This work was supported by the NIH grant (5R01 CA178386-03) and the USC ASPIRE-1 grant to HC. We would like to thank Vrushab Gowda for his assistance with manuscript.

Bu çalışma Kurumsal Gözden Chonnam Ulusal Üniversitesi Hwasun Hastanesi Yönetim Kurulu ve Güney Carolina Üniversitesi tarafından onaylanmıştır. Meme kanseri ve eşleşen bitişik normal meme doku oluşan TMA örnekleri Chonnam Ulusal Üniversitesi Hwasun Hastanesi, Kore Biobank Ağının bir üye Biobank tarafından temin edildi. 1. Solüsyon Hazırlama <ol><li> DNaz ve RNaz içermeyen su 1 L hazırlayın. diethylpyrocarbonate (DEPC) arıtılmış su kullanan tüm kimyasal çözümler olun. DEPC 500 ul su ile 1 L tedavi ve oda sıcaklığında 3 saat inkübe edilir. DEPC inaktive su Otoklav. Dikkat: karsinojen bilindiğinden DEPC teneffüs etmeyin. </li><li> DEPC arıtılmış su kullanılarak 10x PBS 1x PBS hazırlayın. </li><li> 20x SSC (NaCl 175.3 g ve 800 ml su içinde sodyum sitrat 88.2 g çözülür hazırlanması HCI 14 N çözelti bir kaç damla 7.0 pH ayarlamak ve wat 1 L ses seviyesini ayarlamakER),% 4 paraformaldehit,% 95 etanol,% 80 etanol DEPC işlenmiş su ile. </li><li> 8 trietanolamin ilave edildi ve DEPC ile muamele edilmiş su 590 ml HCI 1.05 ml ilave edilir ve asetilasyonu için asetik anhidrid 1.5 ml ilave ediniz. </li><li> tampon maddesi içinde 50 ug / ml proteinaz K hazırlama (50 mM Tris-HCI, DEPC pH 8.0 su muamele edilmiş). </li><li> formamid 500 ul 20x SSC 250 ul, 50x Denhardt çözeltisi, 100 uL, t-RNA 12.5 ul, ringa sperm DNA&#39;sı 2.5 ul, RVC 30 ul, bloke reaktif ve 50 ul 0.02 g oluşan hibridizasyon tamponu hazırlayın DEPC su. taze hazırlayın. </li></ol> 2. Meme Doku Bölümler <ol><li> Mikrotom ile 5 mikron formalin ile fikse parafine gömülü bölümleri kesmek ve slayt 14 şarj etmek bölüm uygulayın. Not: Burada kullanılan meme dokusu Kore Biobank Network önceden hazırlanmıştır. </li></ol> 3. Doku Hazırlanması <ol><li> fro parafin kaldır10 dakika boyunca, taze ksilen Coplin kavanoza 2x her bir slayt daldırarak doku m ve GKD 2 O etanol (% 100,% 95 ve% 80) ve azalan konsantrasyonlarda 5 dakika inkubasyon doku bölümü rehidrasyonu Davlumbaz bu adımı gerçekleştirin. </li><li> DEPC doku 5 dakika arıtılmış su bırakın. Bu noktada hidrofobik bariyer kalemle doku bölümünde etrafında sınırları yapmak. </li><li> PBS ile yıkama, ardından 15 dakika boyunca% 4 PFA doku çözmek. </li><li> PBS ile yıkama, ardından 10 dakika boyunca% 0.3 Triton X-100 / PBS doku bırakın. </li><li> 37 ° C&#39;de 15 dakika boyunca 50 ug / ml Proteinaz K çözeltisi ile örnek inkübe edin. Daha sonra PBS ile bir yıkama ve ardından, oda sıcaklığında 5 dakika için, trietanolamin