This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) (26461185 and 25460137).

注：この研究は、適切な倫理委員会によって承認された。 ヒト肺線維芽細胞の1。初代培養<ol><li>肺組織のコレクション： <ol><li>直接の外科手術室からのヒト肺組織のサンプルを得る。できるだけ離れ腫瘍から収集した非癌性サンプルと、癌性及び非癌性肺組織から約1cm 3ブロックを収集します。 </li><li> 100単位/ mlペニシリン、100μg/ mlのストレプトマイシンおよび0.25 / mlのアンホテリシンB（無血清培地）を補充したダルベッコ改変イーグル培地（DMEM）中でサンプルを懸濁する。無菌50mlチューブ中の浮遊サンプルを維持し、実験室に移す。 NOTE：線維芽細胞は、上皮細胞、内皮細胞および平滑筋細胞などの他の細胞型と比較して高度回遊あり、増殖性。成長方法は、線維芽細胞のこれらの機能を活用し、かつoutgro組織切片から翼細胞は、線維芽細胞に豊富に存在する。いくつかの細胞継代後に、他の細胞型は、精製された線維芽細胞培養物中で得られる、生存し、10％ウシ胎児血清（FBS）を含むDMEM中で伝播することができない。細胞は、初代​​培養後3~7通路を使用することができる。 </li></ol></li><li>細胞成長のための移植片培養およびコンディショニング： <ol><li>修正5と以前に報告されたプロトコルを使用して、線維芽細胞の初代培養を行います。 </li><li>研究室では、培地なし10cmの組織培養皿に組織サンプルを配置し、小さな切片に切断〜滅菌ピンセットとメスを用いて大きさ2〜3mmの。このプロセスの間、部分乾燥を避ける、そうでなければ細胞伸長の効率が損なわれる。 </li></ol></li><li>上皮細胞層および結合組織の分離。 <ol><li> 4℃で16時間培養2000 PU / mlのディスパーゼIを含む培地と培養中の切断組織切片を浸し。&lt;/ LI&gt; <li>皿に組織切片を転送し、隣接する結合組織から上皮細胞層を分離する。微生物の汚染を避けるために、クリーンベンチ内でセクションを処理する。 注：上皮細胞の初代培養が必要な場合は、ステップ1.3を行う。唯一の線維芽細胞を単離することがある場合は、その後の移植片培養し、細胞の継代は、精製線維芽細胞集団をもたらす上皮細胞、のために不利であるため、ステップ1.3を省略します。 </li></ol></li><li>組織切片の添付ファイル： <ol><li> 2メスを使用して1ミリメートル片に組織をミンチ。片が十分に小さくない場合は、皿からより容易に切り離して、細胞をディッシュ表面に脱却することができないであろう。 </li><li>各ピースを囲む空の領域を維持するために、互いに離間して小肺組織切片を置く。組織切片の取り付けを容易にするために、手術用メスの刃を用いて組織培養皿の表面に傷を作る。 注：スクラッチまた、細胞が移動することができ、それに沿ってトラックを提供し、細胞成長を促進するために表示されます。 <ol><li>代替的に、個別に6ウェルプレートの各ウェルに細分化した組織片を置き、カバースリップでそれらをカバー。シリコーングリースを使用したプレートの表面にカバースリップを取り付けます。 </li><li>無菌のピンを使用して、各ウェルに1.5センチメートル離れた2点でのシリコーングリースを配置します。組織片のカバレッジは、いくつかの継代後の細胞成長および細胞増殖の効力を増強する。 </li></ol></li></ol></li><li>その後の移植片培養： <ol><li>サンプルが完全に乾く場合は、効率的な細胞成長は大部分が失われているように、組織切片は、完全に乾く前に静かに皿に培養液を追加します。あまりにも急速に組織切片を切り離すと同じようにメディアをかけないでください。 </li><li> 10％のFBSを含むDMEM中で培養細胞。追加の浮力から脱離を促進するようにそれは、浮動可能にすることをあまりにも多くのちょうど組織をカバーするために十分なメディアを使用してではなく、一品。 </li></ol></li><li>細胞継代： <ol><li>培養皿の繰り返し運動が組織切片の剥離につながることができように、常に注意して培養液を処理します。 </li><li> 5-7日間、37℃のインキュベーターで培養皿を置きます。一日おきに培地を更新し、培地交換の際に最小限の培養皿を扱う。線維芽細胞は、今後数週間にわたって組織切片の端から脱却する。 NOTE：〜2-3週間後、外部増殖線維芽細胞は、ほとんどの表面積の量に依存して、コンフルエンスに達する。 </li><li> 1×PBSで細胞を洗浄し、私はプレートにトリプシンの1ミリリットルを追加します。 </li><li>トリプシン処理後、10％FBSを補充したDMEMの9ミリリットルを用いて細胞を切り離す。組織切片と一緒に、10ミリリットルチューブ内の培地に懸濁した細胞を収集します。細胞ペレットを形成するために遠心分離した後、新しい培地で得られたペレットを再懸濁する。 </li><li>新しいCUL上に細胞や組織切片をシードトゥーレの料理は、その時点で組織は、細胞が付着し、成長しながら、皿に​​取り付けることができない。翌日、吸引により、浮遊組織片を除去し、培地を変更してください。