クリステン·クワンと後期カイビン本研究で使用したプラスミドの贈答用チェン;、抗体染色の支援のためのジェームズ·リスターとDavid Raible、およびのためのジェイムズNeiswender我々は、その研究室、これらの技術が最初に開発されたもので博士はレナードI.ゾンに感謝マイクロインジェクションビデオ。この作品は、CJCにNIHの助成金R00AR056899-03によって賄われていた

1。メラノーマ発症の修飾のためのスクリーニング<ol><li> PCRは、目的の遺伝子の完全長のオープンリーディングフレーム（GOI）を増幅し、BPクロナーゼII（Invitrogen）を用いてpDONR 221に再結合によりゲートウェイ中間エントリークローンを作成します。 miniCoopRベクター（ 図1A）にmitfaプロモーター下に目的の遺伝子を配置する再結合p5E_mitfa、pME_GOI、Tol2kit 302位p3E_SV40polyA 6とminiCoopR 5にマルチゲートウェイ技術（Invitrogen）を使用します。 </li><li> P53（LF）;; mitfa（LF）は前述したように7三重ホモ接合ゼブラフィッシュ胚：1セルTG（BRAF V600E mitfa）にTol2のトランスポザーゼmRNAは25ピコグラムと一緒に、それぞれのクローンの25ピコグラムを注入します。 28.5で注入した胚をインキュベート℃に24 HPFで任意の死亡胚を取り出します。 </li><li>解剖microscopに注入された胚を含むペトリ皿を置くことによって、72 HPFで救出メラノサイトを有するトランスジェニック動物を選択する白い背景（ 図1A）に対する入射光の下でのe。 </li><li>前述の8として4 DPFにおけるゼブラフィッシュ施設の保育園で3リットルタンクに動物を移します。 </li><li> 2ヶ月で、4ミリメートル2（ 図1A）を超えるメラノサイト救助の少なくとも1つの領域を持つ動物を選択します。 72 HPFにおけるメラノサイト救助の程度、2ヵ月後のメラニン細胞救助の程度との間には強い相関関係がある。メラノサイト救助した胚を72 HPFで拾われた場合、それらの大半（約80％）が2ヶ月で4ミリメートル2より大きいメラノサイト救助の少なくとも1つの領域を持つことになります。 </li><li>画面腫瘍の存在（ 図1B）のための週刊選択された動物。組織病理学的および形態学的変化との間には強い相関関係があります。良性から悪性への遷移は、病変が動物の表面から浮き上がるとなる形態変化として認識されています。担癌アニマを隔離研究については、ls。 </li><li>縦軸横軸とパーセント黒色腫、無増悪生存期間に週齢でメラノーマフリー生存曲線を描きます。目的とする遺伝子を発現メラノサイトを持つ動物は、対照動物と比較されるメラノサイトにおける急行EGFP（緑色蛍光タンパク質）（ 図1C）。 miniCoopR-EGFPを注射した動物のための平均腫瘍の発症は約18週間です。 2つの曲線は、ログランク検定を用いて統計的に異なっているかどうかを決定します。 </li></ol> 2。腫瘍浸潤アッセイ<ol><li>背びれ（ 図2A）の後脳と前縁の後方境界との間の領域では、背側に腫瘍を発症孤立ゼブラフィッシュを選択します。 </li><li>黒色腫の発症は、マサチューセッツ大学医学部アドインの大学によって安楽死でアメリカ獣医学協会のパネルで指定し、承認されたガイドラインに従って魚を安楽死二週間後titutional動物管理使用委員会（IACUC）。鰓の動きが止まるまで、具体的には、魚は0.6 mg / mlのtricaineと皿に置かれます。 </li><li>メスで魚の腹腔内に切開を加えてから、固定のために4℃で一晩、4％パラホルムアルデヒドで魚（PFA）を配置します。 </li><li> 4℃で一晩、0.5M EDTAを用いて治療℃で石灰を除去するために。 </li><li>一晩、60℃で2時間ごとに2回℃で1時間、キシレンで3回クリアで1時間脱水し、パラフィンで治療カセット内のホルマリンで固定</li><li>金型内にパラフィンの組織を埋め込むと、それが30分〜1時間凝固させる。 </li><li> 5μmの切片、1毎に50μmを作る。のセクションでは、最も深い侵略のポイントを識別することができるように、横方向および病変全体を通してでなければなりません。ヘマトキシリン​​·エオシン染色により、次のとおりです。 </li><li>腫瘍細胞はコラーゲン層緻密外に残っているかどうかを測定することにより、腫瘍の浸潤を評価する、背側に侵入筋肉や脊柱（ 図2B、C）にさらに侵入する。サンプルの2つのセット間の統計的な差を決定します。 </li></ol> 3。スケールメラノサイトの抗体染色<ol><li> 80ミリリットルの0.1M Na 2 HPO 4および20ミリリットル0.1MのNaH 2 PO 4でPO4バッファを構成しています。 pHが7.3であることを確認します。 8.0グラムのスクロース、0.15ミリリットル0.2M CaCl 2及び90ミリリットル0.2M PO 4緩衝液、pH 7.3を含む2倍修正バッファを構成しています。