Name: preparation and analysis of in vitro three dimensional breast carcinoma surrogates
Uploaded: 2016-05-09T15:00:00.000+00:00
Duration: 8 min 16 s
Description: Watch this Scientific Journal Video about Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates at JoVE.com

The University of Alabama at Birmingham Center for Metabolic Bone Disease performed the histologic processing and sectioning of surrogates. Southern Research (Birmingham, AL) provided support for the manufacture of the bioreactor system. Funding was provided by the United States Department of Defense Breast Cancer Research Program (BC121367).

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The authors declare that they have no competing financial interests.

本明細書において、3D培養の方法は、より密接にモデルヒト乳癌は概括的な3D形態の開発を可能にする量で、細胞外マトリックス（ECM）およびヒト間質線維芽細胞を含む組織の微小環境の成分を組み込むことが記載されています。記載の三次元培養法は、ECMの三次元ボリュームに組み込まれている、複数の細胞型における従来の2D細胞培養物よりも、ヒト疾患のより代表的です。組織構造、微?...

より正確にインビボでの腫瘍アーキテクチャおよび微小環境を模倣する三次元（3D）培養モデルは、細胞とその微小環境との間の複雑な相互作用を分析するために候補治療法の有効性を試験することを目的とした研究のために重要です。腫瘍次元への影響の酸素と栄養勾配、薬物暴露、間質圧/血液の流れ、および3Dアーキテクチャ1-4の均一性。適切な間質微小環境の存在は、腫瘍の次元と影響細胞のECMシグナリングおよびストローマ細胞と悪性上皮細胞間の傍分泌シグナル伝達に貢献しています。細胞機能に対する腫瘍の次元と微小環境の影響はよく両方の要因が、薬物応答1,3,5-8を変更することで、確立されています。さらに、細胞増殖動態、代謝率、および細胞シグナル伝達は、これらの要因がaffeで、3Dで2次元（2D）文化と文化の間で異なります細胞応答1,3,8-10を cting。 インビトロでは 、腫瘍微小環境代理代表ECM成分および間質細胞の集団を含むことによって調節することができます。悪性上皮細胞はさらに、腫瘍の伝播5,6,10を阻害するためにECMおよび癌関連間質細胞のいずれかによって腫瘍の進行を促進するための相乗/保護方法で、または抑制の方法で影響を受けています。いずれかの文脈では、基質は、薬物送達1,6を減少パラ分泌シグナル伝達を介して、および/ ​​または、その結果、腫瘍内の間質圧力を増加させることによって、治療応答および薬物送達に影響を与えることができます。したがって、前臨床モデルへのECMとの間質細胞の添加は、2D培養で十分にモデル化することができない腫瘍の特徴を再現するのに役立ちます。 ECM成分とsを含む概括的微小環境を組み込む乳癌サロゲートを確立するために、本明細書の方法3D体積tromal細胞は、記載されています。乳癌では、間質細胞集団は主に癌関連線維芽細胞（CAF）および間質ECMから構成されているが、主としてラミニンおよびコラーゲンIV型を含む基底膜に見出されるマトリックス成分のより少ない割合でI型コラーゲンで構成されています1,4,11-13。したがって、乳癌の微小環境（ すなわち、CAF、コラーゲンI、および基底膜）のこれらの構成要素は、サロゲートに組み込まれています。この方法は、固体の、非灌流3D代用物（ 図1A）を生成するために使用することができ、またはバイオリアクターシステム（ 図1B）を介してサロゲートを通して培地の灌流を含むように適合させることができます。両方のアプローチは、ここで説明されています。この方法はまた、腫瘍関連マクロファージのような他の間質要素を含むように改変され得る、または必要に応じて、細胞とECMの成分を調整することにより、他の固形腫瘍をモデル化します。 <pクラスここで説明乳癌サロゲートの場合= &quot;jove_content&quot;&gt;我々は、MDA-MB-231を利用している（231）乳癌細胞株、CAFは、以前にヒト乳癌14、及びIは、（90％のコラーゲンからなるECMから単離されました6 mg / mlで）および10％の増殖因子低減基底膜材料（BM）。サロゲートは、いずれか8ウェルチャンバースライド（固体サロゲート）に成長させるか、バイオリアクターシステムは、連続的栄養かん流（灌流サロゲート）を提供するために利用されます。 ECMを含有する細胞の量を収容することができる任意の灌流バイオリアクターシステム15を使用することができます。例として、私たちは私たちのバイオリアクターシステムにおける組織サロゲートの調製を記載します。このシステムは、社内で開発され、市販されていないました。ここで我々の焦点は、3D組織代替物の調製および分析上にあるので、私たちは私たちのバイオリアクターシステムの製造及び組み立ての仕様に関する広範な詳細に行っていません。しかしながら、詳細な説明このシステムとその開発は16を発表してきました。このバイオリアクターシステムにおいては、ポリジメチルシロキサン（PDMS）流路は、PDMSフォームでサポートされているサロゲートを収容するために使用される（Calcagnile ら 17によって記載されたものと同様の方法を用いて形成されます）。このボリュームは、連続的に代理に酸素と栄養を供給するためにmicrophysiologicポンプを介して媒体によって灌流されている4マイクロチャンネル（直径各400ミクロン）によって貫通されています。 サロゲートの適切な分析は、治療または他の操作に応答した細胞機能に関する適切な情報を得るために重要です。サロゲートは、固定および処理後の分泌物、または組織切片上での分析のために、共焦点顕微鏡または非侵襲的イメージングの他の手段、馴化培地、または灌流液をアッセイすることによって間接的細胞解析を用いて、無傷のサロゲートの直接画像を含む様々な方法によって分析することができますにパラフィン。組織切片で評価することができるそのようなパラメータは、セル密度です。我々は、ヘマトキシリンおよびエオシン（H＆E）で染色されたサロゲート組織切片の顕微鏡写真に適用される半自動画像処理技術を用いて細胞密度（ すなわち、断面積当たりの有核細胞数）を測定するための一つのアプローチを提示します。