Name: analyzing the communication between monocytes and primary breast cancer cells in an extracellular matrix extract (ecme)-based three-dimensional system
Uploaded: 2018-01-08T13:00:00.000+00:00
Duration: 10 min 55 s
Description: Watch this Scientific Journal Video about Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System at JoVE.c

この作品は、CONACyT FONSEC SSA、IMSS、ISSSTE プロジェクト号 233061 エセキエル ・ m ・ フエンテス-Pananá、フォンド ・ デ ・ Apoyo 危惧、病院 Infantil ・ デ ・ メヒコ フェデリコ ・ ゴメス (プロジェクト番号 HIM-2014-053) アラカルトにサポートされていました。・ エスピノーザ サンチェス NA は、プログラム ・ デ ・ Doctorado en 芸術 Biomédicas、大学国立自治・ デ ・ メヒコ (UNAM) と CONACYT から受信した交わり 231663 から博士課程の学生です。E S NA、また社会保障 (IMSS) のメキシコ協会によって提供される財政支援を認めます。

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著者は、利害の衝突をしていない宣言します。

上皮細胞 3 D 空間構造の成長し、ECM 蛋白質との相互作用は組織の恒常性のため極めて重要です。細胞膜 (2 D) の成長の多くのがん研究に基づいているし、単分子膜が ECM に課す、細胞の特性を要約しない彼らは腫瘍の形成および進行の多くの側面を理解する重要なされているが、インスタンス:増殖、接着依存性細胞の生存、頂基底極性、ECM を改造、細胞分化などを制限します。重要な?...

