizmetleri kabul etmiş oluyorsunuz. Bu eser NIH hibe CA157686 (R.J. Wong) ve P30 CA008748 tarafından desteklenmiştir (Memorial Sloan Kettering Kanser Merkezi destek hibe).

Tüm yordamları hayvan konuları ile kurumsal hayvan bakım ve kullanım Komitesi Memorial Sloan Kettering Kanser Merkezi tarafından kabul edildi.
1. kanser hücrelerinin hazırlanması
<ol><li>Hasat alt Konfluent Panc02-H7 hücrelerle % 0.25 tripsin 37 ° C'de 5 min için 15 mL santrifüj tüpü hücrelerde toplamak. Not: Hücreleri yaklaşık 12 x %10 80 confluency ve 4 mL tripsin şişesi başına6 hücre içeren T-225 şişesi yetiştirilen / şişesi kullanılır.</li>
	<li>Senaryo Özeti 5 dk. hücre resuspending tarafından hücreleri (yukarı ve aşağı iki kez pipetting tarafından) 1 mL PBS cips ve süspansiyon 1.5 mL microcentrifuge tüp buz üzerinde aktarım yıkama için 4 ° C'de 900 g x santrifüj kapasitesi.</li>
	<li>Tekrar hücreleri 5 min için 4 ° C'de 900 g x santrifüj kapasitesi ve süpernatant Pelet bozmadan atın. Buzda pelleted hücreler enjeksiyon kadar tutmak.</li>
</ol>2. fare ve cerrahi hazırlanması
Not: Bu çalışmada 8-hafta-yaşlı, erkek ve dişi C57BL/6J fareler kullanılır. Ameliyat koşulları kuruluşumuzun IACUC kuralları izler. Aletleri sterilize, cerrahi çalışma yüzeyi dezenfekte, hayvan dezenfekte ve cerrah steril eldiven giyer.
<ol><li>Ameliyattan önce gününde % 2 isoflurane kullanarak fare anestezi ve dorsal tarafta femur uzunluğu boyunca kürk ince bir jilet ya da kimyasal saç kaldırma aracı ile kaldır.</li>
	<li>Ameliyat günü, %2 isofluorane bir indüksiyon odasında kullanarak fare anestezi. Anesthetization bir ayak çimdik uyarıcı ve yanıt alınamaması onaylayın.</li>
	<li>Veteriner merhem göz kuruluğu anestezi altında önlemek için uygulayın.</li>
	<li>Ventral yan imzalat fareyi getirin ve yavaşça her bacak bacak enjekte edilir hafif gerginlik yaratmak için hipoalerjenik bant ile güvenli. Anestezi hassas Buharlaştırıcı ve burun konisi yolu ile teslim isoflurane kullanılarak korunur.</li>
	<li>Enjeksiyon yeri ile Betadine, sonra tekrar % 70 alkol ile temizleyin. Bu işlemi iki kez daha tekrarlayın. Hiçbir gevşek saç cerrahi sahada kalır emin olun.</li>
	<li>Küçük makas yaklaşık 2 mm aşağıda ve uyluk kemiği paralel ile 1 cm kesi yapmak. Cilt yanal kas altında ortaya çıkarmak için Forseps ile geri çek.</li>
	<li>Siyatik sinir derin gluteus maximus ve pazı kemiği kasları çalıştırır. Bu iki kaslar Fasial uçağa boyunca küçük makasla ayırmak ve siyatik sinir altında bulaşmasına neden. Künt diseksiyon kullanarak çevresindeki kas sinir ücretsiz.</li>
	<li>Kanser hücrelerinin (yaklaşık 50.000 hücreleri) 3 μL Pelet 10 μL enjektör çizin. Not: Alternatif olarak, bir denetim olarak PBS 3 μL çizin.</li>
	<li>Destek için enjeksiyon noktasında sinir altında küçük bir metal spatula yerleştirin. Diseksiyon mikroskop ile görselleştirme altında sinir sinir üzerine ekleme mümkün olduğunca paralel olarak iğne tutarak metal üssüne iğne takın. Sinir arkadan ponksiyon vermemek dikkatli olun. Sinir bu süreci boyunca mümkün olduğu kadar kullanımı en aza indirmek.</li>