細胞密度は、時間の経過または様々な増殖条件および処理の結果は、細胞数の相対的な変化の指標として用いることができます。 <img alt="図1" src="/files/ftp_upload/54004/54004fig1.jpg" /> 図1の3次元ボリュームとバイオリアクターシステム。 3Dソリッドサロゲートを生成するためのプロセスのA）概略図。トップ：ECMを含む固体3Dボリュームの漫画（ピンク）、上皮癌細胞（黄色）、およびCAF（オレンジ）。下：サロゲートを含む8ウェルチャンバースライドの上面図B）概略工程の灌流3Dのサロゲートを生成します。トップ：CA培地灌流およびECMを含む（ピンク）、上皮癌細胞（黄色）、およびCAF（オレンジ色）を可能にするためのチャネルを有する3Dボリュームのrtoon。中央：PDMSフォーム（黒矢印）を含むPDMS流路の画像は、細胞+ ECMを注入し、直径400μmの測定ポリマー被覆ステンレス鋼線（ピンク色矢印）によって貫通されます。底：サロゲートを含有する連続媒体灌流を可能にするバイオリアクターシステムに接続されたPDMS流路の画像（蠕動ポンプと媒体リザーバ図示せず）とC）培養後、固体及び灌流サロゲート両方の処理ステップの写真。左：ゲルとサロゲートを処理する検体を含むcryomoldの画像。ミドル：固定され、処理サロゲートを含むパラフィンブロックの画像。右：サロゲートのH＆E染色した組織学的セクションとスライドガラスの画像<a href="https://www.jove.com/files/ftp_upload/54004/54004fig1large.jpg" target="_blank">表示するには、こちらをクリックしてください。</a>この図の拡大版。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagel Medium 1x (DMEM)</td><td>Corning CellGro</td><td>10-014-CV</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>0.25% Trypsin + 2.21&amp;nbsp;mM EDTA 1x</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Tissue Culture plates, 100&amp;nbsp;mm</td><td>CellTreat Scientific Products</td><td>229620</td><td>Sterile</td></tr><tr><td>Tissue Culture plates, 35&amp;nbsp;mm</td><td>CellTreat Scientific Products</td><td>229638</td><td>For PDMS foam formation&amp;nbsp;</td></tr><tr><td>9&quot; Glass pipette</td><td>Fisher&amp;nbsp;</td><td>13-678-20D</td><td>Sterile</td></tr><tr><td>10 ml pipette</td><td>CellTreat Scientific Products</td><td>229210B</td><td>Sterile</td></tr><tr><td>1,000 &amp;micro;l piptette tips</td><td>FisherBrand</td><td>02-717-166</td><td>Sterile Filtered</td></tr><tr><td>200 &amp;micro;l pipette tips</td><td>FisherBrand</td><td>02-717-141</td><td>Sterile Filtered</td></tr><tr><td>10 &amp;micro;l pipette tips</td><td>FisherBrand</td><td>02-717-158</td><td>Sterile Filtered</td></tr><tr><td>15 ml conical tubes</td><td>CellTreat Scientific Products</td><td>229410</td><td>Sterile</td></tr><tr><td>50 ml conical tubes</td><td>CellTreat Scientific Products</td><td>229422</td><td>Sterile</td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>FisherBrand</td><td>05-408-129</td><td>Sterile</td></tr><tr><td>Trypan blue</td><td>Corning Cellgro</td><td>25-900-CI</td><td>Sterile</td></tr><tr><td>Sylgard 184</td><td>Electron Microscopy Sciences</td><td>24236-10</td><td>PDMS elastomer and curing agent. Used for our in-house bioreactor.</td></tr><tr><td>PDMS Foam</td><td></td><td></td><td>Made in-house for use in our in-house bioreactor.</td></tr><tr><td>High Concentration Bovine Collagen Type I</td><td>Advanced Biomatrix</td><td>5133-A</td><td>FibriCol ~10 mg/ml</td></tr><tr><td>Growth Factor Reduced Matrigel (Basement Membrane)</td><td>Corning</td><td>354230</td><td>Basement membrane material</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S8761</td><td></td></tr><tr><td>Molecular Biology Grade Water</td><td>Fisher</td><td>BP2819-1</td><td></td></tr><tr><td>DMEM 10x&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D2429</td><td></td></tr><tr><td>Nunc Lab-Tek Chamber Slide System&amp;nbsp;</td><td>Thermo Scientific</td><td>177402</td><td>8-well</td></tr><tr><td>Bioreactor</td><td></td><td></td><td>Made in-house.