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1。手術標本からのヒト前立腺癌組織の収穫と処理<ol><li>ロズウェルパーク記念研究所（RPMI）1640を15mlの10％ウシ胎児血清（FBS）を補充した培養培地および1％ペニシリン/ストレプトマイシンを含む二つの50ミリリットルポリスチレンコニカルチューブを準備する。 </li><li>治験審査委員会は、プロトコルを承認の下で病理サービス担当者は、それぞれ、前立腺切除および緩和手術標本から原発性および転移性前立腺癌試料を収穫。転移性の試料は、一次外科手術中リンパ節郭清から得ることができる、とりわけ単転移病変の診断除去、緩和的外科手術の間、脊髄減圧などの手順、および肺中の椎骨。 <ol><li>ステップ1.1からの第50ミリリットルのポリスチレンコニカルチューブに臨床診断のために必要とされない余分なバルク組織を採取し、4℃ですぐに保存harvesteD組織は、少なくとも面積4〜5 cm 2であり、厚さ1〜2センチ測定する必要があります。 </li></ol></li><li>研究室の担当者は、細胞懸濁液の組織学的検査および生成の準備のために病理サービスから得られたバルク組織を処理します。 <ol><li>バルク組織の処理は、最大の細胞生存率を保証するために外科的切除から24時間を超えてはならない。 </li><li>滅菌メスや鉗子でバイオセーフティキャビネット内で作業し、巨視的腫瘍結節から2〜4ミリメートルの厚さの水平組織切片を取る。すぐにステップ1.1からの第50ミリリットルポリスチレンコニカルチューブ内のセクションを格納します。 </li><li>巨視的な結節が前立腺切除サンプルから可視化されていない場合、後方ローブと遷移ゾーンからランダムに2〜4ミリメートルの厚さの水平組織切片を収集する。 </li></ol></li><li>滅菌メスとピンセットを用いて外科的組織の一部を分離し、前の属に10％ホルマリンで固定細胞懸濁液のTiON。アーカイブおよび前立腺癌組織の存在を確認するために、この材料組織学的に分析する。 </li></ol> 2。組織切片からの細胞懸濁液の生成<ol><li>無菌のバイオセーフティキャビネット内で作業し、滅菌1×リン酸緩衝生理食塩水（PBS）1 mlを含む60×15mmの培養皿に組織切片を転送する。 <ol><li>機械的に滅菌メスや鉗子を使用してサンプルを粉砕する。 </li><li>いいえ、酵素消化は、このプロトコルではありません。 </li></ol></li><li>無菌50mlコニカルチューブに1ミリリットルサスペンションを転送します。 <ol><li>組織切片が完全に解離されるまで3〜4倍、通常、培養皿に1ミリリットルを加え、チュレーションの手順を繰り返します。 </li><li>すべての組織切片が解離されるまで連続的に、この手順を繰り返します。 </li></ol></li><li> 5ミリリットル血清学的にπの50mlポリスチレンコニカルチューブの内容物を再懸濁1分間最高速度（約3,200 rpm）でpetteと渦。 </li><li> 35μmの細胞濾過器、得られた懸濁液を濾過し、第二の滅菌50ミリリットルポリスチレンコニカルチューブに集める。 </li><li> 10分間450×gでの遠心分離によってペレットを生成します。 </li><li>上清を捨て、赤血球溶解緩衝液5mlでペレットを再懸濁し、そしてRTで5分間溶液をインキュベートする。 