我々は、細胞株のための試薬とフレッドMillerのケン岩田（OSIファーマシューティカルズ、ニューヨーク、アメリカ）（バーバラアンカルマノスがんIntitute、デトロイト、米国）に感謝しています。

1。メトセル溶液（500ml）の準備<ol><li>マグネチックスターラーを含む500mlのボトルにメチルセルロースのオートクレーブ6グラム。 </li><li> 20分間血清（60℃）なしで予熱DMEM/F12 250ml中にメチルセルロースを溶解する。 </li><li> 10％ウマ血清を含むDMEM/F12 250mlの（RT）を追加します。混ぜて一晩（4℃）。 </li><li>遠心分離（5000グラム、2時間、4℃）でソリューションをクリアします。 </li><li>新しいチューブ（ストック溶液の90〜95％程度）に透明な高粘度液を取る。 </li><li> 4℃、使用するまでC。 </li></ol> 2。スフェロイド培養<ol><li> 20パーセントメトセル溶液（10mlメトセル、40 mlの増殖培地）を調製する。 </li><li> PBSで細胞を2回洗浄、37℃で0.1％トリプシンとインキュベートセルを追加° C </li><li>増殖培地中で細胞を再懸濁し、細胞を数えます。 </li><li> 20％メトセルにmlあたり10 000細胞の懸濁液を準備します。 </li><li> 96ウェル丸底のために細胞懸濁液を追加します。uspensionプレート（ウェルあたり100μl）。 </li><li>翌日、サスペンションプレートの回転楕円体の形成を確認してください。スフェロイドは1-3日後に使用できるようになります。 </li></ol> 3。コラーゲンの調製<ol><li>氷上に8mlのコラーゲン、1mlの10倍PBS、1 mlの0.1 N NaOHおよび3滴0.1NのHClの溶液を調製します。 pHがpH指示薬ストリップ上の解の一部をスポッティングを7.4かどうかを確認します。範囲外の場合pHを調整する。 </li><li> 96ウェルプレートのウェル内で中和コラーゲン溶液50μlを加え</li><li>コラーゲンが固化されるまで37℃でインキュベート（90分）。 </li><li>氷の上メトセル - コラーゲン-リガンド溶液を調製：コラーゲン溶液のそれぞれのリガンドピペット1部のために。コラーゲンTGF -βの20ngのあたりのmlを加える。異なるレイヤー上のメトセルと拡散で希釈した後、TGF -βの最終濃度は、ボルテ​​ックスで5 ng / mlのミックスになります。 METHOCEL -ソリューションの1部を追加。ボルテックスにより混和する。 </li></ol> 4。回転楕円体を埋め込む鼎<ol><li> 200μlのピペットに、カットオフ先端から約5 mmを持っている200μlの先端を置きます。ピペットを使用すると、ワンスフェロイドを吸うと慎重に空のプレートに培地を分注する。回転楕円体の先端の内側にとどまる場合観る。回転楕円体は、白いドットとして表示されます。プランジ​​ャーを解放せずに、100μlのコラーゲンメトセルの混合物を吸い取ると、コラーゲンコート96ウェルにコラーゲンの混合物中の回転楕円体を分注する。各条件に12回転楕円体に対してこれを行います。 </li><li>コラーゲンは37℃で少なくとも30分を凝固させて° C </li><li> 200μlの先端で空気の泡を取り除く。 </li><li> DMSOに溶解した1.6％の血清と阻害剤と培地50μlを追加。 SB - 431542のためにストック溶液の4μlの（10mM）を培地1mlに追加します。拡散した後、阻害剤の最終濃度は10μMとなる。 TGF -βおよび阻害剤は​​、実験の過程であり続けるでしょう。 40X倍率を用いて顕微鏡下でスフェロイドの写真を撮ります。 </li><li> 4の後にTGF -βおよび/または阻害剤による刺激の8時間は、40倍の倍率を用いて顕微鏡下でスフェロイドの写真を撮る。 </li><li> 2日後に侵入する回転楕円体の面積を測定し、スタートエリアを引きます。の面積を測定する回転楕円体：Adobe Photoshopの拡張（図2A）の回転楕円体からの映像やクイック選択ツール（図2B）を選択し[開く。 &quot;プラス&quot;（+）記号（図2C）の円にマウスポインタの形が変化。回転楕円体の上にカーソルをドラッグしながら、Photoshopは回転楕円体の境界を認識します。 （ - ）ツールバーの[記号回転楕円体の外側の領域が選択されている場合は、この地域は、Altキーを押すかによって示される負の選択ツールを使用して、選択から除外することができます。 （ - ）&quot;マイナス&quot;の円にカーソルが変化記号と誤って選択した領域の上にカーソルをドラッグすることによっては、このエリアは、選択（図2D）から削除されます。