	<li>Yavaş yavaş sinir üzerinde 5 enjekte s. Bir ampul enjeksiyon alanında bir oluşumu iyi bir enjeksiyon gösterir. Sonra yerde 3 için iğneyi bırak iğneyi yavaşça kaldırmadan önce s. İğne 3 için yerinde tutmak s geri dışarı sinir hücrelerinin en aza indirir. Not: Enjeksiyon distal sinir veya spinal kord doğru gerçekleştirilebilir. Deneyler bir dizi içinde tutarlı kalması önemlidir. Hücreleri dışında dökmek, hayvan deney analize dahil edilmemesini. Deneyim ile bu olaylar çok nadirdir.</li>
	<li>Sinir özgün konumuna geri dönün. Üstteki kaslı sinir kapsar. Fareler uygun analjezi ile tedavi ve Cilt 5-0 naylon sütür ile kapatın.</li>
	<li>Tam anesteziden uyanır kadar fare kurtarma sırasında gözlem için temiz bir kafeste tek başına koyun. Not: Tam kendine gelmek hayvan için 5-15 dakika sürer. Sternal recumbency korumak için yeterli kendine geldi kadar hayvan katılımsız sol değil. Hayvan tamamen iyileşti kadar diğer hayvanlar şirkete döndürülür. Bundan sonra kurtarma en az bir kez her 24 saat 72 h için değerlendirin.</li>
</ol>3. siyatik sinir çıkarma
<ol><li>Ameliyat günü #7, CO2fareler ötenazi. Fare ventral onun yanına koyun ve iğne kullanarak distal ekstremitelerde stabilize.</li>
	<li>Enjekte bacak ve gövde dorsal tarafta cilt kaldırın.</li>
	<li>Künt diseksiyon kullanarak, siyatik sinir kas için derin ortaya çıkarmak.</li>
	<li>Sinir kursları Ilium ve sakrum arasında. Sinir omurilik bölge erişimi için iki kemik ayırın. Siyatik sinir olduğu dar alanda kapalı makas yerleştir ve makas fareyi basılı tutarak açın. Hassas ve diseksiyonu sırasında siyatik sinir bütünlüğünü korumak.</li>
	<li>Siyatik sinir klemple sonuna femur ve proksimale omurilik dikkatle incelemek.</li>
	<li>Not: Son derece kırılgan ve gerilim altında bilgileri veya güçlü işleme eğilimli baskına sinirler vardır.</li>
	<li>Sinir distal sonuna keserek hasat. Bitişik doku boşaltma sırasında sinir çýkýntýdan. Sonunda proksimal, omurga üzerinden onun çıkış için mümkün olduğunca yakın sinir kesti.</li>
	<li>Kayıt sonra bir Vernier Kaliper kullanarak işgalinin brüt uzunluğu. Bu makroskopik tahmin sadece göstergesidir.</li>
</ol>4. işlem ve miktar cesareti
<ol><li>Disseke sinir Ekim bileşik gömün. Yapmak emin sinirler boyuna yerleştirmek ve mümkün olduğunca kalıp alt gibi dümdüz.</li>
	<li>Kasette mektup P ve ö. işaretleyerek sinir proksimal ve distal tarafı belirtmek</li>
	<li>Hemen kesitli değil, gömülü sinirler kuru buz, üstüne koyun. Örnek-80 ° C'de birkaç hafta için korunmuş.</li>
	<li>Cryostat microtome cam slaytlara 5 mikron kalınlık ve yer bölümleri de kullanarak bölümü örnekleri. Mümkünse, slayt 2 sinir bölüm uygun. Proksimal sinir tarafında gösterir.</li>
	<li>H & E12boyama kullanarak slaytları leke.</li>
	<li>Dijital olarak lekeli sinir bölümleri ile yüksek çözünürlüklü dijital verileri sağlayan bir slayt inceden inceden inceye gözden geçirmek.</li>
	<li>Görüntüleme yazılımı kullanarak ölçmek işgalinin uzunluk ölçü mesafe düğmesini tıklatarak, işgal, ya da diğer parametreleri istenen. İstila uzunluğunu iyi bir tahmin için birden çok bölüm (2-4) aynı sinir kullanın.</li>
</ol>