添付線維芽細胞は、その後の文化に伝播する。 </li></ol></li></ol>ヒト肺癌線維芽細胞と癌細胞の2三次元共培養<ol><li>細胞および試薬の調製<ol><li>約2×10 5上皮細胞およびウェルあたり2.5×10 5線維芽細胞を使用しています。コラーゲンタイプIA（3 mg / mlの、pHが3）、FBS（100％）、再構成緩衝液（50mMのNaOH、260ミリモルのNaHCO 3、200mMのHEPES）、5×DMEMを調製し、DMEMは、線維芽細胞培養10％FBSを補充した3次元共培養培地（1：癌細胞に対する線維芽細胞培養培地および培地の1：1混合物）。 </li><li>培養A549肺腺癌細胞、線維芽細胞およびDMEM中でA549の三次元共培養は、10％FBSを補充した。 </li></ol></li><li>コラーゲンゲルfはormation： <ol><li> 1×PBSで10cmディッシュ上で培養した線維芽細胞を洗浄し、プレートにトリプシンの1ミリリットルに私を追加します。 37℃で約5分間トリプシン処理した後、10％のFBSを添加した9ミリリットルのDMEMを用いて細胞を切り離す。 10ミリリットルチューブに培地に懸濁した細胞を収集し、細胞ペレットを形成するために、200〜300×gで、それらを遠心する。 </li><li> 5×10 5 / mlの密度で、100％FBS中で得られたペレットを再懸濁する。 </li><li>氷上のコラーゲンゲルを準備します。次の手順の前に冷蔵庫での試薬、ピペット、チューブクール。偶数氷上で、混合物をある程度固化し、全量を全成分の合計よりも小さい。 </li><li>各ウェルに、（2.5×10 5細胞）、線維芽細胞懸濁液を0.5mlとを混合してコラーゲンゲルを調製FBS、2.3ミリリットルタイプIAコラーゲン、670μlの5×DMEM、および330μlの再構成緩衝液中で。ゲルは、室温で容易に設定することができます。 <ol><li>激しく均質マイルを確実にするためにピペット固定装置。ペッティングプロセス中に気泡を作成しないでください。 </li></ol></li><li> 6ウェルプレートの各ウェルに、混合物（3 ml）を添加し、30〜60分間妨害することなく、37℃のインキュベーター内でゲル化することを可能にする。 </li></ol></li><li>コラーゲンゲル上の癌細胞培養： <ol><li> 6変更を加えて、以前に報告されたプロトコルを使用して、3次元共培養を行います。 </li><li> 3D共培養培地に再懸濁癌細胞（またはA549細胞の場合は10％FBSを含むDMEM中で）1×10 5 / mlの密度で。 </li><li>各ゲルの表面上に再懸濁した細胞溶液2ml（2×10 5細胞）を注ぐ。実験条件に依存して、共培養中の癌細胞または線維芽細胞の細胞数を変更する。 </li></ol></li><li>ゲル収縮： <ol><li>癌細胞がコラーゲンゲルに付着することができるように、37℃で一晩ゲルをインキュベートする。各ゲルを切り離し、およびOを各ウェルに「浮遊培養 &#39;を生成6ウェルプレートfは。 NOTE：浮遊培養を使用することにより、ゲルの大きさの様々な変更は、共培養癌細胞と線維芽細胞との間の細胞の相互作用によって媒介される機械的張力およびコラーゲン分解によって誘導される。 </li><li>ウェルの端から各ゲルを分離するために角度の付いた21 G針や小さなへらを使用してください。 2〜3日おきに、3次元共培養培地2mlでウェルをリフレッシュ。 5日間毎日各ゲルの大きさを測定します。 </li></ol></li><li>コラーゲンゲルの分析： <ol><li>このようSircolアッセイコラーゲン定量法を用いて、各ゲルのコラーゲン含有量を測定する。ゲル5からRNAを収集した後、トランスクリプトーム解析または定量的RT-PCRを行います。 </li></ol></li></ol> 3.気液界面文化と浸潤アッセイ<ol><li> 5日間37℃でコラーゲンゲルを培養した後、新たな6-メッシュ（70μmの孔サイズ）に配置して空気に収縮したゲルを暴露ウェルプレート。フローサイトメトリーのために使用される市販の細胞ストレーナーからメッシュを作成します。 </li><li>静かに3D共培養培地の11ミリリットルで、各ウェルを埋める。 </li><li>無菌のスプーンを使用してメッシュ上にゲルを置き、ゲルから、残りの培地を除去。空気にゲルの上面を露出しながら、この状態では、媒体中のゲルを水没。気液界面、この培養条件を定義する。 注：インキュベーター中、37℃で気液界面培養を維持5-7日後に、浸潤性癌細胞がゲル中に移動する。細胞型および細胞の相互作用の結果として依存して、癌細胞の細胞形態変化の可変度は、気液界面培養によって誘導される。 </li><li>組織学的評価のために、室温で一晩ホルマリン溶液中でゲルを固定し、パラフィンに埋め込む。ヘマトキシリン​​およびエオシン（H＆E）で垂直セクション（4μm）を染色する。免疫組織化学的分析のOを実行nはセクション4。 </li></ol>

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The authors declare that they have no competing financial interests.

