この作品はヒラリークラフトと卿ダグラスマイヤーズに特別な言及にインペリアルBRC、ヘルスリサーチ研究所、およびアクション対がんによってサポートされています。

以下の手順では、倫理的にインペリアル·カレッジ、ロンドンによって承認されている。 ヒト乳癌細胞株からの一次マンモスフィアの1世代注：無菌培養フードの下で、次の手順を実行します。 <ol><li>マンモスフェアメディア2mMのL-グルタミン、100 U / mlペニシリン、100 U / mlストレプトマイシンを補充したDMEM / F12を含む準備する。 、10ng / mlの組換えヒト塩基性線維芽細胞増殖因子（bFGF; R＆D Systems社）及び1×B27サプリメント、20ng / mlの組換えヒト上皮細胞増殖因子（シグマEGF）を添加することによって使用直前に完全培地を調製する。 </li><li>接着性のMCF-7またはMDA-MB-231細胞（または任意の乳癌細胞株; 70〜80％コンフルエンス）を含むフラスコから培地を吸引除去は、1×PBSで2回洗浄し、細胞をトリプシン処理。 </li><li> 5分間室温で200×gで遠心分離細胞。マンモスフェアメディア1-5 mlの上清と再懸濁細胞をデカントします。ピペット40μmのセルストレーナーキャップフィルタ上下に、必要に使用した場合、単一細胞懸濁液を得た。単一細胞懸濁液が形成された（ない場合は、3回まで細胞懸濁液を注射器25 G針を使用）していることを確認するために血球計数器を使用してください。 </li><li> FACS経由必要な分離株のCD44 + CD24-細胞のサブセットは、抗CD24-フィコエリトリン（PE）および抗CD44フルオレセインイソチオシアネート（FITC）モノクローナル抗体9を使用している場合。また、磁気活性化細胞ソーティング抗CD44及び製造者の指示に従って、抗CD24ビオチン結合されたマイクロビーズとの（MACS）システムを使用してこのような集団を単離。 LDカラムを使用して、LSカラムを用いて正の選択、および負の選択を行い、17フローサイトメトリーにより、すべての単離された細胞の表現型を確認する。 </li><li>トリパンブルーを用いて1mlあたり生存細胞の数を計算します。 注：細胞生存率はhemocy上のグリッド内のセルの総数で割った生存細胞の数として計算される光度計。パンブルーを取る細胞は非生存と考えられている。以下の手順を正確に生存細胞の割合を決定するために使用される。 <ol><li> PBS中トリパンブルーの0.4％溶液を調製する。細胞の1ミリリットルのパンブルーストック溶液0.1ミリリットルを追加します。血球計数器をロードし、低倍率で顕微鏡下ですぐに調べる。総細胞数及び青色染色細胞の数を数える。 生存細胞= [1.00 - （青色細胞の数÷全細胞の数）]×100。 </li><li>培養物1mlあたりの生存細胞数を計算するには、以下の式を使用します。希釈係数を補正することを忘れないでください。 10 4×1.1 =細胞/ mlの文化×生細胞の数</li></ol></li><li> 6ウェルの超低付着性プレートの各ウェルに完全なマンモスフェア培地2ml中の細胞の予め決定された量を再懸濁する。播種密度は、通常、ウェル当たり500-4,000細胞/ cm 2の細胞である。必要に応じてLOを使うワットアタッチメント24ウェルプレートは、培地の0.5mlの同じ濃度で細胞を播種した。 注：私たちは、対象となる各細胞株について播種密度と文化の時間の最適化をお勧めします。 </li><li>特に最初の5日間の増殖期の間に、プレートを乱すことなく（細胞株およびマンモスフィアのサイズに応じて）5~10日間、37℃、5％CO 2での超低接着6穴プレートをインキュベート。 </li></ol>ヒト乳癌臨床サンプルからの一次マンモスフィアの2世代注：ヒト乳癌組織は、乳房腫瘍を除去するための手術を受けた患者から得ることができる。 100 U / mlペニシリンおよび100 U / mlのストレプトマイシンを含む培地50mlの滅菌チューブ中で24時間まで氷上店組織。無菌培養フードの下に、次の手順を実行します。 <ol><li>メディアの少容量で100ミリメートル組織ペトリ皿にサンプルを転送します。 Remov無菌のはさみ、scapelとピンセットを使用して電子脂肪組織。 </li><li> DMEM / F12 2-3 mlを加え大きな片がなくなるまで、無菌scapelやカミソリの刃で1ミリメートル3枚にサンプルをミンチ。 </li><li>タンパク質分解酵素（3,000 U / mlコラゲナーゼ1,000 U / mlのヒアルロニダーゼ）を含む予め温めておいた10ミリリットルDMEM中で組織片を再懸濁し、すべての組織断片が消化されるまで、ロータリーシェーカーで37℃でインキュベートする。通常、完全な消化は1-3時間かかる。腫瘍の病理学的な文字によると消化時間を調整します。 （例えば、乳腺癌はより壊れやすく、短い消化時間を必要と粘液癌に比べて消化することが通常困難です）。血球計数器の下に20μlのサスペンションを観察することによって消化ごとに1/2時間の程度を評価。 </li><li>断片はその後、5分間沈降室TEMで10分間200×gで15ミリリットルコニカルポリプロピレンチューブと遠心分離機で上清を転送できるようにする温度。マンモスフェアメディア5mlの - 慎重に1上清および再懸濁細胞をオフにデカント。 </li><li>単一細胞懸濁液を得て、メッキする細胞株について上記のステップ1.1から1.4に従ってください。必要に応じて25〜Gの細胞が損傷した細胞を制限するために2回の最大シリンジ渡します。 </li></ol> 3.マンモスフェア効率（％）計算の形成<ol><li>培養期間の後、40倍の倍率で顕微鏡下でマンモスフィア（より大きい40μm）の直径を数える。光学顕微鏡にデジタルカメラを使用してデジタル画像5ランダムフィールドおよび取得ソフトウェアを使用して、マンモスフィアのサイズを決定する。任意の画像解析ソフトウェアを使用してください。 </li><li>マンモスフェアは、以下の式を使用して効率（MFE％）を形成する計算する： MFE（％）=（ウェル当たりマンモスフィアの＃）/（ウェル当たり播種した細胞の＃）×100 </li></ol>自己再生の評価のためのマンモスフィアの4連続継代<ol><li&gt;ピペット15ミリリットルチューブに各ウェルからマンモスフィアを含む培地。 PBSでウェルを洗浄し、収集したメディアに追加する。室温で10分間、115×gで遠心する。 </li><li>上清を捨て、予め温めておいた0.5％トリプシン/ 0.2％EDTAを500μlにペレットを再懸濁。 2-3分間インキュベートする。 </li><li>トリプシンを中和するためにFCSの500を添加する。 5分間500×gで遠心分離する。 、マンモスフィアメディアの100μlの上清と再懸濁ペレットを廃棄ピペッティング球を脱凝集する。 </li><li>血球計で細胞をカウントし、細胞を単一細胞懸濁液にするかどうかを判断。 25 Gを通過しない場合は、単一細胞を得るために3回まで注射器。主要生成に使用したのと同じ密度で新しい超低接着6ウェルプレートに細胞を播種する。 </li><li> 5-10日後、&gt;40μmの球をカウントし、前に報告された式を使って球形成効率を計算。 </li></ol>