ve asetik anhidrid ile doku bölümleri inkübe edin. Hiçbir PBS yıkama Proteinaz K tedavisi ve trietanolamin / asetik anhidrit tedavisi arasında gereklidir. </li></ol> 4. Melezleme <ol><li> İyice ve incu doku yıkayınOda sıcaklığında 2 ila 3 saat için hibritleme tampon maddesi ile asitleme doku kesiti. Tipik 120 ul hibridizasyon tamponu doku bölümünü karşılamak için yeterli olduğunu. </li><li> Seyreltik 5&#39;-DIG 120 ul hibridizasyon çözeltisi içinde 3 uM nihai konsantrasyona kadar LNA etiketlenmiş prob. stok konsantrasyonu genellikle 40 ng / | il olduğu için, 120 ul hibridizasyon tamponu stok 3 ul ilave et ve 5 dakika boyunca 95 ° C&#39;de karışım inkübe edin. </li><li> nemli bir oda içinde 54 ° C&#39;de (Tm) prob gece boyunca doku bölümü inkübe edin. Tipik 120 ul bir bölüm karşılamak için yeterlidir. İki ıslak kağıt havlu kullanılarak nem oluşturun. odasının ideal boyutu 8 inç 10 inç. </li><li> Not: Tm Farklı miRNA için farklı olabilir. 54 ° C miR-489 için söz olduğunu. </li></ol> 5. sıkılık yıkama <ol><li> 7 dakika boyunca 5x SSC ile doku yıkayın (melezleme aşamasından sonra, RNaz ücretsiz koşullar artık gerekli vardır). </li><li> doku kesiti wit yıkayın57 ° C &#39;de iki kez 7 dakika süre ile H, 1x SSC. </li><li> 57 ° C &#39;de, 7 dakika için 0,2 X SSC ile doku kesiti yıkayın. </li><li> Oda sıcaklığında 7 dakika boyunca 0,2 X SSC ile doku kesiti yıkayın. </li><li> 10 dakika boyunca PBS içinde doku kesiti inkübe edin. </li></ol> 6. Engelleme <ol><li> (Tampon PBS 18 m 2 mi FBS, Tween-20, 10 ul BSA 3 ul bloke yapmak için. Mağazası 4 ° C&#39;de tampon bloke nemli bir oda içinde, 1 saat boyunca oda sıcaklığında bloke edici tampon ile doku kesiti inkübe ° C). </li></ol> 7. Birincil Antikor Kuluçka ve Kalkınma <ol><li> bloke edici tampon içinde 300, anti-DIP-AP antikor ve gece boyunca 4 ° C &#39;de antikor ile doku kesiti inkübe: 1 seyreltin. </li><li> Her biri 5 dakika (geliştirme kiti tarafından sağlanan tampon maddesi) için, yıkama tamponu ile 3 kez bölümleri yıkayın. </li><li> CY-3 floresan ışık üretir alkalin fosfataz için bir ticari kit ile doku bölümü geliştirin. </li> &lt;li&gt; 5 dakika boyunca yıkama tamponu ile bölüm yıkayın. Yıkama tamponu ile 5 dakika yıkama ve ardından, 5 dakika boyunca 2.5 ng / ml Hoechst boya, 100 ul Leke bölümü. </li><li> üreticinin protokolüne göre bir tampon montaj bölümüne monte edin. Doku Bölüm başına 15 ul kullanın ve doku ile aşırı montaj çözümü silin silin. sızıntı önlemek için şeffaf tırnak cilası ile slayt mühür. </li><li> 10X objektif yani toplam büyütme kullanarak bölüm floresan mikroskop gözlemleyin 100X olduğunu. </li></ol>