腫瘍生物学は以前考えられていたよりもはるかに複雑です。がん細胞が、単なる一括制御不能な増殖細胞のより多くを示しています証拠むしろ、異なる腫瘍細胞は高い組織と腫瘍1内の階層を表示するさまざまな機能を実行するようです。腫瘍細胞は、細胞の非変形と親密なコミュニケーションも: マクロファージ、線維芽細胞、リンパ球、脂肪細胞、血管内皮細胞は、他のセルの間ですべての没頭している足場蛋白質および ECM を構成する多糖類。多数の直接的および間接的な相互作用は、トランス フォームおよび非変形細胞と疾患の結果2,3以上の強力な影響力を発揮する ECM に確立されます。BrC の特定のケースでは、Tam、4, 再発・転移のリスクを増加させる, 腫瘍の進化に重要な役割を果たすため、Tam が発見されていることを考慮した BrC 細胞のコミュニケーションを分析するが特に重要です。 ,5。
内腫瘍の相互作用とその結果を分析する、新しい 3 Dの in vitroアプローチがはるかに複雑な微小環境を提供する ECM エキスの使用に基づいて開発されているとの比較で、腫瘍生物学の現実に近い従来の単層培養細胞が増えるは、プラスチックに接続されています。ピーターセンとビッセル6提供悪性の最初のモデルと悪性の乳腺上皮細胞ラミニン豊富な基底膜上で培養した悪性の人間を差別する 3 D 脊髄構造を記述する第 1 だった乳腺上皮細胞悪性の対応。10 年後、Debnath、Muthuswamy、によって開発されたモデルとブルージュ7,8,9悪性腺房の中に危険生物学的経路を解明する貴重なツールの提供など制御不能な増殖、腺腔アノイーキス、セル抵抗の結果としての損失と障害細胞の極性形成の証拠としてタイト結合蛋白質の非局在化による大きな腺形成としてプログラムされた細胞死の種類で発生します。付着依存性細胞周囲の ECM から切り離す。Sameni モデル、Jedeszko、スローンは侵襲, 腫瘍悪性腫瘍10、11,12のもう一つの重要な特徴に関連して細胞イメージングのプロテアーゼ活性に焦点を当てています。これらのモデルは異なる蛍光焼蛋白基質と混合タンパク質行列に依存 (DQ ゼラチン、DQ コラーゲン I と DQ コラーゲン IV)、蛍光信号のコラーゲンの分解を示唆しています。3 D モデルは非変形両方の茎セルのプロパティ、セルの集計、また呼ばれる回転楕円体の腫瘍細胞を研究にも使用され、懸濁液または非対称細胞分化のメカニズムに対する尋問 ECM のようなタンパク質では培養することができます。細胞分裂、細胞間付着、細胞運動13,14。浸潤アッセイは、腫瘍の本質的な攻撃性と侵襲的プロセス15中走化性因子として機能する分子の同定をテストできるようにします。全体としては、3 D モデルは、通常、発癌性の組織形態をより密接に反映の in vitro細胞培養の手頃な多様化を表します。
我々 は両方の人間商業 BrC 知られている積極的なポテンシャル (内腔・ トリプル ネガティブ タイプ)、プライマリの細胞ラインを使用して前述のモデル7,10,11に基づく 3 D 細胞培養システムを設計しています。BrC 患者から仔のセルです。我々 はまず非積極的な (MCF-7) または積極的な (MDA-MB-231) BrC 細胞が細胞外マトリックス抽出物 (ECME) U937 単球との共培養モデルを開発-ベースの 3 D システム直接細胞相互作用を許可します。これらの共培養は、一連のがんの積極的な行動に関わる遺伝子の転写に影響を与えたこれら 2 つの細胞系譜の間の通信を決定するために使用されました。1 つの製品、プロスタグランジン E2 (PGE2) 炎症癌の進行に於いての役割を強調表示されます見つける増産と一致するシクロオキシゲナーゼ 2 (COX-2) 成績証明書の大幅な増加が観察されました。MMP の高められたトランスクリプションが積極的な MDA MB 231 細胞共同 DQ コラーゲン IV を含む文化の U937 単球培養したときよりコラーゲン分解と相関が認められました。注記のうち、私たちの共培養したコラーゲン分解の細胞間相互作用メカニズムが必要であると仮定をサポートしていません。むしろ 2 つセル血統間の通信は分泌された分子によって調停されるようお勧めします。さらに、これらの共同培養の試金から収穫される清には13未変換 mcf-10 a の細胞によって形成された腺房を解体可溶性因子が含まれています。積極的なことが分かったし、分泌される主の BrC 細胞単球走化性分子 MCP 1、GM-CSF と RANTES の上昇します。したがって、我々 は細胞が細胞間相互作用を防ぐために細胞文化の挿入に分けられた 3 D 文化概説。これらの文化は、BrC 細胞と単球の間の間接的な通信に対処するために使用されました。これらの試金、非積極的かつ積極的な商業 BrC 細胞と初代の BrC 細胞ひと単球の 3 種類に: 商業 U937 と THP 1 セル、およびプライマリ単球 (PMs) (すべて健康なドナーの末梢血から分離しました。単球は非活性化状態で使用されていた) に使用されました。共同文化の豊かさに炎症性サイトカイン il-1 と IL-8 の濃度の増加が認められました。同様に、MMP 1、MMP-2、および MMP 10 も BrC 細胞単球共培養、およびこうして補強前調査結果14に増加したことがわかった。本稿では、プライマリの BrC 細胞分離および 3 D 培養テストのポイントによってワークフローは代表の結果と一緒に提示されます。この作品は、腫瘍生物学の特定の側面を調査する 3 D の細胞の in vitroシステムを提供する大きな可能性の良い例を構成します。