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The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this article.

プライマリとセカンダリのマンモスフィアの成功評価は、マンモスフィアは、最小限の球凝集に、単一のクローンから形成する十分に低い密度でメッキされる細胞に依存しています。しかし低すぎる密度で、数が少なすぎるマンモスフィアは、統計学的治療の効果を区別するために形成することができる。播種密度は、彼らは彼らの球の形成効率が大幅に変化することができるので、使用される各細胞株について最適化する必要があります（低E-カドヘリンを発現するものが少なく安定しており、短期のマンモスフェア培養18を形成することができる）。この最適化段階は、単一細胞のクローン成長からほとんどのプライマリとセカンダリの球形成の結果を確認する必要があります。ウェルごとに1つの細胞をプレーティングすることは、完全にクローンマンモスフェア形成を確実にする唯一の方法であり、新たに得られた試料13の自己再生/分化率を定量化するために推奨することができる。しかし、このアプローチは、複雑な要因に苦しんでいる：マンモスフェアが形成1％の効率は、96ウェルプレートあたり単一マンモスフェアが得られると、効率が懸濁された細胞間のシグナリングパラクリン成長因子の非存在下で形成されているより少ないマンモスフィアを過小評価することができる。最後に、継代後の二球の頻度は、自己再生のレベルを推定することができ、真にがん幹細胞特性を有する細胞を選択した場合、一次球の形成よりも二次のより高い効率が期待されるべきである。再凝集は、二球及び細胞型に適切な非常に低い細胞密度を導出するに向かって最適化されるべき特定の問題である。 &gt; 40μmの成長するマンモスフィアのにかかる時間も変化し、臨床サンプルからマンモスフィアは、球の成長速度に応じて、5-12日の間にカウントする必要があるかもしれません。球体の凝集、融着が必然的にメディアの周りの細胞の固有の移動に起因し発生するが、このプロセスは、実質的に元によって増強されるようである増殖期の間にプレート19を移動させるperimentersは、したがって、何かが回避される。 大きな球体は、日常この仮定は慎重に扱われるべきであるより高い長期の複製能を有する幹細胞に由来すると考えられている。大きな球は、前駆細胞の増殖能力を反映し、増殖因子に対する応答、または凝集または融合の産物であり得る。両方の細胞型の合成周波数は依然として元の組織の腫瘍形成能を反映することができるが、両方の幹細胞およびそれらのトランジット増幅娘細胞は、マンモスフィア20を生じさせることが可能である。 マンモスフェアアッセイは、それらのインビボニッチ中の癌幹細胞の形成および動作の複雑性を捕捉する障害によって制限される。真にインビトロおよびインビボ自己再生およびプロスペクティブ単離さ乳癌幹細胞の分化、マンモに長期を評価する球アッセイは、したがって、理想的に系統のトレースと異種移植実験と併せて行われるべきである。系譜トレースが正確にがん幹細胞サブセットを反映したマーカーに依存しているが、これらは「骨の善意」がん幹細胞の動的な形成に寄与、生体内の状態を保持から大きな恩恵を受ける。まだヒト免疫および間質部品21を欠いている間免疫不全マウスにおける異種移植実験で腫瘍形成を評価することはまた、マンモスフェアアッセイから欠落している多くのニッチなコンポーネントを提供します。もっと技術的に厳しいが、これらの技術は、乳癌幹細胞の特性と腫瘍形成のより完全かつ有益なビューのために試みるべきである。 マンモスフェアアッセイは、有益で便利なツールを提供し、それは慎重に行われ、上記の制限を考慮提供：確かに、生体内コンディの我々の知識としてがん幹細胞の進歩によって経験ン、マンモスフェアアッセイは、分子と環境因子の増加範囲を組み込むことができる。上記で概説したアプローチを用いて、球形成率は、異種移植片2,15,16における腫瘍イニシエーション·レートに対応し、球の自己再生は、より攻撃的な乳癌の22の増加と化学療法23などの後の幹細胞の増加介入を行う。一方、球体自体が発現差および幹細胞遺伝子発現のアーキテクチャに関与する新規因子を研究する際の価値ある資源を提供することができる。