必要に応じて、複数の領域はSHを押すことで選択できます。IFTのボタン。より正確に動作するように、マウスポインタのサイズは、ツールバーにあるブラシの直径（図2E）を変えることによって調整することができます。全体の回転楕円体が選択されている場合、選択の面積は、メニューバーの分析- MEASUREを介して、またはCtrl + Shiftキー+ M（図2F）を押すことによって計算されます。測定の記録ウィンドウには、ファイル名とピクセル単位で選択の領域だけでなく、他のデータ（図2G）を示すが表示されます。複数の選択が測定されている場合、最初の行はすべての領域の合計を示しています。個々の選択の測定は、下記の行で表示されます。すべての測定のデータはテキストフ​​ァイルにエクスポートし、Excelで開くことができます。 </li></ol> 5。代表的な結果： MCF10A1（M1）、正常な乳房上皮細胞とスフェロイドアッセイの例、H - RASはMCF10AneoT（M2）細胞を形質転換し、M2由来MCF10CA1a（M4）は、図3に示します。 M1細胞はなくだけ弱い侵攻を示した刺激が、TGF -βによる刺激により有意に優れて侵入した。対照的に、M2は、刺激の添加なしに効率的にすでに侵入したM1 -派生RAS -変換、およびこれをさらにTGF -β治療時に4〜5倍に増加した。 M4の細胞が両方でとTGF -βを添加していない、最強の侵略を示した。 次に、私たちは化学物質の阻害剤を使用して、この回転楕円体モデルではTGF -βによって誘導される侵攻を妨害する可能性があるかどうかを検討した。この目的のために我々は、SB - 431542、ATPのアナログとTβRIのキナーゼ活性の選択的阻害剤だけでなく、アクチビン型IB受容体（ActRIB）とアクチビン受容体様キナーゼ（ALK）7〜12を使用していました 。予想通り、SB - 431542は強力M1のTGF -β誘発性浸潤、そのTGF -β誘発性浸潤を示すM2とM4の細胞（図4）、TβRI-キナーゼ依存を抑制した。さらに、スフェロイドの基底侵攻も強く基礎侵攻もDEがいることを示唆し、阻害されたTGF -βで独立。 <img alt="図1" src="/files/ftp_upload/3337/3337fig1.jpg" /> 3D回転楕円体アッセイの図1。フローチャート。最初に、細胞はU字型サスペンションプレートの非接着性の条件下で播種されています。細胞はmultcellularスフェロイドにaggregrateとコラーゲンマトリックスに埋め込むことができます。このコラーゲンマトリックス中に、彼らは侵入することができます。 <img alt="図2" src="/files/ftp_upload/3337/3337fig2.jpg" /> 図2：Adobe Photoshopを使用してスフェロイドの面積の定量化が拡張。 （A）回転楕円体の画像が使用wongly含まれる領域の回転楕円体（D）除去の領域を選択する回転楕円体の上にカーソルをドラッグしてクイック選択ツールのAdobe PhotoshopのExtendend（B）の選択（C）で開かれますのより正確に領域を選択するには、クイック選択ツールのブラシの大きさの負のクイック選択ツール（E）調整（F）の選択測定は、コマンド（G）測定記録の例を記録する。 <img alt="図3" src="/files/ftp_upload/3337/3337fig3.jpg" /> 図3。自発的に不死化MCF10A1（M1）（A）のスフェロイドのTGF -β誘発性浸潤、MCF10AneoT（M2（B）とその転移性微分MCF10CA1a（M4）（C）。M1、M2およびM4スフェロイドが埋め込 ​​まRAS -変換示されるようにコラーゲンに48時間のためにTGF -βの5ng/mlで処理した。AC代表画像は埋め込 ​​みの一日で撮影し、二日後。D相対侵入が2日目マイナス0日目に面積の差として定量した。結果が表現されているとして平均値± SDは、13から脚色。 <img alt="図4a" src="/files/ftp_upload/3337/3337fig4a.jpg" /> <img alt="図4b" src="/files/ftp_upload/3337/3337fig4b.jpg" /> 図4基礎とTGF -βによって誘導される回転楕円体の侵入は、Bを阻害されるY SB - 431542。 M1、M2とM4球状体がコラーゲンに埋め込まれ、示されるように5ng/ml TGF -βおよび/または10μMSB - 431542で処理した。 2日間（）後に採取した代表的な写真。相対的な侵略は、2日目マイナス0日目（B）上の領域の差として定量化した。結果は平均± SDは、13から脚色として表現されています。