</td></tr><tr><td>Spring-Back 304 Stainless Steel&amp;mdash;Coated with PTFE polymer</td><td>McMaster-Carr</td><td>1749T19</td><td>Stainless steel wires to generate microchannels in our in-house&amp;nbsp; bioreactor system. 0.016&quot; Diameter</td></tr><tr><td>BioPharm Plus platinum-cured silicone pump tubing, L/S 14</td><td>Masterflex</td><td>EW-96440-14</td><td>For use in our in-house bioreactor system. Tubing ID: 1.6 mm, Hose barb size: 1/16 in.</td></tr><tr><td>2-Stop Tubing Sets, non-flared PVC, 1.52&amp;nbsp;mm ID</td><td>Cole-Parmer&amp;nbsp;</td><td>EW-74906-36</td><td>For use in our in-house bioreactor system (with microperistalitic pump).&amp;nbsp;</td></tr><tr><td>Six Channel precision micro peristaltic pump</td><td>Cole-Parmer</td><td>EW-74906-04</td><td>For use with our in-house bioreactor system</td></tr><tr><td>Tuberculin Syringes</td><td>BD Medical</td><td>309625</td><td>26 gauge 3/8 in. needle; Sterile</td></tr><tr><td>Dissecting Tissue Forceps</td><td>FisherBrand</td><td>13-812-36</td><td>5.5 inch</td></tr><tr><td>Mini Tube Rotator</td><td>Boekel Scientific</td><td>260750</td><td>Equipment option for surrogate rotation. Used with carousel for 50 ml tubes (model number 260753)</td></tr><tr><td>50 ml tube carousel</td><td>Boekel Scientific</td><td>260753</td><td>Used with mini tube rotator</td></tr><tr><td>Bambino&amp;nbsp; Hybridization Oven</td><td>Boekel Scientific</td><td>230301</td><td>Equipment option for surrogate rotation</td></tr><tr><td>HistoGel Specimen Processing Gel</td><td>Thermo Scientific</td><td>HG-4000-012</td><td>Specimen Processing Gel described in Step 5.2</td></tr><tr><td>Cryomold</td><td>Andwin Scientific</td><td>4566</td><td>15 mm x 15 mm x 5 mm</td></tr><tr><td>Tissue Marking Dye</td><td>Cancer Diagnostics, inc.&amp;nbsp;</td><td>03000P</td><td>Can be used to mark surrogates,&amp;nbsp; allowing multiple samples to be included in one tissue cassette&amp;nbsp;</td></tr><tr><td>Hinged tissue cassettes&amp;nbsp;</td><td>FisherBrand</td><td>22-272-416</td><td></td></tr><tr><td>Formalin</td><td>Fisher</td><td>23-245-685</td><td></td></tr><tr><td>GoldSeal Plain Glass Slides</td><td>Thermo Scientific</td><td>3048-002</td><td></td></tr><tr><td>Xylene</td><td>Fisher</td><td>X3P-1GAL</td><td></td></tr><tr><td>Ethanol, 200 proof (100%), USP</td><td>Decon Laboratories, Inc.</td><td>2805M</td><td></td></tr><tr><td>Hematoxylin</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7211</td><td></td></tr><tr><td>Clarifier</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7401</td><td></td></tr><tr><td>Bluing Solution</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7301</td><td></td></tr><tr><td>Eosin Y</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7111</td><td></td></tr><tr><td>Cytoseal XYL mounting media</td><td>Thermo Scientific Richard-Allan Scientific</td><td>83124</td><td></td></tr><tr><td>Coverslips</td><td>Fisher Scientific</td><td>12-548-5G</td><td></td></tr></tbody></table>

