Bu çalışmada, DOD Meme Kanseri IDEA Ödülü kısmen desteklenen W81XWH-08-1-0583 ve NIH / NCI CA141348-01A1 (DZ) ve FAMRI klinik bilim adamı ödülü (DS). Görüntüleme altyapı U24 CA126608 ve Simmons Kanser Merkezi (P30 CA142543) ve NIH 1S10RR024757-01 tarafından kısmen desteklenen Uluslararası Küçük Hayvan Görüntüleme Araştırma Programı tarafından sağlanmaktadır.

Bütün hayvan prosedürleri Teksas Üniversitesi Güneybatı Tıp Merkezi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. 1 in vitro HIF-1α biyoparlaklık tahlil <ol><li> Gereç ve Yöntem: <ul><li> İnsan metastatik meme kanserli hücre hattı MDA-MB231, HIF-1-bağımlı bir roman muhabiri geni ile transfekte, 5HRE-ODD-luc Dr. Harada tarafından üretildi. </li><li> Hipoksik durum, gelişmiş, oksijen bağımlı bozulması etki alanı (ODD) Lusiferaz füzyon proteini ifade hipoksi-yanıt elemanı 5 kopyaya (5HRE) tarafından tahrik edilir. ODD varlığı normoksik koşullarında son derece düşük arka plan luciferase aktivitesi ile sonuçlanan ODD-Luc protein bozulmasına neden olur. Bu nedenle, bu yeni sistem, gerçek zamanlı görüntüleme ile bir tümör hipoksik bölgeleri tespit etmek için kullanılabilir. Harada ve ark 1,2 ile inşaat 5HRE-ODD-luc ifade vektör bildirilmiştir. </li></ul></li><li> Kültür normoxia veya hipoksi altında hücreleri: <ul><li> % 10 fetal sığır serumu (FBS)-DMEM orta% 1 glutamin, 400 mikrogram / ml G418 antibiyotik ve% 1 penisilin / streptomisin içeren rekombinant MDA-MB231 hücreleri koruyun. </li><li> In vitro HIF-1α biyoparlaklık deneyi, plaka 3 x 10 5HRE-ODD-luc vektör her iki altı iyi yemeğin iyi ifade 5 MDA-MB231 hücreleri. </li><li> Normoksik durum (% 21 O 2) altında diğer çanak tutmak hücreleri, gecelik inkübasyondan sonra çanak duvara takın ve daha sonra hipoksi hipoksi çalışmaları için bir odası (Billups-Rothenberg, Inc. Del Mar, CA) içine bir çanak transferi için izin verin . </li><li> % 0.1 O 2 oda ve gaz odasına bir gaz silindiri ile giriş deliği boru bağlayarak monte edin. Not: Bu işlem sırasında her ikisi de giriş ve çıkış bağlantı noktası kelepçeler açık olmalıdır. </li><li> Plastik kelepçeler kapatarak tasfiye kamara ve sızdırmazlık odaları olmuştur sonra gaz kaynağı bağlantısını kesin. </li><li> % 5 CO 2 ile 37 ° C inkübatör odasına yerleştirin. Not: odasına kültürlerin aşırı buharlaşma önlemek için nemlendirilmiş olmalıdır. - 20 ml steril su odasında 10 yerleştirerek başarılı olabilir. </li></ul></li><li> Biyoparlaklık tahlil: <ul><li> 24 saat inkübasyondan sonra, orta çıkarın ve buz PBS (2X) ile hücrelerin hızlı bir şekilde yıkayın. </li><li> 100 ul luciferase (Altın Biyoteknoloji, St Louis, MO), soğuk PBS 1 ml ekleyin. </li><li> (1, 30, 60, 180 s) farklı pozlama süreleri ile BL görüntüleme (IVIS Spektrum sistemi, kaliper Yaşam Bilimleri, Hopkinton, MA) edinin. </li><li> Her Yaşayan Image yazılımı (Kaliper Yaşam Bilimleri) kullanarak ışık yoğunluğunu ölçün. </li></ul></li></ol> 