This work was conducted with funds to M.B. from the Canadian Institutes of Health Research (CIHR) grant MOP 107972. M.B. is a recipient of a CIHR New Investigator Salary Award. D.C. is a recipient of studentships from the Translational Breast Cancer Research Unit and the CIHR Strategic Training Program in Cancer Research and Technology Transfer, London Regional Cancer Program. C.G. is recipient of studentships from the Translational Breast Cancer Research Unit, London Regional Cancer Program and from the CIHR- Strategic Training Program in Cancer Research and Technology Transfer. SGD.

基底膜マトリックス中の乳癌細胞の1。三次元培養（埋め込み技術） <ol><li>取り扱いマトリゲル基底膜マトリックス：4℃で一晩を氷上で溶かす基底膜マトリックスは、低温で液体であるが、室温で固化する。氷（ 図1A、B）に基底膜マトリックスを保管してください。 </li><li>螺旋パターン（ 図1C）で、P-200ピペットマンのチップを用いて、マトリックス拡散による基底膜マトリックス50μlの共焦点No.1のガラスボトムディッシュをカバーしています。気泡の形成を回避するために、基底膜マトリックスを拡散する場合は注意が必要です。同様に、メニスカスの形成を防止するために、ガラスボトムディッシュの国境近くまで行列を広げないようにしてください。経験の浅い取り扱い基底膜マトリックスは、次いで氷上に配置でき、皿、P-200ピペットマンやピペットを予冷した場合（または4℃で冷蔵庫に一晩放置）。このステップの提供固形化の前に、基底膜マトリックスを広めるために追加の時間。 （ 図1D）を固化する基底膜マトリックスを可能にするために、少なくとも30分間（5％CO 2、37℃で）、細胞培養インキュベーター中皿（複数可）を配置します。 </li><li>行列が凝固を受けている間に、細胞の70〜80％コンフルエント100 mmのプレートをトリプシン処理。細胞がプレートから離れ持ち上げ始めたら、トリプシン（ 図1E）を不活性化するために、10％（v / v）のウシ胎児血清（FBS）を含むRPMI 1640培地10mlに再懸濁。その後、15ミリリットルコニカルチューブ（ 図1F）に再懸濁した細胞を移す。 </li><li>専用の細胞培養遠心分離機（ 図1G-H）で3分間、100×gで（円錐管中に存在する）の細胞をスピン。 </li><li> 1ミリリットルマイクロ遠心チューブに細胞がスピンダウンされている間、一定分量行列50μlの（注：各ガラスボトムディッシュを1マイクロチューブが割り当てられ、それ故に、もし実験は）それは3マイクロ遠心管が同様に必要であるということに、3料理を必要とし、その後、氷（ 図1B）にチューブを配置します。 </li><li> （ 図1I）乱さペレットを残しながら、ステップ1.4からのコニカルチューブから培地を吸引する。 FBSを補充したRPMI培地の1ミリリットル（ 図1J）に（スピンダウンしてきた）細胞を再懸濁する。 </li><li>細胞をマイクロ遠心チューブに血球計数器（ 図1K）、または細胞粒子カウンタのアリコートを2.5×10 4個の細胞を用いて計数し、50μl（ 図1L）の総容積を得るように適切なメディアを使用してそれを先頭したら。 </li><li> 1:1の比率のステップ1.5からのマトリックスを含むマイクロチューブにステップ1.7（50μl中25,000細胞）から細胞を混合する;最終容量は100μL（ 図1M）になります。 </li><li> Sにステップ1.8から細胞混合：優しく行列100μlのプレートステップ1.2からolidified基底膜マトリックスでコーティングされたディッシュ（ 図1N）。これは、基底膜マトリックスに埋め込まれるべき細胞を可能にする。 </li><li> （5％CO 2で37℃で）、細胞培養インキュベーター中皿（複数可）を転送できるようにし、マトリックス：細胞混合物は、少なくとも30分間凝固させる。 </li><li>行列はかつて：細胞混合物が固化し、ディッシュ（ 図1O）にFBSを補充したRPMI培地の2ミリリットルを追加し、実験（ 図1P）の残りのために保存されるインキュベーターをお皿を取り戻す。 </li><li> 5日間毎日、メディアを変更（またはアッセイに必要なに従って、MDA-MB-231細胞のアッセイの持続時間の長さは5日である）。 </li><li>光学顕微鏡を用いて、微分干渉コントラスト基底膜マトリックス（ 図3A）に懸濁MDA-MB-231コロニーの（DIC）画像を撮影する。一日一回10倍の対物レンズでの像20代表のエリア5日間のコロニーの形態形成（ 図3C）を決定する。 </li><li>細胞コロニー星状形成（ 図3B）を決定するために盲目的に画像を分析。コロニー細胞のスフェロイドからの1つ以上の突起が知覚される場合星状であると見なされる。 、侵襲性星状コロニーの割合を決定取得画像あたりの細胞コロニーの総数で星状コロニーの数を分割し、各20日の画像の星状コロニーのパーセンテージを平均化する。 </li></ol><img alt="図1" fo:content-width="5in" src="/files/ftp_upload/51341/51341fig1highres.jpg" width="500" /> 図1基底膜マトリックス（埋め込 ​​み技術）中のMDA-MB-231乳癌細胞の3次元培養。 AB）ドラフト内で行わ実験の模式図（）。C）コーティングされたガラスボトムディッシュのほか基底膜マトリックスを50μlと少なくとも30分間凝固させるマトリックスを可能にする（）、5％CO 2、37℃で細胞培養インキュベーター中に置いた。D）ディッシュ（複数可）E）細胞をトリプシン処理した。F ）細胞を円錐管に存在する。GH）細胞を（）組織培養遠心機で3分間100×gでスピンダウンした15ミリリットルコニカルチューブに再懸濁した。i）細胞ペレットをスピンダウンされています。J）細胞（）マイクロ遠心チューブに分注し4個の細胞を2.5×10）L。K）細胞を血球計を用いて計数した。FBSを補充したRPMI培地1mlに再懸濁し、50μlの総体積を得るように適切なメディアを使用してオフに突破した。M 1:1の比率のステップ1.5からのマトリックスを含むマイクロチューブにステップ1.7からの）ミックス細胞（50μl中25,000細胞）;最終容量は100μなります。。、L、N）静かに行列を100μlプレート：ステップ8からの細胞混合ステップ1.2から固化したマトリックスでコーティングされたディッシュにO）行列たら：細胞混合物が固化するが、に、FBSを補充したRPMI培地の2ミリリットルを追加一品。P）は 、それが実験の残りのために保存されるインキュベーター内の皿を置きます。 免疫蛍光で3D培養の形態形成機能の2。検討<ol><li>セットアップ製氷皿、冷リン酸緩衝溶液（PBS）、20％アセトン：80％メタノール（固定液）、および3％ウシ血清アルブミン（BSA;ブロッキング溶液）皿を取り出す前に（ 図2A）（複数可）インキュベーターから。 </li><li>製氷皿と吸引メディアにディッシュ（ES）を配置し、冷却したPBSの2ミリリットル（ 図2B）で3回洗浄します。 </li><li> 図2C（皿（複数可）中に80％メタノール溶液：最後のPBS洗浄を吸引した後、20％の2mlのアセトンを追加</st）&gt;栄4℃（氷上で、または冷蔵庫内のいずれか）（ 図2D）で20分間細胞を固定します。 </li><li>固定の20分が終了すると、室温での料理を持ち帰る固定液を吸引し、その後、PBSで3回洗浄します。最終的なPBS洗浄を吸引した後、室温（ 図2E）で少なくとも30分間ブロックするディッシュ（ES）への3％BSAの2ミリリットルを追加します。 </li><li>ブロック期間中にブロッキング緩衝液3％ウシ血清アルブミン（BSA）中に溶解し（適切な希釈で）一次（ラミニンV 1:100）および二次抗体の希釈液を調製する。細胞混合物：「BSA +一次抗体」ソリューションμL400〜500は、マトリックス上に直接追加する必要がありますのでご注意ください。 </li><li>ブロッキングの30分の有効期限が切れた後、一次抗体を添加して、室温（ 図2F）で少なくとも1時間インキュベートする。何PBS洗浄は30分blockinに続いて実行されるべきではないことに注意してくださいG期間。 </li><li>ステップ2.6が完了すると、「BSA +一次抗体」溶液を除去し、PBSで3回洗浄します。 </li><li>直接マトリックス上に（適切な希釈時）の3％BSAに溶解した二次抗体を追加します。細胞混合物、料理をカバーして、室温（ 図2G）で1時間インキュベートする。 </li><li> 「BSA +二次抗体」溶液を除去し、PBSで3回洗浄します。 </li><li> （ 図2H）核染色のためにPBSに溶解し、ヘキスト33258（1:10,000）の2ミリリットルを追加し、アルミホイルの下で5分間インキュベート（または光退色を制限する不透明の容器で覆い）。 </li><li>ヘキスト33258トンとのインキュベーションの5分の有効期限が切れた後、PBSで（ES）5倍をお皿を洗う。 </li><li>直接マトリックス上に封入剤を追加し、共焦点ディッシュ中の細胞混合物と基底膜matriにおけるコロニーの完全性に任意の混乱を誘発防止するために、慎重にカバーガラスでカバーX：細胞混合物（ 図2I）。 </li><li>ディッシュ（ES）は、室温（ 図2I）で一晩（または24時間）乾燥させます。乾燥したら、お皿（ES）は、-20℃で保存することができますが、それは非常に彼らは可能な限り迅速に画像化されるべきであることをお勧めします。 </li><li> （ 図3D-E）使用される抗体の適切なレーザー波長を有する蛍光顕微鏡を用いて画像を取得する。 </li></ol><img alt="図2" fo:content-width="5in" src="/files/ftp_upload/51341/51341fig2highres.jpg" width="500" /> 図2。免疫蛍光法によって3次元培養の検討。であって、a）実験装置の模式図は、B）ディッシュ（ES）製氷皿の上に置き、培地を吸引。 。続いて、細胞を冷PBS C）20％の2mlのアセトン2mlで3回洗浄した：80％のメタノール溶液をディッシュ（複数可）に添加しD）のための細胞を固定BSAは、室温で少なくとも30分間ブロックする皿（複数可）に加え、4℃（氷上でのいずれか、または冷蔵庫で）E）、3％、2 mlで20分F）の30分間一旦ブロッキングは、一次抗体を添加し、室温で少なくとも1時間インキュベートし、有効期限が切れているG）の適切な希釈率で3％BSA（）に溶解させた二次抗体は、基底膜マトリックス上に直接添加した：細胞混合物; 。続いて皿を覆い、室温で1時間インキュベートH）、ヘキスト2mlの（1:10,000; PBSに溶解）をアルミホイルで5分間インキュベートしdishe（ ​​複数可）、細胞に添加I）装着培地に直接添加した。マトリックス上：共焦点皿と皿中の細胞混合物をガラスカバースリップで覆われている。ディッシュ（ES）を室温で一晩（または24時間）乾燥させ。