RTにおける450×gで5分間遠心分離し、赤血球細胞溶解緩衝液を除去する。 </li><li>上清を捨て、5％FBSを補充した1×PBS中でペレットを再懸濁し、血球計又は自動化細胞カウンターを用いて生存（トリパンブルー陰性）細胞の数を定量化する。 1×10 6個/ mlの懸濁物を生成し、それはもはや、1時間よりも、氷上で保管していません。 <ol><li>細胞の生存率および収率は腫瘍検体との間でかなり変化し得る。前立腺切除の設定では、生存率は45から70パーセント、かつfの間の範囲であり得る肉眼で見える腫瘍小結節から2〜4ミリメートルの厚さの水平組織切片が約3×10 6個の生細胞を得ることが期待できる見る。 </li></ol></li></ol> 3。 FACSのための細胞の抗体染色<ol><li>以下に示すように5 15ミリリットルポリスチレンコニカルチューブにラベルを付けます。それぞれ、造血および内皮の要素を除外するために、CD45およびCD31抗原のラベルを使用してください。 <ol><li>汚れのない</li><li>のIgG 2aは κ-PE +のIgG 1κ-FITC </li><li> HLAI-PE +のIgG 1κ-FITC </li><li>のIgG 2aは κ-PE + CD31-FITC + CD45-FITC </li><li> HLAI-PE + CD45-FITC + CD31-FITC </li></ol></li><li>ステップ3.1において5管に前のセクション（ステップ2.7）から定量細胞懸濁液を配布します。 1:250の希釈で抗体を追加し、30分間氷上で細胞懸濁液をインキュベートする。 </li><li> 4℃で3分間450×gで細胞を遠心分離し、上清を捨てる。 </li&gt; <li>滅菌1×PBSで各ペレットを再懸濁することにより細胞を洗浄し、4℃で3分間、450×gで遠心分離し、続いて10％FBSを補充した</li><li> 10μgの/ mlの濃度で、4 &#39;、6 - ジアミジノ-2 - フェニルインドール（DAPI）を含有する1×PBS 1mlの溶液で細胞を再懸濁する。 </li><li> 12ミリメートル×75ミリメートルのポリスチレンチューブに35μmのストレーナーキャップを通じて最終的な解決策をフィルタリング。 </li></ol> 4。 FACSによる前立腺のCSCの分離<ol><li>細胞選別のためのフローサイトメーターを利用している。 </li><li>管＃1、＃2、＃3、＃4からの細胞を用いて補償制御を設定する。 </li><li>前方および側方散乱パラメータを用いて破片および細胞のクラスターを除外ゲートを作成する。 </li><li>それぞれ、チューブ＃3とチューブ＃4からの懸濁物を使用して、FITCおよびPEゲートを確立する。 </li><li>パシフィックブルー·チャネルを使用して実行可能なゲート（DAPI陰性）細胞。 </li><li>チューブ＃5廃棄CD31陽性およびCD45陽性細胞を使用し、のRPMI 2ミリリットルを含む滅菌15 mlのポリスチレンコニカルチューブにHLAI陰性とHLAI陽性集団の両方を収集し、10％FBSを添加した。 </li><li>遠心機で5分間450×gで細胞懸濁液をソートした後、10％のFBSを補充したRPMI 200〜500μlにペレットを再懸濁する。 </li><li>血球計数器または自動化細胞カウンターを使用して実行可能な（パンブルー陰性）細胞を定量。 </li></ol>