幹様がん幹細胞が率いる腫瘍細胞系列の存在が大幅に腫瘍の不均一性の我々の理解に追加されました。腫瘍におけるいくつかの表現型の多様性は遺伝的に異なるクローンのクローンの成長から生じませんが、かなりのコンポーネントは、エピジェネティックな違いに起因すると表示されます。癌細胞は、特定の遺伝子の活性化または抑制を経由して幹、前駆、および分化した状態の間で（時には可逆的に）遷移することができる表現のプログラム1から3。これは、現在調節因子を提供する隣接癌、間質、または免疫細胞の傍分泌シグナル伝達と関連して、その結果自己分泌シグナル伝達を細胞内で発現された遺伝子発現プログラムを反映して、細胞内因性または外因性の要因を反映して、そのようなの程度microenviromental条件よい低酸素症2,4,5。 革新的な系統は、アプローチをトレースするが、それらのインビボニッチ6で推定される癌幹細胞を研究する当社の能力進めています- 8、球形成アッセイは、少なくとも使用されるアッセイ条件下では、幹細胞のように振る舞うように乳癌細胞」の可能性を推定するための普及した、便利なアプローチのまま。これは、しばしば膜マーカーCD44およびCD24 9、及び酵素ALDH（アルデヒドデヒドロゲナーゼ）10の活性レベルの発現によって精製癌幹細胞について遡及的な方法は、よりmesenchymal-上皮に対応するために提案されたマーカーと一緒に使用され様がん幹細胞をそれぞれ11。球形成のアプローチは、最初の上皮成長因子（EGF）12を添加して非接着、無血清条件下で単一クローン由来の推定幹細胞の増殖を可能にする、ニューロスフィアアッセイとして開発された、後で有用正常および癌に適用される乳房組織。 骨善意幹細胞は、 生体内で活性化を有利に働く要因の正確な組み合わせは発生しません。マンモスフェアアッセイは、代わりに有糸分裂に構えたり、すでに13を分割するいずれかの細胞の成長を可能にします。これらの前駆細胞ではなく、真に静止細胞が、アッセイにおいて使用されるEGFおよび塩基性線維芽細胞増殖因子（bFGF）マイトジェンで増殖する細胞段階であってもよい。それにもかかわらず、それらは活性化された幹細胞に関連するシグナル伝達経路14の範囲を含む。また、それらの形成の速度は、マウス異種移植片2,15,16の中で限界希釈アッセイにおけるそれらの効力により測定し、それらから採取された組織の腫瘍形成に関連する&lt;/ SUP&gt;。 ここでは、単一細胞を単離し、ヒト乳癌細胞株および乳房腫瘍の臨床試料の両方からの一次マンモスフィアを生成するための詳細なプロトコルを提供する。我々はまた、自己再生を評価するために主要なマンモスフィアの連続継代を実行する方法を説明し、どのように異なる播種密度間での比較は（ 図1のスキームを参照）を可能にする効率を形成する球を計算する。

<table border="0" cellpadding="0" cellspacing="0" width="1049">
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			<td height="21" style="height:21px;width:625px;">Name of Material/ Equipment</td>
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			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12</td>
			<td style="width:139px;">Lonza</td>
			<td>CC-3151</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:625px;">2mM L-Glutamine</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>G8540</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:625px;">100U/ml Penicillin &#38; Streptomycin</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>P4083</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:625px;">20ng/ml recombinant human epidermal growth factor (EGF)</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>E9644</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:625px;">20ng/ml recombinant human basic fibroblast growth factor (bFGF)</td>
			<td style="width:139px;">R&#38;D systems</td>
			<td>233-FB-025</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:625px;">1x B27 supplement&#160;</td>
			<td style="width:139px;">Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Phosphate buffered saline (PBS);</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td>12399902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;0.5% trypsin-0.2%EDTA;</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>59418C</td>
			<td></td>
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			<td height="21" style="height:21px;">Fetal Calf Serum</td>
			<td style="width:139px;">First Link UK</td>
			<td>02-00-850</td>
			<td></td>
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			<td height="21" style="height:21px;">&#160;Trypan Blue</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>93595</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">&#160;Low attachment 6 well plates</td>
			<td style="width:139px;">Corning</td>
			<td>CLS3814</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase type 1A</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>C9891</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hyaluronidase</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile razor blades</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td>12443170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:625px;">Sterile scalpel</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">11758353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:625px;">Sterile micro-dissecting scissors</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>S3146</td>
			<td></td>
		</tr>
	</tbody>
</table>