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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=beta4+integrin-dependent+formation+of+polarized+three-dimensional+architecture+confers+resistance+to+apoptosis+in+normal+and+malignant+mammary+epithelium.">beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium.</a> Cancer Cell. 2, 205-216 (2002).</li><li>Cvetkovic, 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=KISS1R+Induces+Invasiveness+of+Estrogen+Receptor-Negative+Human+Mammary+Epithelial+and+Breast+Cancer+Cells.">KISS1R Induces Invasiveness of Estrogen Receptor-Negative Human Mammary Epithelial and Breast Cancer Cells.</a> Endocrinology. , 1999-2014 (2013).</li><li>Alemayehu, 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=beta-Arrestin2+regulates+lysophosphatidic+acid-induced+human+breast+tumor+cell+migration+and+invasion+via+Rap1+and+IQGAP1.">beta-Arrestin2 regulates lysophosphatidic acid-induced human breast tumor cell migration and invasion via Rap1 and IQGAP1.</a> PLoS One. , (2013).</li></ol>

我々は、開示することは何もありません。

三次元細胞培養技術の開発は、研究者たちは、劇的な形態学的変化を視覚化できるように、乳房上皮細胞の形質転換を研究することを可能にした。細胞浸潤を分析するほか、単一または多細胞スフェロイドは乳房上皮細胞の接着、増殖、サイズ、および基底頂端極性の変化を評価するために使用することができる。細胞は、ECM 8を重ねている、以前に報告された方法論とは対照的に、...

個人または集団の細胞の移動および浸潤癌の2特徴である、癌細胞1-4の転移拡散のために必要。転移を開始する癌細胞の能力は、遊走細胞の基底膜を分解するために浸潤突起を使用して、隣接組織に侵入するそれらの能力に依存する。浸潤突起はマトリックス分解プロテアーゼ5の放出を介して細胞外マトリックスの分解を可能にする動的なアクチンが豊富なマトリックス分解突起である。癌細胞浸潤は、癌細胞の移動、続いてマトリックスの分解を伴い、これは三次元（3D）マトリクス環境2の再編を伴う。このようにして、マトリックスを貫通するように、セルは、その形状を変形し、細胞外マトリックス（ECM）2と対話する必要があります。 乳房組織の完全性の維持は、緊密に依存controlled個の組織構造の細胞ECMおよび細胞-細胞接着接合は、上皮極性の遺伝子発現および破壊に影響を与えるので6-10癌の発症につながる可能性がある。しかし、このようなトランスウェルチャンバーアッセイまたは創傷スクラッチアッセイなどのほとんどのin vitroでの遊走および浸潤アッセイは、2次元（2D）であり、したがって、これらは、細胞とその隣接する環境3,6,8,11-14間の複雑な相互作用を無視。細胞形態における変化、細胞分化、細胞-マトリックス接着、遺伝子発現パターンを含む、かなりの形態学的および機能的多様性は、一般に、2D 2,6,8,11をアッセイに欠けている3D培養物中で細胞を培養することによって検出されている。このように、診療所6月10日に、基礎研究における画期的な成果のより良い翻訳につながる、3Dアッセイの生体状態のより生理学的にrecapitulatingで有意に有益であり、使用しています。しかしながら、留意すべきである、3D培養物を用いて得られる多くの利点にもかかわらず、このモデルは、種々の細胞型を含むインビボでの腫瘍微小環境の複雑さのすべてをキャプチャすることはできません。しかし、癌細胞の接着および浸潤15-17上の腫瘍間質の相互作用の効果を研究するために、3Dモデル（例えば、線維芽細胞、白血球、およびマクロファージ）への間質細胞を組み込むことが可能である。 培養中の乳房上皮細胞は、ラミニンおよびコラーゲンなどのECMタンパク質が存在する場合に最も効果的に成長する。これは公知で、市販のマトリックス混合物は、エンゲルブレス-ホルム-スウォーム（EHS）マウス腫瘍から誘導され、マトリゲル基底膜マトリックス2,8として知られている。多数の技術が基底膜マトリックス2,8に3次元のコロニーとして上皮細胞を成長させるために確立されている。三次元基底膜マトリックスモデルは、悪性および非悪性の両方の乳房細胞を確立するために有効である成長、 生体内環境18,19で発生しているものに似ている。 MCF10A細胞は、非悪性乳腺上皮細胞である。基底膜マトリックス中で増殖させたときに、これらの細胞は、正常な乳房細胞のin vivo形質の展示および細胞増殖、細胞極性、及び管腔スペース8,12,20を確立するために、アポトーシスを制御受ける。さらに、MCF10A細胞の細胞核の外観はより密接21単層で培養されたものよりも組織中の乳房上皮細胞のものに似ている三次元培養物中で腺房を形成する。ビッセルおよび同僚による研究は、悪性細胞が高度に無秩序な表現型を示すので、ラミニンに富む環境の中で増殖させ、増殖を増加させ、減少したときに悪性の乳房細胞は非悪性乳房細胞から分化することができることを明らかにした最初の細胞 - 細胞接着、間葉マーカーの発現増加及び侵襲性構造体の数の増加は3,6,2を形成2。 セル環境の異常は、腫瘍形成に影響を与えることができる20。三次元培養法を効果的に腫瘍細胞とその周囲の環境との間で生じる通信を研究し、このような通信方法14,20,21,23タンパク質発現の影響を決定するために使用することができる。この記事では、侵襲性を分析するために、3D培養物におけるMDA-MB-231乳癌細胞を成長させるため、および上皮マーカーラミニン、細胞基底膜18,19,24の成分を用いて、上皮形態の損失を研究するための詳細な方法論を提供25。詳細な手順は、正確かつ再現性の任意の浸潤癌細胞により星状（侵襲性）構造形成を定量化する能力を提供し、そのようなMDA-MB-231、Hs578T細胞、MCF-7、またはT47Dなどの一般的な乳癌細胞株（に限定されない）。従って、このアッセイは、タンパク質とどのように細胞内で発現または治療を評価するためのプラットフォームとして機能することができるプロまたは抗侵襲性化合物は、単一又は複数のセルにより、細胞外マトリックスの分解を調節する。