必要がNaOHまたはHClで7.3にpHを調整する場合は、PO 4緩衝液で100mlにする。 </li><li> 300mgの固体PFAまで重量を量るとマイクロ遠心チューブに追加します。 PFAの4.5 x質量、mgで、（ 例えば、450 ulの5 mMのNaOHを100 mgのPFAに）に等しい容積に5mMのNaOHを追加します。 60から70℃の熱でPFAが溶解するまで揺れ、時折持つ。残りの微粒子をスピンダウンし、20％のPFA、上清を回収。毎回新鮮なことを確認します。 </li><li> 1X fiを含む固定液を準備xのバッファと4％PFA。 </li><li> 0.17 mg / mlのtricaineで魚を麻酔。 </li><li>スケールのメラニン含有半分（ 図3A）を損傷しないように注意しながら、シャープな先の細いピンセットを用いて背側のスケールを摘み取る解剖顕微鏡下で、入射光を用いた。場所の鱗直接鉗子から室温で1 mlの固定液とインキュベートに、≥2時間、回転させながら。魚の水に魚を移し、正常な回復を確認するために魚を監視します。 </li><li>漂白剤着色されたメラノサイトは、室温で5分間PBST（1×PBSのpH 7.4、0.1％Triton X-100）を2回に固定されたスケールを洗浄する。のdH 2 Oで5 mlに0.4ミリリットル10％KOHと0.15ミリリットルの30％H 2 O 2を含む1 ml加え作りたて漂白溶液管が開いて破裂からガスを防ぐためにパラフィルムやキャップロックをかけた。顔料は（ 図3B、C）（10分が一般的です）なくなるまで漂白続ける。室温で5分間PBSTで4回洗浄するerature。 </li><li> 1X PBS pHは7.4、0.2％トリトンX-100、2 mg / mlのBSA、1％DMSO、0.02％NaN 3を 、2％子羊血清で構成されたブロック溶液中で少なくとも30分間ブロック。 HRP反応を使った開発がNaN 3を残す。 </li><li>室温で一晩ブロック溶液中で一次抗体とインキュベートします。あなたは彼らが水没維持する以上のサンプル溶液の少なくとも400μlを持っている必要があります。 </li><li>スケールを室温でPBST中で30分間、3回洗浄する。 </li><li> 2時間から室温でブロック溶液中で一晩に二次抗体とインキュベートします。 </li><li> PBST +0.1μMのDAPIで10分間染色する。 </li><li> PBSTで5分間3回洗浄する。 </li><li>スケールの凹面側がスライドに向かって下向きになるようvectashieldのドロップでスライドガラス上にスケールを取り付け、その上にガラスカバースリップを置く。明確なマニキュアでスライドの端をシールし、蛍光顕微鏡下でスライドを観察する（ 図3D、E、F、G </stronグラム&gt;）。 </li></ol> 4。移植アッセイ<ol><li>移植10以前のガンマ線照射25 Gyの1日を照射することにより、受信者2から3ヶ月のキャスパー 9魚を準備します。魚は魚の水で回復することができます。 </li><li>前述のように、担癌魚（セクション2.2）を安楽死させる。 </li><li>腫瘍を切り取り、5％FBSを含むPBSで滅菌0.9x約5mlのフィルターを用いてペトリ皿に入れてください。溶液中でしばらくカミソリで腫瘍をサイコロ。 </li><li>室温で働いて、単一細胞を得るためにP1000ピペットでひいて粉にする。 5％のFBSを持つフィルター滅菌0.9x PBSで25mlにボリュームを構成しています。 </li><li> 40μMのメッシュフィルターでろ過。 </li><li> 10分間1,500回転（453 RCF）でエッペンドルフ5810R卓上遠心機でスピン。 </li><li>上清を除去し、およそ所望の濃度（100ミリメートル3腫瘍のため10 7個の細胞を想定しています）に細胞懸濁液を作る。 </li><li> tを算出彼の正確な細胞数を血球計数器を用いて、最終的な注入量は約5μLになるように細胞懸濁液を希釈します。 50,000個の細胞を注入することになっている場合は、細胞懸濁液を10,000細胞/μlの最終濃度に希釈する必要があります。細胞の凝集を防ぐために細胞懸濁液の入ったチューブごとに数分をはじく。 </li><li> 100％エタノールと0.9x PBSで26Sゲージ（ベベルチップ）701 N10μlのハミルトンシリンジを2〜3回洗ってください。細胞懸濁液の5μlでシリンジをロードします。 </li><li> 0.17 mg / mlのtricaineの照射レシピエント魚を麻酔。湿らせたキムワイプでその側の上に置いて魚（ 図4A）。 </li><li>片手で魚を安定させ、約半分の菱脳や背びれの前縁の後方境界との間の腹膜腔上記の魚の脇腹に45°の角度で上向きにベベルに針を挿入します。ゆっくりプランジャー（ 図4B）を踏み込みます。&lt;/ LI&gt; <li>魚は新鮮な魚を水に回復し、腫瘍移植（ 図4C）のために毎日魚を観察することができる。生着が発生した場合は、継続的な成長と病気の発症も観察することができます。 </li></ol>