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利害の衝突は。

我々はMCF10A1細胞とその悪性誘導体はTGF -β依存的にコラーゲンマトリックス内に侵入した回転楕円体モデルを確立した。最初に、スフェロイドは、メチルセルロースの存在下で96ウェル丸底サスペンションプレートに形成されています。もちろん、またU字型のポリ- HEMAコートしたプレートは、この目的のために使用することができます。細胞はメチルセルロースのない状態で死んでしまうの?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（省略可能） </td></tr><tr><td>メチルセルロース</td><td>シグマアルドリッチ</td><td> M0512 </td><td></td></tr><tr><td> PureCol </td><td>高度なニッピ</td><td> 5005 - B </td><td></td></tr><tr><td> pH指示薬のストリップ</td><td>メルク</td><td> 9533 </td><td></td></tr><tr><td> 96ウェルサスペンション プレート</td><td>グライナーバイオワン</td><td> 650 185 </td><td></td></tr><tr><td> TC接着96ウェル プレート</td><td>グライナーバイオワン</td><td> 655 180 </td><td></td></tr></tbody></table>

spheroid assay to measure tgf-&#946;-induced invasion

TGF -βは、初期段階の腫瘍抑制因子として作用するが、後の段階の1,2で浸潤や転移を刺激することによって、乳がんの進行の役割を反対している。また、TGF -βは、頻繁に乳癌において過剰発現し、その発現は予後不良と転移3,4と相関している。 TGF -β誘導の侵入はよく理解されていないメカニズム。 TGF -βはTGF -βII型（TβRII）とI型（TβRI）受容体を介してその細胞応答を誘発する。 TGF -βによって誘導されるヘテロ複合体形成時には、TβRIIはTβRIをリン酸化する。活性化TβRIはすなわちSmad2およびSmad3、受容性Smad（R - Smadの）をリン酸化することによって、その細胞内の標準的なシグナル伝達経路を開始します。これらの活性化R - Smadは核に蓄積し、標的遺伝子5の転写を調節であるSmad4とヘテロ複合体を形成。以前にDに加えていくつかの他の非のSmadシグナル伝達経路の活性化の傍接したのSmad経路、受容体の活性化の結果は、例えば、マイトジェン活性化プロテインキナーゼ（MAPK）は6パスウェイ。 乳癌の異なる段階におけるTGF -βの役割を研究するために、我々は、MCF10A細胞システムを利用していました。このシステムは、自発的に不死化MCF10A1（M1）乳房上皮細胞7から構成され、H - RASは、マウス8で前癌病変を生成するM1 -派生MCF10AneoT（M2）、、およびから設立されたM2 -派生MCF10CA1a（M4）を、変換M2異種移植片およびフォーム肺9に転移する能力を持つハイグレードの癌腫。このMCF10Aシリーズは、異なる遺伝的背景によってバイアスされていない悪性の異なるグレードの細胞の応答を研究する可能性を提供しています。 TGF -β誘発性浸潤の分析のために、我々はホモタイプMCF10Aスフェロイド培養細胞のembeを生成試験管内 （図1） における 3次元コラーゲンマトリックスにdded。このようなモデルは、密接に回転楕円体10の内部に外側と静止あるいは壊死細胞上でアクティブと浸潤細胞で、その結果、酸素や栄養素の勾配を確立することによりin vivoでヒトの腫瘍に似ています。回転楕円体ベースのアッセイはまた、単層培養11時薬剤耐性を向上を再現することが示されている。このMCF10 3Dモデルのシステムでは、私たちは悪性腫瘍のさまざまな段階で乳がん細胞の浸潤特性に関するTGF -βシグナル伝達の影響を調査することができました。