<ol>
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Yazarlar ifşa gerek yok.

Bu protokol için pankreas kanseri hücreleri tarafından siyatik sinir işgalinin miktar sağlar perineural işgalinin bir vivo içinde fare modeli açıklar. Bu model sinir işgalinin moleküler mekanizmaları çalışma sağlar. Bu tekniği kullanarak başarılı deneyler üç kritik adımı sürecinde dikkatli bir yaklaşım gerektirir: 1) enjeksiyon (Adım 2.7, 2.8), kanser hücrelerinin baskına sinirler (Adım 3.4) 2) çıkarımı ve 3) hasat sinirler (Adım 4.1) işlenmesi.
En...

Sinirleri uyarır kanser büyüme ve geçiş1,2,3belirli tümör microenvironment oluştururlar. Perineural işgali (PNI) içinde ve çevresinde sinirler hangi kanser hücreleri istila işlemidir. Sinirler boyunca kökenli siteleri uzak kanseri istila genişletir beri metastaz benzersiz bir yol olarak düşünülebilir. PNI % 22'i % 1001,2' ye kadar değişen bir insidans ile pankreas, prostat, baş ve boyun, tükürük, servikal ve kolorektal kanserler gibi çeşitli kanser türleri bulunur. PNI ağrı ile ilişkili olan ve kötü prognoz ve kötü hayatta kalma oranları1,2ile karşılıklı olarak ilişkilendirir.
Modelleri perineural işgalinin geliştirmeye, hücresel ve moleküler mekanizmaları bu sürecin aydınlatmak için ve aday terapötik ajanlar PNI azaltmak için test etmek esastır. Kanser hücrelerinin sinir explants4, dorsal kök ganglions5,6,7veya belirli hücreleri ile ortak kültür vitro kanserleri ve sinirler arasındaki etkileşimler eğitim yöntemleri şunlardır sinir7microenvironment gibi Schwann hücreleri. Vivo yaklaşımlar, ancak, daha fizyolojik ilgili, hangi kanser indüklenen nakledilen veya kanser fare modelleri kullanımını içerir ve tüm sinir microenvironment için muhasebe avantajına sahip. Orthotopic pankreas veya Prostat Kanser, PNI modelleri bildirilen8,9,10 olmuştur ve PNI insidansı kaydedilebilir, ancak bu organlar sinirler küçük boyutu nedeniyle, görmek zordur Tüm sinir ve bu nedenle PNI kapsamını ölçmek için. Biz burada tarif hangi kanser hücreleri-basit bir cerrahi prosedür11farelerin siyatik sinir içine enjekte edilir PNI vivo içinde modelin modelidir. Heterotopik transplantasyon sinir omurilik doğru içinde işgal etti. Sinir işgali sitesinden enjeksiyon spinal kord uzunluğu, yanı sıra sinir içinde kanser hacmi ölçülecek. Önemlisi, baskına sinir deneyleri mikroskobik ve moleküler analizleri de dahil olmak üzere çeşitli için toplanabilir. Kanser hücrelerinin çeşitli test ve genetik olarak değiştirilmiş veya belirli bileşikler ile tedavi ana bilgisayar fare de kullanılabilir olur. Bu güçlü tahlil kanser hücreleri ve ana bilgisayar microenvironment için PNI mekanizmaları içine soruşturma için değişiklik yapılmasına izin verir.

<table border="0" cellpadding="0" cellspacing="0" style="width:973px;" width="973">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:340px;">Mouse</td>
			<td style="width:123px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;">Number and age variable depending on experimental needs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell culture media (PBS, Trypsin, and DMEM+10% FBS)</td>
			<td>Any</td>
			<td></td>
			<td>Steps 1.1, 1.2, 1.3.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Conical centrifuge tube, 50 mL</td>
			<td>Falcon</td>
			<td>352098</td>
			<td>Step 1.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microcentrifuge tube 1.5 mL</td>
			<td>Axygen</td>
			<td>MCT-150-C-S</td>
			<td>Step 1.2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electric razor</td>
			<td>WAHL</td>
			<td>9962</td>
			<td>Step 2.1. Can be substituted with commercial hair removal agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane, 250 mL</td>
			<td>Baxter</td>
			<td>1001936060</td>
			<td>Step 2.2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hypoallergenic surgical tape</td>
			<td>3M Blenderm</td>
			<td>70200419342</td>
			<td>Step 2.3</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Betadine Swapsticks</td>
			<td>PDI</td>
			<td>SKU 41350</td>
			<td>Step 2.4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Webcol Alcohol Preps</td>
			<td>Covidien</td>
			<td>5110</td>
			<td>Step 2.4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sterile surgical tools (scissors and forceps)</td>
			<td></td>
			<td></td>
			<td>Steps 2.4, 2.5, 3.3, 3.4, 3.5</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">10 &#956;L Hamilton syringe</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td>Steps 2.7, 2.8</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Steel Micro spatula</td>
			<td>Fisher Scientific</td>
			<td>S50823</td>
			<td>Step 2.7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td></td>
			<td></td>
			<td>Step 2.7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bupivacine, 1 g</td>
			<td>Enzo Life Sciences</td>
			<td>BML-NA139-0001</td>
			<td>Step 2.9. Reconstitute to 0.5%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">5-0 Nylon suture</td>
			<td>Ethicon</td>
			<td>698H</td>
			<td>Step 2.9</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue-Tek O.C.T. Compound</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Step 4.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue-Tek Cryomold Molds</td>
			<td>VWR</td>
			<td>25608-916</td>
			<td>Step 4.1</td>
		</tr>
	</tbody>
</table>