<ol>
	<li>Beroukhim, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+landscape+of+somatic+copy-number+alteration+across+human+cancers.">The landscape of somatic copy-number alteration across human cancers.</a> Nature. 463, 899-905 (2010).</li><li>Curtin, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+sets+of+genetic+alterations+in+melanoma.">Distinct sets of genetic alterations in melanoma.</a> N. Engl. J. Med. 353, 2135-2147 (2005).</li><li>Lin, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+genomic+diversity+and+tumor+dependency+in+malignant+melanoma.">Modeling genomic diversity and tumor dependency in malignant melanoma.</a> Cancer Res. 68, 664-673 (2008).</li><li>Patton, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRAF+mutations+are+sufficient+to+promote+nevi+formation+and+cooperate+with+p53+in+the+genesis+of+melanoma.">BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.</a> Curr. Biol. 15, 249-254 (2005).</li><li>Ceol, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histone+methyltransferase+SETDB1+is+recurrently+amplified+in+melanoma+and+accelerates+its+onset.">The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset.</a> Nature. 471, 513-517 (2011).</li><li>Kwan, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev. Dyn. 236, 3088-3099 (2007).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide of the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>White, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2, 183-189 (2008).</li><li>Traver, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nat. Immunol. 4, 1238-1246 (2003).</li></ol>

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miniCoopRメソッドは、ゼブラフィッシュメラノサイトにおいて、目的の遺伝子の発現を可能にします。このアプローチは、ゼブラフィッシュmitfa遺伝子は細胞自律的役割を果たしているという事実を利用しています。このような理由から、miniCoopRベクトルによって救出メラノサイトは、それが物理的に結合されているミニ遺伝子と関心のある任意の遺伝子を含んでいるために確信?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td> Gateway組換え試薬</td><td>インビトロジェン</td><td></td><td></td></tr><tr><td> miniCoopR </td><td></td><td></td><td>参考5 </td></tr><tr><td> Mitfa抗体</td><td></td><td></td><td>参考5 </td></tr><tr><td> FITC標識ヤギ抗ウサギIgG抗体</td><td>インビトロジェン</td><td></td><td></td></tr><tr><td> Vectashield </td><td>ベクトルラボ</td><td> H-1000 </td><td></td></tr><tr><td> キャスパーゼブラフィッシュ</td><td></td><td></td><td>文献[9] </td></tr><tr><td> 701N 10μLシリンジ</td><td>ハミルトン/フィッシャー</td><td> 14から824 </td><td> ＆NBSPは、 </td></tr><tr><td> 40μmフィルタ</td><td> BDファルコン/フィッシャー</td><td> 352340 </td><td></td></tr><tr><td> FBS </td><td>インビトロジェン</td><td> 26140079 </td><td></td></tr></tbody></table>