<table border="0" cellpadding="0" cellspacing="0" width="1062">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Material/ Equipment</td>
			<td style="width:265px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:355px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL tubes</td>
			<td>VWR</td>
			<td>CA10011-700</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100-mm culture dish BD353003</td>
			<td>VWR</td>
			<td>CABD353003</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Falcon tube</td>
			<td>VWR</td>
			<td>CA21008-918</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL filtered tips</td>
			<td>VWR</td>
			<td>10011-350</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-016</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-008</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35-mm glass-bottomed Confocal No.1 culture dishes&#160;</td>
			<td style="width:265px;">MatTek Corporation</td>
			<td>P35G-1.0-14-C</td>
			<td>Precooled before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>BioShop</td>
			<td>ALB003.100</td>
			<td>Used at 3% for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody, highly cross-adsorbed</td>
			<td>Life Technologies</td>
			<td>&#160;A11029</td>
			<td>1:250 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td>Life Technologies</td>
			<td>&#160;A11011</td>
			<td>1:1200 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-Beta-Catenin&#160;</td>
			<td>BD Transduction Laboratories</td>
			<td>610153</td>
			<td>Mouse-monoclonal; Used at 1:100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F1051</td>
			<td>Used at 10% (v/v)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33258, Pentahydrate (bis-Benzimide) - 10 mg&#8260;mL Solution in Water</td>
			<td>Life Technologies</td>
			<td>H3569</td>
			<td>Used at 0.1% (1:10000 dilution)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InVivo Analyzer Suite&#160;</td>
			<td style="width:265px;">Media Cybernetics</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kisspeptin-10 (KP-10)</td>
			<td style="width:265px;">EZ Biolabs</td>
			<td style="width:119px;">PT0512100601</td>
			<td>Used at 100nM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Laminin&#160;</td>
			<td>Cedarlane</td>
			<td>AB19012(CH)</td>
			<td>Rabbit-polyclonal full length human; Used at 1:100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSM-510 META laser scanning microscope&#160;</td>
			<td style="width:265px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>Used at 63X objective; oil immersion lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel phenol red free (BD356237)</td>
			<td style="width:265px;">VWR</td>
			<td>CACB356237</td>
			<td>&#160;Lot No.2180819; 10.4 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus IX-81 microscope&#160;</td>
			<td style="width:265px;">Olympus</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Antibiotic (added to media; used at 0.01%)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL pipet&#160;</td>
			<td>VWR</td>
			<td>CA53300-523</td>
			<td>Sterile, disposeable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium with Glutamine</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td>Used for culturing of MDA-MB-231 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA (1X), Phenol Red</td>
			<td>Life Technologies</td>
			<td>25200-072</td>
			<td>Used to trypsinize MDA-MB-231 cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">MEGM (bullet kit): MEBM (CC3151)+Single quots (CC4136)</td>
			<td style="width:265px;">Lonza</td>
			<td style="width:119px;">CC-3150</td>
			<td>Used for culturing of MCF10A cells</td>
		</tr>
	</tbody>
</table>

quantification of breast cancer cell invasiveness using a three-dimensional (3d) model