an in vivo murine sciatic nerve model of perineural invasion

Kanser hücrelerinin hangi kanser hücreleri çoğalırlar ve sinir microenvironment göç perineural işgali (PNI) olarak adlandırılan bir süreç sinirleri istila. İstila bu tür kanser türleri çeşitli tarafından sergilenen ve çok sık pankreas kanseri bulundu. Sinir lifleri fare pankreas içinde mikroskobik boyutunu PNI çalışmanın orthotopic fare modellerinde zor işler. Burada, nerede biz syngeneic pankreas kanseri hücre kültürünü Panc02-H7 fare siyatik sinir enjekte PNI, heterotopik vivo içinde modeli açıklar. Bu modelde, siyatik sinir imzalat farelerin maruz ve kanseri hücreleri enjekte. Kanser hücrelerinin sinirler omurilik enjeksiyon noktasından proksimale doğru istila. Baskına siyatik sinirleri sonra ayıklanır ve donmuş kesit için OCT ile işlenebilir. H & E ve bu bölümleri ayirt boyama işgali ve değişiklikleri her iki derece miktar protein ifadede izin verir. Bu model çok yönlü verilen PNI üzerinde çalışmalar çeşitli uygulanabilir. Farklı genetik değişiklikler ve/veya kanser hücrelerinin farklı türleri ile fare kullanarak PNI hücresel ve moleküler mekanizmaları incelenmesi ve farklı kanser türleri sağlar. Ayrıca, tedavi edici ajanların etkileri sinir işgali üzerinde bu farelerde tedavi uygulayarak okudu.

Bu yöntem pankreas kanseri hücreleri cerrahi implantasyon ölçülebilir sinir işgalinin vivo içinde model oluşturmak için fare siyatik sinir içine açıklar. Siyatik sinir anatomik konumunu ve Enjeksiyon Makinası şekil 1 gösterir. Şekil 2 bir çıplak fare iki siyatik sinirleri gösterir. Damarına enjekte PBS (solda) MiaPaCa-2 kanser hücreleri (sağda) enjekte bir sinir ile karşılaştırıldığında. Ka...

Watch this Scientific Journal Video about An In Vivo Murine Sciatic Nerve Model of Perineural Invasion at JoVE.com

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Vivo fare siyatik sinir modeli Perineural işgali

Biz bir vivo içinde fare modeli perineural işgalinin siyatik sinir syngeneic pankreas kanseri hücreleri enjekte edilerek tanımlamak. Model ölçüde sinir işgalinin miktar için sağlar ve araştırma perineural işgalinin hücresel ve moleküler mekanizmaları destekler.

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

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Research

JoVE Journal

Cancer Research

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

11.9K Görüntüleme.  Memorial Sloan Kettering Cancer Center. Biz bir vivo içinde fare modeli perineural işgalinin siyatik sinir syngeneic pankreas kanseri hücreleri enjekte edilerek tanımlamak. Model ölçüde sinir işgalinin miktar için sağlar ve araştırma perineural işgalinin hücresel ve moleküler mekanizmaları destekler.

Video: An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Coculture Tahliller Glioblastoma Invasion Makrofaj ve Mikroglia Stimülasyon Eğitim için

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Kanser Araştırmaları

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Tümör metastaz üzerinde Hipoksi Test Etkisinin Vivo Modeli

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Baş ve Boyun Skuamöz Hücre Karsinomu içinde perinöral invazyon İçin Bir Model

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Periferik Radyasyonla Bağlı Yorgunluk A Fare Modeli

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Yukarı doğru hareket: Bir basit ve esnek Vitro üç boyutlu işgali tahlil iletişim kuralı

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

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 ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Orthotopic Homogref (Syngeneic) fare birincil KPC pankreas duktal adenokarsinom, akış sitometresi tarafından Immunophenotyping

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Otomatik bir canlı hücre Imager kullanarak basit geçiş/işgali iş akışı

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

Retroperitoneal Lenfadenopati li Ortotopik Endometriyal Kanser Modeli In Vivo Propagated ve Kültürlü VX2 Hücrelerinden Yapılmıştır

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

Mutant melanosit kök hücrelerinden Murine Melanoma başlangıcı uzamsal ve temporal kontrol

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...