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144, 646-674 (2011).</li><li>Magee, J. A., Piskounova, E., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+impact,+heterogeneity,+and+uncertainty.">Cancer stem cells: impact, heterogeneity, and uncertainty.</a> Cancer Cell. 21, 283-296 (2012).</li><li>Nguyen, L. V., Vanner, R., Dirks, P., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+an+evolving+concept.">Cancer stem cells: an evolving concept.</a> Nat. Rev. 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利害の対立が宣言されていません。

このプロトコルは、新鮮なヒト前立腺癌組織からのCSCの単離を記載している。いくつかの重要な考慮事項は、このプロトコルの成功結果に影響を与える。 生存可能な前立腺癌細胞の高い数の回復は、外科的試料を慎重に肉眼的評価に依存する。巨視的腫瘍結節が観察され、10を処理する際に我々の経験では、腫瘍形成性前立腺細胞の正常なアイソレーションが最?...

腫瘍内不均一性癌1の特徴であると考えられている。実際、腫瘍内の不均一性のいくつかのメカニズムは、遺伝子変異と微小環境との相互作用を含む、記載されている。さらに、いくつかの癌は、連続移植アッセイ2-5における腫瘍開始能力および自己再生の性質を示す癌幹細胞（CSCが）の亜集団を有する細胞の階層を含むことができる。最初に血液学6、7乳房、および脳8に記載の悪性腫瘍、CSCはまた、前立腺癌の9-12、ならびに他の腫瘍タイプ2-5に研究されている。 CSCは、一般的に不均一な集団2-5内細胞画分と見なされます。したがって、CSCが機能的分子特性は、バルク集団からの濃縮条件とする。したがって、最後の二十年の間に数が満たさCSCの濃縮のhodologiesは、典型的には、フローサイトメトリーによって標識された集団の分離を伴うが考案されてきた。また、CSCが研究で重要な考慮事項は、ヒト組織の階層的な組織がそのような文化や免疫不全マウスでの連続継代として実験操作によって破壊され得ることである。その結果、ヒト組織からのCSCの直接単離は、CSCの分野で重要な方法論として浮上している。 前立腺癌は、癌の罹患率および米国および世界中13の周り死亡の主要な原因である。従って、前立腺のCSCの単離および生物学的特徴付けは重要な関心である。前立腺CSCは、以前の細胞株、患者に由来する異種移植片、および低継代患者派生懸濁培養9,10,12,14から濃縮されています。 我々は最近、直接人間の外科sから前立腺のCSCの単離を報告そのHLAI陰性細胞表面表現型10のおかげでamples。ここでは詳細は、これらの細胞の単離のための手順を実装WE。前立腺腫瘍は、手術標本から採取し、細胞懸濁液中に形成されている。次いで、細胞をHLAIに対する抗体ならびに間質および生存度マーカーを用いて染色し、CSCは、蛍光活性化セルソーティング（FACS）により単離される。単離されたCSCはその後、生細胞を必要とするアッセイに用いることができる。

<table border="0" cellpadding="0" cellspacing="0" width="779">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RPMI</td>
			<td style="width:195px;">Gibco Life Technologies</td>
			<td style="width:187px;">11875-093</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Fetal bovine serum</td>
			<td style="width:195px;">Gibco Life Technologies</td>
			<td style="width:187px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Penicillin Streptomycin</td>
			<td style="width:195px;">Gibco Life Technologies</td>
			<td style="width:187px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">PBS</td>
			<td style="width:195px;">Corning cell gro</td>
			<td style="width:187px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">60 mm-15 mm cell culture dish</td>
			<td style="width:195px;">Corning</td>
			<td style="width:187px;">3295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">35 &#181;m Cell Strainer</td>
			<td style="width:195px;">BD Falcon</td>
			<td style="width:187px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">50 ml conial tube</td>
			<td style="width:195px;">Crystalgen</td>
			<td style="width:187px;">23-2263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">15 ml conical tune</td>
			<td style="width:195px;">Crystalgen</td>
			<td style="width:187px;">23-2265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Red blood cell lysing buffer</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:187px;">R7757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">HLA class I (W6/32) PE antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:187px;">ab43545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">CD31 FITC antibody</td>
			<td style="width:195px;">ebioscience</td>
			<td style="width:187px;">11-0319-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">CD45 FITC antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:187px;">ab27287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">IgG2a&#954; PE antibody</td>
			<td style="width:195px;">BD Pharminogen</td>
			<td style="width:187px;">555574</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">IgG1&#954; FITC antibody</td>
			<td style="width:195px;">BD Pharminogen</td>
			<td style="width:187px;">551954</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">DAPI</td>
			<td style="width:195px;">Invitrogen&#160;</td>
			<td style="width:187px;">d3571</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">12 x 75 mm polystyrene tubes with cell strainer cap</td>
			<td style="width:195px;">BD Falcon&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:187px;">352235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Vortex Mixer</td>
			<td style="width:195px;">Crystalgen</td>
			<td style="width:187px;">CG-BV1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">10% Neutral Buffer Formalin</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:187px;">RBBP-0480</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of cancer stem cells from human prostate cancer samples