<table><tbody><tr><td>U937</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>CRL-1593.2</td><td>Monocytic cell line/histiocytic lymphoma&amp;nbsp;</td></tr><tr><td>THP-1</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>TIB-202</td><td>Monocytic cell line/acute monocytic leukemia&amp;nbsp;</td></tr><tr><td>MCF-10A</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>CRL-10317</td><td>Non-transformed breast cell line</td></tr><tr><td>MCF-7</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>HTB-22</td><td>Breast Cancer Cell line</td></tr><tr><td>T47D</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>HTB-133</td><td>Breast Cancer Cell line</td></tr><tr><td>HS578T</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>HTB-126</td><td>Breast Cancer Cell line</td></tr><tr><td>MDA-MB-231</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>HTB-26</td><td>Breast Cancer Cell line</td></tr><tr><td>RPMI 1640 medium</td><td>GIBCO BRL Life Technologies</td><td>11875-093</td><td></td></tr><tr><td>DMEM High Glucose&amp;nbsp;</td><td>GIBCO BRL Life Technologies</td><td>11964-092</td><td>4.5 g/L glucose</td></tr><tr><td>DMEM/F12</td><td>GIBCO BRL Life Technologies</td><td>11039-021</td><td></td></tr><tr><td>Antibiotic/Antimycotic</td><td>GIBCO BRL Life Technologies</td><td>15240-062</td><td>100 U/mL penicillin, 100 &amp;micro;g/mL streptomycin, and 0.25 &amp;micro;g/mL Fungizone</td></tr><tr><td>Fetal Bovine Serum</td><td>GIBCO BRL Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>Horse serum&amp;nbsp;</td><td>GIBCO BRL Life Technologies</td><td>16050114</td><td></td></tr><tr><td>0.05% Trypsin-EDTA 1X</td><td>GIBCO BRL Life Technologies</td><td>25300-062</td><td></td></tr><tr><td>PBS 1X (Phosphate Buffered Saline</td><td>GIBCO BRL Life Technologies</td><td>20012-027</td><td></td></tr><tr><td>Epidermal Growth Factor (EGF)</td><td>PeproTech</td><td>AF-100-15 (1.00mg)</td><td></td></tr><tr><td>Insulin</td><td>SIGMA-ALDRICH</td><td>I1882-100MG</td><td></td></tr><tr><td>Hydrocortisone</td><td>SIGMA-ALDRICH</td><td>H088-5G</td><td></td></tr><tr><td>Cholera toxin Vibrio cholerae</td><td>SIGMA-ALDRICH</td><td>C8052-1MG</td><td></td></tr><tr><td>Matrigel&amp;nbsp;</td><td>Corning Inc</td><td>356237</td><td>Engelbreth-Holm-Swarm (EHS) mouse sarcoma, extracellular matrix extract, Store at -20&amp;deg;C until use at 4&amp;deg;C</td></tr><tr><td>GM-CSF</td><td>PeproTech</td><td>300-03</td><td></td></tr><tr><td>MCP-1</td><td>PeproTech</td><td>300-04</td><td></td></tr><tr><td>RANTES</td><td>PeproTech</td><td>300-06</td><td></td></tr><tr><td>IL-8</td><td>PeproTech</td><td>200-08</td><td></td></tr><tr><td>IL-1&amp;beta;</td><td>PeproTech</td><td>200-01B</td><td></td></tr><tr><td>Crystal-violet</td><td>Hycel M&amp;eacute;xico</td><td>541</td><td></td></tr><tr><td>Paraformaldehyde</td><td>SIGMA-ALDRICH</td><td>P6148</td><td></td></tr><tr><td>Transwell permeable supports (inserts)</td><td>Corning Inc</td><td>3422</td><td>6.5 mm diameter, 8 &amp;micro;m pore size/24-well plates&amp;nbsp;</td></tr><tr><td>Transwell permeable supports (inserts)</td><td>Thermofisher Scientific</td><td>140620</td><td>6.5 mm diameter, 0.4 &amp;micro;m pore size/24-well plates&amp;nbsp;</td></tr><tr><td>Monocyte Isolation Kit II Human&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-091-153</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>Human Cytokine/Chemokine Magnetic Bead Panel Kit 96 Well Plate Assay</td><td>EMD Millipore</td><td>HCYTOMAG-60K</td><td></td></tr><tr><td>Magpix with xPonent software (laser based fluorescent analytical test instrumentation)</td><td>Luminex Corporation</td><td>40-072</td><td></td></tr><tr><td>Lab-Tek chamber slide with cover (8 well)</td><td>Nalge Nunc International</td><td>177402</td><td></td></tr><tr><td>Glass bottom microwell 35mm petri&amp;nbsp; dishes</td><td>MatTek Corp.</td><td>P35G-1.5-14-C</td><td></td></tr></tbody></table>

analyzing the communication between monocytes and primary breast cancer cells in an extracellular matrix extract (ecme)-based three-dimensional system