2. Bir meme kanseri beyin metastazı model oluşturulması <ol><li> MDA-MB231/5HRE-ODD-luc hücrelerin hazırlanması <ul><li> % 10 FBS,% 1 glutamin ve% 1 penisilin / streptomisin içeren Deme orta MDA-MB231/5HRE-ODD-luc hücreleri almak ve kültür. </li><li> 2-3 günde bir orta değiştirin. Tripsinize ve% 80 izdiham ulaştığınızda hücreleri yıkayın. </li><li> Uygun sayıda hücre sayımı ve serum serbest Deme orta 4 ul hacmi 10 5 hücre nihai bir konsantrasyon ile% 25 Matrigel (BD Biosciences, San Jose, CA) ile tekrar süspansiyon haline getirin. </li><li> Intrakranial enjeksiyon öncesinde buz üzerine yerleştirin hücreleri. </li></ul></li><li> Cerrahi implantasyon <ul><li> Bu çalışmada Bayan çıplak farelerin (BALB / c nu / nu, Ulusal Kanser Enstitüsü, Bethesda, MD) eski 4-6 hafta kullanılır. </li><li> Anestezisi (indüksiyon odasında O2 / izofluran% 3, Baxter International Inc, Deerfield, IL, ABD izofluran) ve oksijen (1 dm 3 / dak) izofluran (% 1), cerrahi işlem 3 sırasında hayvanları korumak . </li><li> Sağ parietal cilt betadin ve sonra insizyon öncesi% 70 alkol ile prepped olmalıdır. </li><li> Yüksek devirli kullanarak, çapak, kafatası, 1mm ön sağ yarımkürede koronal sütür ve saggital dikiş lateral 2 mm 1 mm delik. </li><li> 4 ul hücre karışımı (10 5 hücreleri) 10 ul Hamilton şırınga (Hamilton Company, Reno, NV) kullanarak çizin. Hücreleri doğrudan sağ kaudal diensefalon, ısmarlama bir 32G Hamilton iğne kullanarak duramater altında 1,5 mm içine enjekte edilir. Çekilme önce yaklaşık 30 iğne tutun. 32G ince iğne kullanımı, doku hasarı en aza indirir. </li><li> Burr hole kemik balmumu ile doldurun ve kafa derisi emilebilir dikişlerle yakın. </li><li> % 70 alkol ile kesi bölgesi hazırlayın. </li><li> Buprenorfin analjezi iki gün boyunca her 12 saat uygulayın. </li></ul></li></ol> 3, meme kanseri beyin metastazı tümör hipoksi dinamikleri in vivo biyoparlaklık görüntüleme <ul><li> 8 haftada bir kez intrakranial implantasyon ve tekrar sonra uzunlamasına biyoparlaklık görüntüleme başlatın (IVIS Spektrum sistemi) iki hafta-10 Hafta. </li><li> Bir seferde üç fareler anestezi (% 3 O 2 / indüksiyon odasında izofluran) </li><li> Daha önce 4 ayrıntılı olarak açıklandığı gibi her bir fare boyun bölgesinde subkutan D-luciferase (Altın Biyoteknoloji 120 mg / kg toplam hacminin 80 ul PBS) Yönetici bir çözüm . </li></ul> D-luciferase non-toksik ve sağlam kan-beyin bariyeri (KBB) ve hücre zarlarının 3,5 etme yeteneğine sahip olduğu gösterilmiştir . <ul><li> Görüntüleme odasında 3 fareler yerleştirin ve görüntüleme sırasında oksijen (1 dm 3 / dak) izofluran (% 1) ile korunur. </li><li> Beş dakika luciferase enjeksiyon sonra, bir dizi çeşitli pozlama süresi (1, 30, 60, 180 s) BL görüntüleme kazanır. </li></ul> Gözlemlerimiz tepe ışık yayma süresi boyun bölge 3,4 luciferase subkutan uygulamadan sonra yaklaşık 5 dakika olduğunu gösteriyor. <ul><li> Living