癌幹細胞（CSC）のモデルはかなり過去20年間に再訪されています。この時間の間のCSCを同定し、直接単離されたヒト組織から直列典型的にフローサイトメトリーによる細胞分画の亜集団の抗体標識を介して、免疫不全マウス中で増殖されている。しかし、前立腺癌の独特の臨床的特徴は、かなり新鮮なヒト腫瘍試料から前立腺のCSCの研究が限られている。我々は最近、HLAクラスI（HLAI）のおかげで、直接ヒト組織由来の前立腺のCSCの分離陰性の表現型を報告した。前立腺癌細胞は、手術標本から採取し、機械的に解離される。細胞懸濁液が生成され、蛍光共役HLAIおよび間質抗体で標識される。 HLAI陰性細胞の亜集団は、最終的にフローサイトメーターを用いて単離される。このプロトコルの主な制限は、頻繁に微視的および多発性性質である前立腺切除標本における原発性癌。それにもかかわらず、孤立したライブ前立腺CSCは免疫不全マウスでの移植による分子特性および機能検証に適しています。

<img alt="図1" fo:content-width="5in" fo:src="/files/ftp_upload/51332/51332fig1highres.jpg" src="/files/ftp_upload/51332/51332fig1.jpg" /> 図1。ヒト前立腺癌腫瘍を収穫し、ヒト前立腺癌の特定の集団を示すフローサイトメトリープロット。（A）腫瘍結節細胞懸濁液を生成するために収穫される。処理された試料の腫瘍含有量は、従来のヘマトキシリン​​及びエオシン染色を用?...

Watch this Scientific Journal Video about Isolation of Cancer Stem Cells From Human Prostate Cancer Samples at JoVE.com

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

直接ヒト組織からがん幹細胞（CSCが）の単離は、それらの生物学的特性評価のために必要である。また、難しい手順のトラブルシューティングのヒントを提供しながら、この原稿は、ヒト組織からの前立腺のCSCを単離するための方法論を説明します。

ヒト前立腺癌サンプルからがん幹細胞の単離

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

isolation-of-cancer-stem-cells-from-human-prostate-cancer-samples

Research

JoVE Journal

Medicine

14.0K ビュー.  Icahn School of Medicine at Mount Sinai. 直接ヒト組織からがん幹細胞（CSCが）の単離は、それらの生物学的特性評価のために必要である。また、難しい手順のトラブルシューティングのヒントを提供しながら、この原稿は、ヒト組織からの前立腺のCSCを単離するための方法論を説明します。 

ビデオ: Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

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

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

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

生体内イメージングを使ってリアルタイムで腫瘍におけるナノ粒子の取り込みの評価

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

医学

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

単離、特徴付けと二つの異なる方法を使用して永久歯に由来するヒト歯髄幹細胞の分化の比較

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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.

乳がんの遺伝子改変マウスモデル由来の初代前癌乳腺上皮細胞のタイムラプスイメージング

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

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

CD133の患者由来の細胞培養および分離<sup&gt; +</supメラノーマから&gt;推定されるがん幹細胞

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

齧歯尿生殖洞間充織とヒト幹細胞の組織の組換えを用いたヒト前立腺上皮の形成

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

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

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

ヒト前立腺癌転移の同所マウスモデル

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

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

ヒト細胞株から化学療法抵抗性の卵巣がん幹細胞の濃縮

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

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.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

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

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

マウス癌モデルからの循環腫瘍細胞の単離と伝播

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

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).