Watch this Scientific Journal Video about Spheroid Assay to Measure TGF-ÃŽÂ²-induced Invasion at JoVE.com

Spheroid Assay to Measure TGF-&#946;-induced Invasion

定量的形質転換成長因子（TGF）-β誘導性3次元コラーゲンゲル内侵入を測定するアッセイが記載されている。このアッセイでは、乳がんの開発のさまざまな段階を表現する細胞株のMCF10Aシリーズ、を活用しています。このメソッドは、他の細胞株で使用できるように採用することが可能と侵略の他の潜在的な活性化剤または阻害剤を調査するために使用される可能性があります。

TGF -β誘発性浸潤を測定する回転楕円体アッセイ

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

spheroid-assay-to-measure-tgf-induced-invasion

Research

JoVE Journal

Medicine

21.8K ビュー.  Leiden University Medical Centre. 定量的形質転換成長因子（TGF）-β誘導性3次元コラーゲンゲル内侵入を測定するアッセイが記載されている。このアッセイでは、乳がんの開発のさまざまな段階を表現する細胞株のMCF10Aシリーズ、を活用しています。このメソッドは、他の細胞株で使用できるように採用することが可能と侵略の他の潜在的な活性化剤または阻害剤を調査するために使用さ?...

ビデオ: Spheroid Assay to Measure TGF-β-induced Invasion

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

癌細胞による内皮細胞単層の侵入を測定するリアルタイム電気インピーダンス基づく手法

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

医学

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

モデリングとイメージング、3次元の集団細胞浸潤

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Method to Measure Tone of Axial and Proximal Muscle

The control of tonic muscular activity remains poorly understood. While abnormal tone is commonly assessed clinically by measuring the passive resistance of relaxed limbs1, no systems are available to study tonic muscle control in a natural, active state of antigravity support. We have developed a device (Twister) to study tonic regulation of axial and proximal muscles during active postural maintenance (i.e. postural tone). Twister rotates axial body regions relative to each other about the vertical axis during stance, so as to twist the neck, trunk or hip regions. This twisting imposes length changes on axial muscles without changing the body's relationship to gravity. Because Twister does not provide postural support, tone must be regulated to counteract gravitational torques. We quantify this tonic regulation by the restive torque to twisting, which reflects the state of all muscles undergoing length changes, as well as by electromyography of relevant muscles. Because tone is characterized by long-lasting low-level muscle activity, tonic control is studied with slow movements that produce "tonic" changes in muscle length, without evoking fast "phasic" responses. Twister can be reconfigured to study various aspects of muscle tone, such as co-contraction, tonic modulation to postural changes, tonic interactions across body segments, as well as perceptual thresholds to slow axial rotation. Twister can also be used to provide a quantitative measurement of the effects of disease on axial and proximal postural tone and assess the efficacy of intervention.

We have developed a device (Twister) to study the regulation of tonic muscle activity during active postural maintenance. Twister measures torsional resistance and muscular responses in standing subjects during twisting of the body axis. The device can be flexibly configured to study various aspects of tonic control across the neck, trunk, and/or hips.

軸方向および近位筋のトーンを測定する方法

The control of tonic muscular activity remains poorly understood.  While abnormal tone is commonly assessed clinically by measuring the passive resistance ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

歯科医学、真度と精度を測定するための新しい方法の精度

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

変更された<em&gt;インビトロ</em媒介する癌細胞浸潤におけるホルモン、サイトカインおよび/または成長因子の潜在的な役割を決定するために&gt;浸潤アッセイ

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

リアルタイムで中皮細胞の卵巣癌スフェロイドアタッチメントおよび浸潤の評価

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

マウス収縮圧力を測定するために子宮内テレメトリ<em&gt;インビボ</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

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 ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

テストのためのひよこ心臓浸潤アッセイ癌細胞の浸潤性および潜在的に抗浸潤化合物の活性

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

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 ...