Görüntüleme yazılımı (Kaliper Yaşam Bilimleri) ile ilgi bir bölgede mutlak foton sayısı (fotonlar / s) kullanarak verileri analiz (ROI), beyin BLI sinyal anahat elle çizilmiş. </li><li> Foton sayıları Arsa zaman içerisinde eğrisi tümör hipoksi dinamiği göstermek için. </li><li> Son BLI hemen sonra, pimonidazole, hipoksi marker ve daha sonra 1 saat yönetmek fareler kurban ve beyinleri teşrih. Optimal Kesme Sıcaklık (OCT) orta ve donma -80 ° C derin dondurucu bütün beyinleri gömün. Daha sonraki histolojik kresil violet boyama ve lucifeares, hipoksi işaretleyici pimonidazole karşı immünohistokimyasal ve HIF-1α görüntüleme gözlemlerini doğrulamak için yapılır. </li></ul> 4. Temsilcisi sonuçları: Şekil 1&#39;de gösterildiği gibi, önemli ölçüde daha yüksek BLI sinyal, 24 saat hipoksik odası (% 1 O 2) inkübe transfekte hücreler sonra gözlenmiştir. Zayıf ışık yayma normoxia altında kontrol hücreleri gözlendi. Bu, hücrelere bir stres sinyali HIF-1α eksprese uyarılan 3 x 5 10 hücreleri, 24 saat kültürden sonra kalabalık hücre popülasyonu sonucu olabilir . Bununla birlikte, in vivo BLI sonuçları immünohistokimyasal boyama ile tümör hipoksi ve lusiferaz ifade arasındaki ko gösteren onaylandı . Pozlama süreleri otomatik bir dizi uzun pozlama süresi ile zayıf bir sinyal yakalama kolaylaştırır dizi görüntüleri, doyurarak CCD olmadan kısa bir süre kullanarak güçlü sinyaller sürekli alımlar sağlar. <img alt="Şekil 1" src="/files/ftp_upload/3175/3175fig1.jpg" /> In vitro HIF-1α biyoparlaklık tahlil Şekil 1. sıra: 24 hipoksi bir odasında 6-iyi çanak (% 0.1 O 2) her bir kuyunun içinde inkübe, 3 x 10 5 MDA-MB231/5HRE-ODD-luc hücreleri saat orta önce kaldırılır yıkanmış ve 1 ml PBS ile değiştirilir. 100 ul luciferase her kuyuya eklendi hemen sonra, BLI maruz kez bir dizi (1, 30, 60, 180 s) elde edildi. Güçlü lüminesans temsilcisi kuyulardan gözlendi Alt satır: Kontrol olarak, 3 x 10 5 hücre normoxia (% 21 O 2) zayıf ışık yaydığı altında inkübe. <img alt="Şekil 2" src="/files/ftp_upload/3175/3175fig2.jpg" /> In vivo tümör hipoksi dinamikleri biyoparlaklık görüntüleme Şekil 2 A) fare beynin sağ tarafında zayıf ışık sinyali MDA-MB231/5HRE-ODD-luc meme kanseri hücrelerinin intrakranial implantasyonu ilk görüntülenmiştir 5 hafta sonra. Artan optik sinyal, ek 6 hafta içinde artan tümör hipoksi belirten gözlendi B) arsa, ışık sinyali kantitatif foton sayımı zaman ders eğrisi gösterdi . <img alt="Şekil 3" src="/files/ftp_upload/3175/3175fig3.jpg" /> Şekil 3 ve immun boyama ile kimyasal boyama tespit hipoksi lusiferaz Colocaliztion OCT gömülü bir metastaz meme kanseri MDA-MB231/5HRE-ODD-luc taşıyan bir donmuş bir farenin beyin kesitli oldu . 10 mikron bölümü, hipoksik işaretleyici, pimonidazole ile boyanmış anti-lusiferaz boyama ile tespit lusiferaz ifade ile mekansal olarak ilişkili bulunmuştur hipoksi intratümöral heterojenite saptandı. Ölçeği bar, 100 mm.