現在では周知の細胞および組織の微小環境は、腫瘍の開始と進行に影響を与える重要な調節因子であることが知られている。さらに、細胞外マトリックス（ECM）は、in vivoでの培養およびホメオスタシスにおける細胞の挙動の重要な調節因子であることが実証されている。二次元（2D）上で細胞を培養する現在のアプローチは、細胞及びそれらの微小環境との間の複雑な相互作用の妨害及び損失のプラスチック表面をもたらす。三次元（3D）培養アッセイの使用を介して、細胞-微小環境との相互作用のための条件は、in vivoでの微小環境に似ている確立されている。この記事では、周囲の環境への細胞浸潤の評価における3D培養の可能性を例示する、三次元基底膜タンパク質マトリックス中に乳癌細胞を成長させるための詳細な方法を提供する。加えて、我々はこれらの3Dアッセイはmolecシグナリングの喪失を検査する可能性を有する方法について説明手続きを免疫染色することにより上皮形態を規制ULES。これらの研究は、乳癌の広がりに必要な浸潤を調節するプロセスに重要な機構の詳細を識別するために助ける。

3Dマトリックスに侵入するMDA-MB-231細胞の例は、 図3Cに示されている。細胞は、マトリックス（1日目）に埋め込 ​​まれ、3日目で、侵襲性（星状）構造の形成を開始し、完全に5日目（ 図3C）によってマトリックスに侵入している。星状に形成されたコロニー数をカウントし、（侵襲性及び非侵襲性）ディッシュあたりのコロニーの総数のパーセンテージとして表す?...

Watch this Scientific Journal Video about Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model at JoVE.com

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

この資料は、乳癌細胞の浸潤を定量化するための3次元（3D）のアッセイの使用のための詳細な方法論を提供する。具体的には、細胞が侵入したときに発生する膜の完全性の喪失を検査するためにこのようなアッセイ、定量化、およびデータ分析だけでなく、メソッドを設定するために必要な手順を説明します。

3次元（3D）モデルを用いた乳癌細胞浸潤の定量化

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

quantification-breast-cancer-cell-invasiveness-using-three

Research

JoVE Journal

Medicine

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

15.7K ビュー.  University of Western Ontario. この資料は、乳癌細胞の浸潤を定量化するための3次元（3D）のアッセイの使用のための詳細な方法論を提供する。具体的には、細胞が侵入したときに発生する膜の完全性の喪失を検査するためにこのようなアッセイ、定量化、およびデータ分析だけでなく、メソッドを設定するために必要な手順を説明します。 

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

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.

磁気共鳴イメージング、体積CT画像と超音波を用いた乳がんの骨転移のヌードラットモデルにおける血管新生のマルチモーダルイメージング

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

医学

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

三次元異<em&gt; in vitroで</em卵巣がんの発生と進行中に間質相互作用の上皮&gt;モデリング

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

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 ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

初代ヒト骨髄およびその悪性腫瘍を研究する三次元組織培養モデル

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

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

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

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

乳癌の新規トランスジェニックマウスモデル：アデノ-Creを持つ乳管上皮の標的とすることにより、転移性乳癌の開始

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

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.

乳癌細胞におけるエストロゲン調節マイクロRNAのプロファイリング

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

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

三次元文化：乳細胞の悪性形質転換正常対腺房アーキテクチャを研究するためのツール

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. 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 ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三次元（3D）腫瘍スフェロイド浸潤アッセイ

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

3Dの設定で侵略を評価するために、癌細胞スフェロイドアッセイ

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

Methods Article

3次元（3D）モデルを用いた乳癌細胞浸潤の定量化