mammosphere formation assay from human breast cancer tissues and cell lines

より分化した子孫を生じることながら健康な組織に似ては、多くの血液および固形の悪性腫瘍は、現在、自己再生幹様癌細胞のサブセットで、階層的に組織化されると考えられている。非幹腫瘍の大きさと比較して強化された化学療法および放射線抵抗性を有することが、乳癌、これらの癌幹細胞を標的と理解とは、重要な研究分野となっている。マンモスフェアアッセイはまた、広く遡及sphere-を同定する能力について用いられるようになっている：CD44、CD24、およびALDH活性を含むマーカーは、将来に向かって、免疫不全マウスに移植したときに増強された腫瘍形成性を示す細胞を単離するために蛍光活性化細胞選別（FACS）を用いて評価することができる単一の幹細胞様のクローンから発生セルを形成する。ここでは、適切な細胞株または一次患者サンプルからマンモスフィアの培養、彼らの継代、及び球体Fを推定する計算のためのアプローチを概説orming効率（SFE）。まず、適切な計画とマンモスフェア実験の解釈の重要な考慮事項と落とし穴を議論する。

異なるサンプルまたは異なる処理を施したものは播種初期セルに対して正規化した後に形成マンモスフィア&gt;40μmの数が変化してもよい。三重で成長し、各治療のためにマンモスフェア形成効率（MFE）を計算します。これは、異なる播種密度を用いた実験を比較することを可能にする。データは、最高の理想的な陽性および陰性対照を、棒グラフに表示し、三つ組のウェルで標準偏差を表示している。一貫して60〜80％コンフルエンスの間に非接着性プレートに付着細胞を移す。慎重な細胞計数は正確に治療の効果を定量化することが不可欠である。この重要なステップはよく同じ処理とは別に、複数の懸濁液を調製することにより、検索結果に寄与している変動率を推定します。測定される細胞濃度が同じウェルと著しく異なる場合には、（ 図2）を計数アプローチを精製考える。 <pキープtogether.withinページ= &quot;常に&quot;&gt;：FOクラス= &quot;jove_content&quot; <img alt="図1" src="/files/ftp_upload/52671/52671fig1.jpg" /> 図1：原発性乳癌及び細胞株試料の処理は、マンモスフェア培養物を得るために、サンプルは、酵素的に消化され、単一の細胞として、検証、および成長因子無血清マンモスフェア培地中で非接着性プレート上にプレーティングする。プレートは、球の融合を回避するために、増殖期中に処理すべきではありません。右の写真は、5日後に撮影した代表的なMCF-7球を示している。 <a href="https://www.jove.com/files/ftp_upload/52671/52671fig1highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/52671/52671fig2.jpg" /> 図2：正上皮エストロゲンから形成されたマンモスフィアの代表的な結果（MCF-7）、および間葉三重negatiVEの（MDA-MB-231）細胞株。最小細胞融合/凝集は、MCF-7、500細胞/ cm 2で、ここで、低密度めっきにより得、1,000細胞/ cm 2 MDA-MB-231のためにした。

Watch this Scientific Journal Video about Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines at JoVE.com

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

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

ヒト乳癌組織および細胞株からマンモスフェア形成アッセイ

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

mammosphere-formation-assay-from-human-breast-cancer-tissues-cell

Nanyang Technological University

Research

JoVE Journal

Medicine

33.1K ビュー.  Imperial College London. Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

ビデオ: Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

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 ...

医学

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

細胞培養のための人間の削減乳房形成や乳房切除組織の処理

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

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 ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

個々の腎細胞がんの中のタンパク質発現変動を探るために逆相タンパク質アレイ（RPPA）の使用

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

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 ...

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 ...

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

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

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

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

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 ...

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, ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

アン<em&gt;エクスビボ</emヒト乳癌でホルモンの作用を研究するために&gt;モデル

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

培養ヒト大腿骨組織外植するための方法は、転移性ニッチの乳癌細胞コロニー形成を研究する

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...