<ol>
	<li>Harada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+combination+of+hypoxia-response+enhancers+and+an+oxygen-dependent+proteolytic+motif+enables+real-time+imaging+of+absolute+HIF-1+activity+in+tumor+xenografts.">The combination of hypoxia-response enhancers and an oxygen-dependent proteolytic motif enables real-time imaging of absolute HIF-1 activity in tumor xenografts.</a> Biochem Biophys Res Commun. 360, 791-796 (2007).</li><li>Ou, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+HIF-1+imaging+for+determining+optimal+timing+of+combining+bevacizumab+and+radiotherapy.">Usefulness of HIF-1 imaging for determining optimal timing of combining bevacizumab and radiotherapy.</a> Int J Radiat Oncol Biol Phys. 75, 463-467 (2009).</li><li>Zhou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+near-infrared+optical+imaging+of+2-deoxyglucose+uptake+by+intracranial+glioma+of+athymic+mice.">Dynamic near-infrared optical imaging of 2-deoxyglucose uptake by intracranial glioma of athymic mice.</a> PLoS One. 4, e8051-e8051 (2009).</li><li>Contero, A., Richer, E., Gondim, A., Mason, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+quantitative+bioluminescence+imaging+for+assessing+tumor+burden.">High-throughput quantitative bioluminescence imaging for assessing tumor burden.</a> Methods Mol Biol. 574, 37-45 (2009).</li><li>Zhao, D., Richer, E., Antich, P. P., Mason, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antivascular+effects+of+combretastatin+A4+phosphate+in+breast+cancer+xenograft+assessed+using+dynamic+bioluminescence+imaging+and+confirmed+by+MRI.">Antivascular effects of combretastatin A4 phosphate in breast cancer xenograft assessed using dynamic bioluminescence imaging and confirmed by MRI.</a> FASEB J. 22, 2245-2451 (2008).</li><li>Chang, E. L., Lo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+management+of+central+nervous+system+metastases+from+breast+cancer.">Diagnosis and management of central nervous system metastases from breast cancer.</a> Oncologist. 8, 398-410 (2003).</li><li>Palmieri, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vorinostat+inhibits+brain+metastatic+colonization+in+a+model+of+triple-negative+breast+cancer+and+induces+DNA+double-strand+breaks.">Vorinostat inhibits brain metastatic colonization in a model of triple-negative breast cancer and induces DNA double-strand breaks.</a> Clin Cancer Res. 15, 6148-6157 (2009).</li><li>Mason, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodality+imaging+of+hypoxia+in+preclinical+settings.">Multimodality imaging of hypoxia in preclinical settings.</a> Q J Nucl Med Mol Imaging. 54, 259-280 (2010).</li><li>Tatum, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia:+importance+in+tumor+biology,+noninvasive+measurement+by+imaging,+and+value+of+its+measurement+in+the+management+of+cancer+therapy.">Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy.</a> Int J Radiat Biol. 82, 699-757 (2006).</li><li>Moeller, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+effects+of+HIF-1+blockade+on+tumor+radiosensitivity.">Pleiotropic effects of HIF-1 blockade on tumor radiosensitivity.</a> Cancer Cell. 8, 99-110 (2005).</li><li>Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dynamics+and+distinct+functions+of+hypoxia+in+primary+tumor+growth+and+organotropic+metastasis+of+breast+cancer.">In vivo dynamics and distinct functions of hypoxia in primary tumor growth and organotropic metastasis of breast cancer.</a> Cancer Res. 70, 3905-3914 (2010).</li><li>Zhao, D., Chang, C. H., Kim, J. G., Liu, H., Mason, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Near-Infrared+Spectroscopy+and+Magnetic+Resonance+Imaging+Monitoring+of+Tumor+Response+to+Combretastatin+A-4-Phosphate+Correlated+With+Therapeutic+Outcome.">In vivo Near-Infrared Spectroscopy and Magnetic Resonance Imaging Monitoring of Tumor Response to Combretastatin A-4-Phosphate Correlated With Therapeutic Outcome.</a> Int J Radiat Oncol Biol Phys. 80, 574-581 (2011).</li></ol>