screening for melanoma modifiers using a zebrafish autochthonous tumor model

ヒト癌のゲノム研究は、腫瘍1,2,3で変更されている遺伝子に関する豊富な情報が得られている。これらの研究から生じる課題は、多くの遺伝子が改変されていることであり、それは、変換時に偶然に生じたものから、腫瘍形成を運転した遺伝的変化を区別するのが困難な場合があります。この区別を描画するには、それを定量的に腫瘍の開始と腫瘍が持続し、普及することができ、他のプロセスで変更された遺伝子の効果を測定できるアッセイを有することが有益である。ここでは、黒色腫の開始と維持のために必要なステップを包含土着腫瘍モデル4を用いてゼブラフィッシュにおける候補メラノーマモディファイ多数を上映するために迅速な手段を提示する。このアッセイにおけるキー試薬がminiCoopRベクトルであり、その中に候補メルGateway組換えカセットに結合mitfaメラノサイト仕様因子の野生型コピーをanoma遺伝子は5を再結合することができる。 miniCoopRベクターは、プロモーター、オープンリーディングフレームと野生型mitfa遺伝子の 3&#39;-非翻訳領域を含むmitfaの救出ミニ遺伝子を持っています。それは私たちが候補メラノーマ修飾子の完全長のオープンリーディングフレームを使用して構造体を作ることができます。 P53（LF）;; mitfa（LF）ゼブラフィッシュの胚：これらの個々のクローンは、単一セルのTg（BRAF V600E mitfa）に注入することができる。 miniCoopRベクトルは Tol2媒介形質転換6によって統合取得し、メラノサイトを救う。それらは物理的にmitfa救出ミニ遺伝子に結合されているので、候補遺伝子が変革し、腫瘍に発展するそのうちのいくつか救出メラノサイト、で表されます。メラノーマ開始とメラノーマ細胞特性に関する候補遺伝子の効果はメラノーマフリー生存曲線、浸潤アッセイは、抗体染色および移植アッセイを用いて測定することができる。

ワンセルTG（mitfa：BRAF V600E）、P53（LF）; mitfa（LF）ゼブラフィッシュの胚はメラノーマ癌遺伝子mitfaプロモーターの制御下SETDB1 5またはEGFP、それぞれを含むminiCoopRベクターを注入した。メラニン細胞救助を伴う胚を選択して成熟させた。 4ミリメートル2より大きいメラニン細胞救助を伴う年齢動物の2ヶ月の時点で選択した。動物は、黒色腫の?...

Watch this Scientific Journal Video about Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model at JoVE.com

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

ゼブラフィッシュ土着腫瘍モデルを用いてメラノーマ修飾するための画面への迅速な方法が提示される。これは、メラノサイト内の候補メラノーマの遺伝子の発現を可能にminiCoopRベクトルを利用しています。メラノーマフリー生存曲線、浸潤アッセイ、スケールメラノサイトおよびメラノーマ移植アッセイの抗体染色のためのプロトコルを取得するための方法が記載されている。

ゼブラ土着腫瘍モデルを用いたメラノーマの修飾のためのスクリーニング

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

screening-for-melanoma-modifiers-using-zebrafish-autochthonous-tumor

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13.6K ビュー.  University of Massachusetts Medical School. ゼブラフィッシュ土着腫瘍モデルを用いてメラノーマ修飾するための画面への迅速な方法が提示される。これは、メラノサイト内の候補メラノーマの遺伝子の発現を可能にminiCoopRベクトルを利用しています。メラノーマフリー生存曲線、浸潤アッセイ、スケールメラノサイトおよびメラノーマ移植アッセイの抗体染色のためのプロトコルを取得するため?...

ビデオ: Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor 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.

口腔扁平上皮癌の同所性マウスモデルにおける腫瘍細胞の浸潤の多光子イメージング

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

医学

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

胎児性アルコール暴露のゼブラフィッシュモデルの催奇形性変化を評価する

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人間の神経内分泌腫瘍療法の研究のための三次元モデルとしての人間の神経内分泌腫瘍細胞株

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

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 ...

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

糖尿病とメタボリックメモリーのゼブラフィッシュモデル

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

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.

新規高分解能<em生体内で&gt;</em腫瘍増殖と治療に頭蓋内構造物の動的応答を研究する&gt;イメージング技術

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

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

多発性嚢胞腎疾患の可能性のあるゼブラフィッシュモデル：のノックダウン<em&gt; WNT5A</em&gt;ゼブラフィッシュ腎臓で嚢胞を引き起こす

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

腫瘍間質相互作用のための三次元共培養モデル

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

ブドウ膜黒色腫の肝転移の治療として単離した肝灌流

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

皮下腫瘍モデルにおける腫瘍浸潤白血球サブセットの評価

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...