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There is nothing to disclose.

Bu çalışmanın amacı, in situ hibridizasyon miRNA kullanarak meme kanseri dokularında MiRNA ekspresyon seviyesini belirlemek için yapıldı. Deneyde, tüm koşullar meme kanseri dokusu için optimize olduğu not edilmelidir. Ayrıntılı optimizasyon diğer doku tipleri için gerekli olabilir. Bütün çözeltiler DEPC su ile yapılmalıdır ve kaplar DEPC işlenmiş, su ile durulandı ve daha sonra otoklavlanmalıdır kullanılır. Hatta hafif RNaz kirlenme deney nihai sonucu etkileyebilir. MiRNA çok kısa bir dizisi olduğu için, hedef daha hibridizasyon kolaylaştırmak için bir LNA prob kullanmak gereklidir. Ayrıca, RNaz içermeyen Proteinaz K doku fiksasyon, tedaviden sonra, aksi proteinler endojen miRNA&#39;nın ile sondanın hibridizasyon müdahale edebilir kesinlikle gereklidir. Buna ek olarak, sonda ile doku kesiti inkübe edildi, ikinci 95 kaynatılması gereken herhangi açılmak C °ikincil yapılar. gece boyunca inkübe edildikten sonra, spesifik olmayan bağlanmayı azaltmak için bir protokol, yukarıda sözü edilen ısıda SSC konsantrasyonları ile yıkamak için önemlidir. floresans geliştirilmesi için, en az 1 saat süre ile alt-tabaka ile doku kesiti inkübe edin. O overdevelopment önlenebilir, böylece, kuluçkalama boyunca bölümü gözlemlemek için tavsiye edilir. kiti ile sağlanan montaj medya ile bölümüne monte edildiğinden emin olun. tek dikkate almalısınız bir sınırlayıcı faktör vardır. Hedef miRNA ifade dokuda düşükse, bu tekniği kullanarak ifade algılamak için zor olacak. Teknik teyit edilebilir ve tüm reaktifler çalışma sağlanacaktır ve ücretsiz RNAaz ari, böylece bu tür U6 olarak bir pozitif kontrol ile bu protokolü gerçekleştirmek için her zaman tavsiye edilir. Zayıf bir işaret söz konusu olduğunda değil, DIG etiketli prob biyotin etiketli sonda kullanmak mümkündürDIG Al etiketlenmiş prob. Sinyal büyütme biyotin-avidin sistemi ile mümkündür. Avidin biotin için güçlü bir yakınlık vardır. Bu afinite yararlanarak, alkalin fosfataz ile bağlamak olduğunu avidin-biotin dubleks ile biyotin probu etiketlemek mümkündür. Bu strateji kullanılarak, hem de düşük bol miRNA için sinyal yükseltmek mümkündür. miRNA&#39;ların önemli hücresel süreçlerde önemli roller oynar ve güçlü bir kanser gelişimi ile bağlantılıdır çünkü miRNA&#39;nın alanı hızla ortaya çıkmaktadır. Ayrıca, bu miRNA&#39;ların kanser tanısında belirteç olarak da kullanılabilir. Çeşitli çalışmalarda kanser prognozu miRNA ifade olası uygulama düşündüren tümör evresi olarak miRNA ifade ve klinik parametreler arasında güçlü bir korelasyon göstermiştir. Bu teknik, diğer kanser türlerinde MiRNA ifade analizi için kullanılabilir. Örneğin, miR-182 ve miR-503 ekspresyonu si kullanılarak kolon kanseri dokularında incelenditu melezleme 15. Geleneksel gen ekspresyon analizi dokusunda miRNA ifade seviyesini ortaya çıkarabilir ancak miRNA ifade ediliyor hangi yapı dan konumu veremiyoruz. Bu tekniği kullanarak, kolayca konumu yanı sıra miRNA ifade seviyesini bulabilirsiniz.