Çıkar çatışması ilan etti.

Meme kanseri, beyin metastazı Evre IV meme kanseri hastalarının% 30&#39;unda ortaya çıkar. Yüksek morbidite ve mortalite ile ilişkili ve medyan sağkalım 13 ay 6 sahiptir. , Intrakranial başlangıcında ve ilerlemesinde gibi patofizyolojik profilleri anlaşılmasını kolaylaştırmak için bu klinik yıkıcı hastalığı taklit etmek için uygun hayvan modelleri için bir ihtiyaç vardır. Burada, insan meme kanseri hücreleri, fare beyninin içine doğrudan enjekte edilerek ortotopik meme kanseri ...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> D-luciferase </td><td> Altın Biyoteknoloji </td><td> L-123 </td><td> 120 in vivo çalışmada toplam hacmi 80 ul PBS mg / kg </td></tr><tr><td> İsofluranın </td><td> Baxter International Inc. </td><td> 1001936060 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354234 </td><td></td></tr><tr><td> Hamilton şırınga </td><td> Hamilton Şirket </td><td> 1701 </td><td></td></tr><tr><td> 32G Hamilton iğne </td><td> Hamilton Şirket </td><td> 7803-04 </td><td></td></tr><tr><td> Hipoksi haznesi </td><td> Billups-Rothenberg, Inc. </td><td> MIC-101 </td><td></td></tr><tr><td> Biyoparlaklık görüntüleme sistemi </td><td> Kaliper Yaşam Bilimleri </td><td> IVIS Spektrum sistemi </td><td></td></tr><tr><td> G418 </td><td> Fisher Scientific </td><td> SV3006901 </td><td></td></tr></tbody></table>

in vivo bioluminescence imaging of tumor hypoxia dynamics of breast cancer brain metastasis in a mouse model