preparation and analysis of in vitro three dimensional breast carcinoma surrogates

三次元（3D）培養は、二次元培養物よりもin vitroでの細胞の挙動をモデル化するために、より生理学的に関連する方法です。乳癌を含む癌は、線維芽細胞および細胞外マトリックス（ECM）を含む、がん上皮細胞および間質成分からなる複雑な3次元組織です。しかし、乳癌のほとんどのin vitroモデルは、したがって、間質を省略し、唯一の癌上皮細胞からなり、そして、in vivoでの腫瘍の3Dアーキテクチャ。癌の適切な3Dモデリングは、腫瘍生物学、行動、および治療に対する反応の正確な理解のために重要です。しかし、3Dモデルの文化とボリュームの持続時間は、培養中の酸素や栄養素の利用可能性によって制限されます。ここで、我々は、乳癌上皮細胞及び間質線維芽細胞は、間質を含むとして培養することができる3D乳癌サロゲートを生成するECMに組み込まれた方法を示します固体の3D構造または酸素および栄養素を送達するための灌流バイオリアクターシステムを使用することによって。セットアップおよび初期成長期に続いて、サロゲートは、前臨床薬物試験のために使用することができます。代替的に、代理の細胞およびマトリックスの構成要素は、生物学的問題の様々な対処するために変更することができます。培養後、サロゲートを固定し、目的のパラメータの評価のために、臨床的乳癌試料の処理と同様に、パラフィンに処理されます。 ImageJのとCellProfiler画像解析ソフトウェアシステムは、サロゲートの組織切片の顕微鏡写真に適用され、説明されているそのようなパラメータ、存在する細胞の密度の評価は、面積当たりの有核細胞の数を定量します。これは、時間の経過とともに細胞数の変化または変動する成長条件および治療から得られた細胞数の変化の指標として使用することができます。

上述の7日間増殖させたような固体及び灌流の両方の3D乳癌代用物を調製しました。その後、サロゲートは、固定パラフィンように処理し、切片、および上記のように、ヘマトキシリン​​およびエオシンで染色しました。各サロゲートの面積当たりの有核細胞の数（両方とも231細胞及びCAF）を測定しました。 図12に見られるように、H＆E染色切片の代表的...

Watch this Scientific Journal Video about Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates at JoVE.com

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

の調製と分析<em&gt;インビトロ</em&gt;三次元乳癌サロゲート

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

preparation-analysis-vitro-three-dimensional-breast-carcinoma

Research

JoVE Journal

Bioengineering

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

10.6K ビュー.  University of Alabama at Birmingham. We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

ビデオ: Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

交感性乳癌モデルを用いたトリプルネガティブ乳癌の研究

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

がん研究

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

患者前立腺癌骨転移標本及びその異種移植片に由来する3次元(3D)オルガノイドの確立と解析

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

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 ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

コラーゲンベースの足場を用いた神経芽細胞腫の3次元 in vitro 生体模倣モデル

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

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 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 ...

医学

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 ...

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 ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

癌細胞の浸潤や衛星腫瘍形成を探検するための三次元培養アッセイ

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

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