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?...

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><tbody><tr><td>DMEM/F12</td><td>Lonza</td><td>CC-3151</td><td></td></tr><tr><td>2 mM L-glutamine</td><td>Sigma Aldrich</td><td>G8540</td><td></td></tr><tr><td>100 U/ml Penicillin &amp;amp; Streptomycin</td><td>Sigma Aldrich</td><td>P4083</td><td></td></tr><tr><td>20 ng/ml Recombinant human epidermal growth factor (EGF)</td><td>Sigma Aldrich</td><td>E9644</td><td></td></tr><tr><td>20 ng/ml Recombinant human basic fibroblast growth factor (bFGF)</td><td>R&amp;amp;D systems</td><td>233-FB-025</td><td></td></tr><tr><td>1x B27 supplement</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Thermo Scientific</td><td>12399902</td><td></td></tr><tr><td>0.5% trypsin/0.2% EDTA</td><td>Sigma Aldrich</td><td>59418C</td><td></td></tr><tr><td>Fetal calf serum</td><td>First Link UK</td><td>02-00-850</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma Aldrich</td><td>93595</td><td></td></tr><tr><td>Low attachment 6-well plates</td><td>Corning</td><td>CLS3814</td><td></td></tr><tr><td>Collagenase type 1A</td><td>Sigma Aldrich</td><td>C9891</td><td></td></tr><tr><td>Hyaluronidase</td><td>Sigma Aldrich</td><td>H3506</td><td></td></tr><tr><td>Sterile razor blades</td><td>Fisher Scientific</td><td>12443170</td><td></td></tr><tr><td>Sterile scalpel</td><td>Fisher Scientific</td><td>11758353</td><td></td></tr><tr><td>Sterile micro-dissecting scissors</td><td>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?...

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

Research

JoVE Journal

Medicine

33.2K 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

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.

The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

マイクロアレイを用いてがん細胞型に及ぼす微小環境への影響を調知る

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

がん研究

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

マウス乳房上皮HC11細胞とEpH4細胞の分化

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

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 ...

医学

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

ヒト乳腺上皮細胞における発癌性転換のインビトロ評価

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

細胞および組織サンプルからのヒト乳がん幹細胞の単離と機能評価

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

微小生理学的システムを用いたヒト乳房組織における乳癌のモデル化

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

マウス乳腺上皮細胞は乳腺刺激分化の過程でMammospheresを形成し、

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

生物学

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

乳がん研究のための潜在的機能 インビトロ モデルとしての乳腺上皮および内皮細胞スフェロイド

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Quantification of Self-renewal in Murine Mammosphere Cultures

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. The role of mammary stem cells (MaSCs) is widely studied both in the physiological/developmental context and with regards to breast carcinogenesis. In this aspect, ex vivo studies focused on MaSC properties are highly sought after. Mammosphere cultures represent a surrogate of organ formation and have become a valuable tool for both basic and translational research. Here, we present a detailed protocol for the generation of murine primary mammosphere cultures and the quantitation of MaSC growth properties. The protocol includes mammary gland collection and digestion, isolation of primary mammary epithelial cells (MECs), establishment of primary mammosphere cultures, serial passaging, quantitation of mammosphere growth parameters and interpretation of the results. As an example, we present the effect of low-level constitutive Myc expression on normal MECs leading to increased self-renewal and proliferation.

Here, we describe the implementation and interpretation of the results of an in vitro mammosphere self-renewal quantitative assay.

マウスマンモスフィア培養における自己再生の定量

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. ...

発生生物学

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

マンモスフィアの自然発生の事前侵襲乳房病変の非酵素的、無血清組織培養

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

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

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この動画の章

マイクロアレイを用いてがん細胞型に及ぼす微小環境への影響を調知る

マウス乳房上皮HC11細胞とEpH4細胞の分化

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

ヒト乳腺上皮細胞における発癌性転換のインビトロ評価

細胞および組織サンプルからのヒト乳がん幹細胞の単離と機能評価

微小生理学的システムを用いたヒト乳房組織における乳癌のモデル化

マウス乳腺上皮細胞は乳腺刺激分化の過程でMammospheresを形成し、

乳がん研究のための潜在的機能インビトロモデルとしての乳腺上皮および内皮細胞スフェロイド

マウスマンモスフィア培養における自己再生の定量

マンモスフィアの自然発生の事前侵襲乳房病変の非酵素的、無血清組織培養

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

要約

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マイクロアレイを用いてがん細胞型に及ぼす微小環境への影響を調知る

マウス乳房上皮HC11細胞とEpH4細胞の分化

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

ヒト乳腺上皮細胞における発癌性転換のインビトロ評価

細胞および組織サンプルからのヒト乳がん幹細胞の単離と機能評価

微小生理学的システムを用いたヒト乳房組織における乳癌のモデル化

マウス乳腺上皮細胞は乳腺刺激分化の過程でMammospheresを形成し、

乳がん研究のための潜在的機能 インビトロ モデルとしての乳腺上皮および内皮細胞スフェロイド

マウスマンモスフィア培養における自己再生の定量

マンモスフィアの自然発生の事前侵襲乳房病変の非酵素的、無血清組織培養

乳がん研究のための潜在的機能インビトロモデルとしての乳腺上皮および内皮細胞スフェロイド