Meme kanseri dünya genelinde kadın nüfusu etkileyen en yaygın maligniteler arasında yer alıyor. Meme kanseri 1.3 milyon vaka dünya çapında 1,2 yılda bildirilmektedir. Tümör hücreleri, geleneksel biyolojik homojen ve yüksek proliferatif olarak kabul edilmiş olmasına rağmen, meme kanseri genetik ve klinik heterojen olduğunu belirgin hale gelmiştir. Yaygın antikanser ilaçlara direnç kanser ilerleme ve mesafe metastazı 3 onun kolaylaştırılması yoluyla hastaların çoğunluğunda mortaliteye neden ilerlemiş meme kanserlerinin bir özelliğidir. MikroRNA&#39;lar (miRNA&#39;ların) mRNA translasyonunu bloke veya mRNA bozulmasını 4 aracılık gen ekspresyonunu düzenleyen yaklaşık 22 nükleotid uzunluğunda küçük RNA molekülleri sınıfıdır. 2.000 &#39;den fazla farklı miRNA&#39;lar kadar insan hastalıkları 5 ile bağlantılı olan, insanları tespit edilmiştir. çalışmalar artan bir dizi havE miRNA&#39;lar onkojenlerin ve tümörleri giderici olarak işlev görebilir ve genellikle tümör 6 düzensiz olduğunu göstermiştir. miRNA&#39;lar kuvvetle tümör hücre hareketi, invazyon ve metastaz 7 olanlarla birlikte hücre döngüsü ilerlemesinin, yaşlanma, apoptoz ve otofaji önemli regülatörleri kontrol ederek birincil tümörogenez tüm etkileyebilir. Aberrant miRNA ifade aynı zamanda sahne, farklılaşma, prognozu ve adjuvan tedavi 8 yanıt olarak klinik etkileri ile ilişkili olmuştur. Özellikle, miR-146 ifadesi akciğer kanserli hastalarda 9 kötü prognoz ile ilişkilidir arttı. Başka bir çalışmada, miRNA let7b kayıp sentezleme Sonuç olarak, zayıf yaşama 6, habis fenotipler ile bağlantılı ve göstermiştir. miRNA&#39;lar ifade eden hücrelerin ve yapı türlerini anlamak olduğunu mekaniğini anlamak önemli bir parçası geçtiği miRNA ifade etkisi hücrede değişiklikler veDoku 10 fenotip. stromal hücrelerde salgılandığı bulunmuştur belirli miRNA&#39;ların epitel hücreleri tarafından alınır ve özel olarak hedef doğrultusunda hareket edilir. Aynı şekilde, miR-212 ve miR-132 meme bezi gelişimi sırasında 11 duktal büyümesi için gerekli epitel-stromal etkileşimleri stroma tarafından salgılanan ve regüle edilir. Bu çalışmanın genel amacı in situ hibridizasyon miRNA ile meme kanseri dokularında miRNA ifade algılamak için ayrıntılı bir protokol açıklamaktır. Biz meme kanseri hasta örneklerinde miRNA 489 ifadesinin seviyelerini analiz etmek için tüm şartları optimize ettik. Bu amaçla, işaretlenmiş DIG eden deneyde nükleik asit (LNA) prob kilitli kullanılır. Bunu nükleotid bazı, yani, bir LNA modifikasyonu sahip 2 &#39;, oksijen atomu ve 4&#39; de hibridizasyon sonrası &quot;yerine kilitli&quot; kalacak şekilde nükleotidi giderir riboz omurga karbon atomu bağlayan bir metilen köprüsü. Bunun bir sonucu olarak, çok daha zorhibridize kompleksi 12,13 denatüre etmek için.

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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ELF-97</td>
			<td style="width:159px;">Life technology&#160;</td>
			<td style="width:119px;">E6604</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50X Denhard&#39;t&#160;</td>
			<td style="width:159px;">Life technology&#160;</td>
			<td align="right" style="width:119px;">750018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">t-RNA</td>
			<td style="width:159px;">Roche</td>
			<td align="right" style="width:119px;">109541</td>
			<td>Reconstitute in DEPC water</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Herring Sperm DNA</td>
			<td style="width:159px;">Promega&#160;</td>
			<td style="width:119px;">D1815</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Roche Blocking&#160;</td>
			<td style="width:159px;">Roche</td>
			<td align="right" style="width:119px;">11096176001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RVC</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:119px;">50-812-650</td>
			<td>Before use spin down it at 16.1 RCF and take supernatant&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">miR-489 probe&#160;</td>
			<td style="width:159px;">Exiqon</td>
			<td style="width:119px;">38599-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nuclease free BSA</td>
			<td style="width:159px;">Roche&#160;</td>
			<td align="right" style="width:119px;">711454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primary antibody-anti DIG</td>
			<td style="width:159px;">Roche&#160;</td>
			<td align="right" style="width:119px;">11093274910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diethyl Pyrocarbonate&#160;</td>
			<td style="width:159px;">Sigma</td>
			<td align="right" style="width:119px;">1609478</td>
			<td></td>
		</tr>
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</table>

clinicopathological analysis of mirna expression in breast cancer tissues by using mirna in situ hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