細胞外マトリックス (ECM) に埋め込まれている正常および腫瘍性上皮細胞は密接に従って大きく正常組織の恒常性と病気の結果に影響を及ぼす造血および非造血細胞と通信します。乳がん、腫瘍関連マクロファージ (Tam)、病気の進行、転移, 再発形式に重要な役割を果たすしたがって、単球より腫瘍微小環境と腫瘍細胞との相互作用のメカニズムを理解することは、病気をコントロールすることが重要です。ここでは、我々 はひと乳癌 (BrC) 細胞およびひと単球の三次元 (3 D) 共培養システムの詳細な説明を提供します。BrC 細胞生産規制活性化の高い基底レベル、通常 T 細胞表現 (RANTES) を分泌、単球走化性因子蛋白質 1 (MCP-1)、および顆粒球マクロファージコロニー刺激因子 (G-CSF)、単球との共培養中炎症性サイトカイン インターロイキン (IL)-1 β (il-1) 及び il-8 はマトリックスメタロプロテアーゼ (MMP)-1、MMP 2、MMP 10 と濃縮しました。この腫瘍間質の微小環境推進アノイーキス mcf-10 a 3 D 腺様構造、単球のより、積極的な BrC 細胞の侵入への抵抗です。ここに示すプロトコル内腫瘍通信を勉強する経済的な代替手段を提供、腫瘍に関連する腫瘍生物学の特定の機能を問い合わせるに 3 D の細胞の in vitroシステムを提供する大きな可能性の例侵略。

BrC 細胞三次元培養における形態素解析:
低・高密度 3 D 文化で育つ主の BrC 細胞の形態を 5 日間にわたって調べた。最初の 48 時間の間に、細胞は、ECME に準拠し、低密度を維持します。この時点でそれが明らかに高く評価される細胞がいくつかの 2 つ以上の長い細胞質突起を有すると明らかな細胞間の接触を示...

Watch this Scientific Journal Video about Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System at JoVE.c

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

単球と細胞外マトリックスの主要な乳癌細胞間コミュニケーションを分析抽出 (ECME)-ベースの三次元システム

ここでは、乳癌、がん細胞同様単球とコラーゲン分解、免疫細胞の動員、セルなど成果の直接的/間接的相互作用の研究としての形態を分析する三次元培養法について述べる侵略、および炎症の癌関連のプロモーション。

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

analyzing-communication-between-monocytes-primary-breast-cancer-cells

Research

JoVE Journal

Cancer Research

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

10.9K ビュー.  Hospital Infantil de México Federico Gómez. ここでは、乳癌、がん細胞同様単球とコラーゲン分解、免疫細胞の動員、セルなど成果の直接的/間接的相互作用の研究としての形態を分析する三次元培養法について述べる侵略、および炎症の癌関連のプロモーション。

ビデオ: Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

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

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

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 ...

医学

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 ...

がん研究

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

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

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

初代ヒト骨髄およびその悪性腫瘍を研究する三次元組織培養モデル

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

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 ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

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, ...

生物工学

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 ...

<meta charset="utf8"></head><body>3Dコラーゲンマトリックスセットアップにおけるマクロファージ-腫瘍スフェロイド相互作用の検討

A Macrophage-Tumor Spheroid Co-Invasion Assay

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

<meta charset="utf8"></head><body>マクロファージ-腫瘍スフェロイド共浸潤アッセイ

Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion.

マクロファージ-腫瘍スフェロイド共浸潤アッセイ

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

上皮癌細胞のスキャフォールドフリー3D培養におけるヘテロ多細胞間質細胞による浸潤の促進

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

単球と細胞外マトリックスの主要な乳癌細胞間コミュニケーションを分析抽出 (ECME)-ベースの三次元システム

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の調製と分析

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

マクロファージ-腫瘍スフェロイド共浸潤アッセイ

マクロファージ-腫瘍スフェロイド共浸潤アッセイ

マクロファージ-腫瘍スフェロイド共浸潤アッセイ

上皮癌細胞のスキャフォールドフリー3D培養におけるヘテロ多細胞間質細胞による浸潤の促進