Bu tümör hipoksi malign ilerlemesini teşvik ve terapötik yanıt olumsuz etkileyen önemli bir rol oynadığı kabul edilmektedir. Bu, derin yerleşimli ortotopik tümörlerin izlemek için verimli bir anlamı olmaması nedeniyle, kötü huylu beyin tümörleri intrakraniyal gelişimi sırasında in vivo, tümör hipoksi in situ hakkında az bilgi vardır. Lusiferaz gen ifade canlı hücreler tarafından yayılan ışık algılama dayalı Biyoparlaklık görüntüleme (BLI), preklinik hayvan çalışmalarında tedaviye yanıt olarak tümör büyüme ya da tümör boyutu değişiklikleri değerlendirmek için, hızla, özellikle, kanser araştırmaları için kabul edilmiştir. Ayrıca, bir muhabir gen promotor dizisinde kontrol altında ifade ederek, belirli bir gen ekspresyonu BLI tarafından non-invaziv takip edilebilir. Hipoksik stres altında, sinyal yanıtları ağırlıklı olarak hipoksi indüklenebilir faktör 1α (HIF-1α) aracılığıyla çeşitli genlerin transkripsiyon sürücü. Bu nedenle, stably insan meme kanseri MDA-MB231 hücreleri (MDA-MB231/5HRE-ODD-luc) in vitro HIF-1α biyoparlaklık tahlil yapılır transfekte HIF-1α muhabiri inşa 5HRE-ODD-luc, normoxia hücrelerin (% 21 O 2) bir kontrol olarak hizmet ederken, BLI 24 saat önce hipoksik odası (% 0.1 O 2) transfekte hücreler kuluçka. Hipoksi altında hücreler için gözlenen anlamlı olarak daha yüksek foton akısı, organizatörü normoxia içinde olanlara oranla bir artış HIF-1α bağlanma (HRE elemanları), göstermektedir. Hücreler bir meme kanseri beyin metastazı modeli kurmak için doğrudan fare beyninin içine enjekte edilir, in vivo tümör hipoksi dinamikleri biyoparlaklık görüntüleme implantasyon sonrası 2 hafta başlatılan ve haftada bir kez tekrarlanır . BLI artmış intrakraniyal tümör hipoksi gösteren, beyin tümör ilerledikçe artan ışık sinyalleri ortaya koymaktadır. Histolojik ve immünhistokimyasal çalışmalar in vivo görüntüleme sonuçları onaylamak için kullanılır. Burada, in vitro HIF-1α biyoparlaklık tahlil, intrakranial tümör hipoksi izlemek için in vivo biyoparlaklık görüntülemede bir çıplak fare ve uygulama bir meme kanseri beyin metastazı cerrahi kurulması yaklaşımları tanıtacak.

Watch this Scientific Journal Video about In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model at JoVE.com

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Hipoksi indüklenebilir faktör-1α aktivite Biyoparlaklık görüntüleme, bir meme kanseri beyin metastazı fare modeli intrakranial tümör hipoksi gelişimini izlemek için uygulanır.

Meme Kanseri Beyin metastaz tümör Hipoksi Dynamics in vivo Biyoparlaklık Görüntüleme Fare Modeli

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

in-vivo-bioluminescence-imaging-tumor-hypoxia-dynamics-breast-cancer

Research

JoVE Journal

Medicine

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

19.5K Görüntüleme.  University of Texas Southwestern Medical Center. Hipoksi indüklenebilir faktör-1α aktivite Biyoparlaklık görüntüleme, bir meme kanseri beyin metastazı fare modeli intrakranial tümör hipoksi gelişimini izlemek için uygulanır.

Video: In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Oral Skuamöz Hücre Karsinomu Ortotopik Fare Modeli Tümör Hücre Invasion Multi-foton Görüntüleme

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Tıp

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Bir Ortotopik Fare Beyin Tümörü Modelinde Kombinasyon Radyoterapi

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Stereotaktik intrakranial implantasyon ve<em&gt; In vivo</emGlioblastoma Multiforme bir Mouse Model Sistemde Tümör Xenografts arasında&gt; Bioluminescent Görüntüleme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Manyetik Rezonans Görüntüleme, Volumetrik Bilgisayarlı Tomografi ve Ultrason Kullanımı Meme Kanseri Kemik metastazı bir Nude Rat Model Angiogenezis Multi-modal Görüntüleme

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Meme Kanseri Fare Modelleri Tümör Mikroçevre İlaç Yanıtlarının Görüntüleme Canlı

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Birincil Preneoplastic Meme Epitel Hücrelerinin Time-lapse görüntüleme Meme Kanseri Genetik Fare Modelleri türetilen

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

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

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

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

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

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

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

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Boyuna MRG Ardından Meme Kanseri Beyin Metastaz bir Fare Modeli geliştirmek için ultrason görüntüleme eşliğinde intrakardiyak enjeksiyon

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Meme Kanseri Bir Roman Transgenik Fare Modeli: Adenovirüs-Kretasedekine ile epiteli Hedefleme ile Metastatik Meme Kanseri Başlatma

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

Floresan SapC-DOPS Nanovesicles kullanma Beyin Tümörleri ve Artrit Vivo Optik Görüntüleme

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...