İnsan göğüs kanseri dokusu miRNA 489 ekspresyonunu belirlemek için kullanılmıştır. Meme bezi kanalı ve epitel hücreler bitişik iki hastada tümör dokusunda (Şekil 1A ve 1B) önemli ölçüde daha yüksek MiRNA-489 seviyelerini ifade bulunmuştur. Bu açıkça miRNA 489 ifadesi miRNA 489 tümör baskılayıcı aktivite düşündüren, tümör dokusunda kayıp olduğunu göstermektedir. daha da aynı hastadan alınan, bu sonuçlar, tümör dokusu ve bitişik normal doku doğrulamak için karşılaştırılmıştır. Şekil 2A ve 2B&#39;de görüldüğü gibi, normal doku bitişik tümör dokusu daha miRNA 489 eksprese olduğu bulunmuştur. Görüntülerde 10 mikron çaplı bar gösterir. Görüntüler 10X lens kullanarak alınmıştır. <img alt="Şekil 1" src="/files/ftp_upload/53928/53928fig1.jpg" /> Şekil 1: normal meme dokusunda miR-489 sentezlenmesi. Hem normal hem de kanal yapısı ve tümör hücreleri, aynı bölümde gösterilir. Dokular, farklı meme kanseri hastalarında vardır. Normal meme kanalı ve meme bezi tümör dokusunda ise yok miRNA 489 ifade eder. Ölçek çubuğu = 10 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53928/53928fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="şekil 2" src="/files/ftp_upload/53928/53928fig2.jpg" /> Şekil 2:. Göğüs kanseri hastasından çifti Mir-489 sentezlenmesi doku mikro dizi, bir göğüs kanseri hasta miR-489 ekspresyonunu incelemek için kullanılmıştır. Bir temsilci hasta çifti burada gösterilmektedir. Ölçek çubuğu = 10 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53928/53928fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization at JoVE.com

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

miRNA kullanarak Meme Kanseri Dokularındaki miRNA İfade Klinikopatolojik Analizi<em&gt; In Situ</em&gt; Melezleme

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

clinicopathological-analysis-mirna-expression-breast-cancer-tissues

Nanyang Technological University

Research

JoVE Journal

Medicine

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

6.7K Görüntüleme.  University of South Carolina. Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

Video: Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

Floresan In-Situ Hibridizasyon ve Konfokal Lazer Tarama Mikroskopi Palatal Genişleticilerden Oral Biyofilm Analizi

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

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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 ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Meme Kanseri Hücrelerinde östrojen düzenlenmiş microRNA profil

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Üç boyutlu (3D) Modeli Kullanılarak Meme Kanseri Hücre invazivliğinin Kantitasyonu

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Farelerde Taksanlar Farmakokinetik ve Farmakodinamik Analiz Karotis Arter İnfüzyonlar

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

İnsan Göğüs Kanseri dokuları ve hücre hatlarından gelen Mammosphere Oluşumu Testi

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

Vivo ve Ex Vivo Periton yağ içinde Samanyolu Nokta Yapıların Yumurtalık Kanseri Metastatik Kolonizasyon Eğitim Yaklaşımlar içinde

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pankreas Kanseri Yeni Tedavi Stratejileri Geliştirme Hücre Özellikleri Kök incelenmesi

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

Prostat Kanseri Klinik Dokularındaki miRNA İfade Analizleri

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

Meme Kanserinde Östrojen Reseptör Sinyali tarafından Metabolik Yönetmelik Sistem Biyolojisi

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...

miRNA kullanarak Meme Kanseri Dokularındaki miRNA İfade Klinikopatolojik Analizi

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