CAFSは、癌細胞の周囲のECMの主要な成分を形成し、腫瘍のための足場を提供するだけでなく、積極的に腫瘍の発達7に参加するだけでなく。証拠を蓄積すると、CAF媒介腫瘍進行8の重要な役割を強調し、最終的な予後上のCAFSまたはその関連分子の影響を解く。 以前の研究では、肺CAFS 4を単離するための成長方法を用いた。この実験では、皿の表面上...

Cancer tissue can be perceived as a type of organ, which evolves through close interactions between the cancer and the tumor stromal microenvironment, composed of cancer-associated fibroblasts (CAFs), immune cells, tumor vessels and the extracellular matrix (ECM). CAFs are the major source of soluble factors (cytokines, growth factors and chemokines) that exert mitogenic, pro-migratory and pro-invasive effects on cancer cells. They also stimulate tumor vessel formation and recruit precursor cells, such as bone marrow-derived cells (BMDC). Activated CAFs are involved in the production and remodeling of the ECM, thereby promoting the growth and spread of cancer cells1. CAFs also provide a niche that facilitates tumor cell colonization and metastasis and are capable of conferring stem cell phenotypes onto neighboring cancer cells. Pathological observations suggest that stromal reactions or fibrotic changes in cancer tissues are indicative of a poor prognosis. Recent studies have also demonstrated that tumor stromal features, such as the gene signature, can predict patient prognosis. Furthermore, CAF-derived factors can modulate sensitivity to chemotherapy, highlighting the role of CAFs in determining drug sensitivity and resistance2.
As CAFs play a multifaceted role in the promotion of tumor progression through signaling pathways that mediate interactions between CAFs and different cell types within the tumor microenvironment, they have attracted increasing attention as novel targets for cancer therapies. The heterogeneity of the cell populations within the cancer microenvironment presents an obstacle for targeting CAFs. Several markers for CAFs have been proposed, such as &#945;-smooth muscle actin (&#945;-SMA), fibroblast activation protein (FAP), and fibroblast specific proten-1 (FSP-1: also called S100A4); however, these molecular markers are not specific for distinguishing CAFs from other cells present in non-cancerous tissues3. Therefore, further studies are needed to obtain more knowledge about the specific properties of CAFs. To this end, it is informative to characterize primary cultured CAFs compared with patient-matched normal fibroblasts.
Recently, analyses on patient-derived CAFs have been reported in several cancer types, revealing unique gene expression patterns and cell behaviors compared with fibroblasts derived from non-cancerous tissues. Using isolated CAFs from human lung cancer tissues, we developed a three-dimensional co-culture method, enabling us to evaluate the properties of lung CAFs. In this model, we investigated the effects of the CAFs on lung cancer cell invasion, proliferation and collagen gel contraction, which experimentally recapitulated the tumor-promoting roles of lung CAFs4.

<table><tbody><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D5796</td><td></td></tr><tr><td>FBS</td><td>GIBCO</td><td>10437</td><td></td></tr><tr><td>Collagen type IA</td><td>Nitta gelatin Inc.</td><td>CELL-1A</td><td></td></tr><tr><td>Reconstitution buffer</td><td>Nitta gelatin Inc.</td><td></td><td></td></tr><tr><td>Cover slip</td><td>NUNC</td><td>174934</td><td></td></tr><tr><td>Silicone grease</td><td>Dow Corning Toray</td><td>High vacuum grease</td><td></td></tr><tr><td>Dispase I</td><td>WAKO</td><td>386-02271</td><td></td></tr><tr><td>6-well plate</td><td>BD Falcon</td><td>353046</td><td></td></tr><tr><td>Cell strainer (70 &amp;mu;m)</td><td>BD Falcon</td><td>352350</td><td></td></tr></tbody></table>

three-dimensional co-culture model for tumor-stromal interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

この共培養法は、腫瘍微小環境を模倣し、コラーゲンゲルに埋め込まれた癌細胞と線維芽細胞との間の相互作用を研究するための有用なツールである。コラーゲンゲル収縮、癌細胞浸潤および形態学的変化：以前の研究では、3つのパラメータは、この実験モデルで評価した。癌細胞の増殖はKi67の免疫染色4を用いて推定した。肺の組織サンプルを切除肺葉（ 図1A）の癌性...

Watch this Scientific Journal Video about Three-dimensional Co-culture Model for Tumor-stromal Interaction at JoVE.com

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

腫瘍間質相互作用のための三次元共培養モデル

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

three-dimensional-co-culture-model-for-tumor-stromal-interaction

Research

JoVE Journal

Medicine

17.3K ビュー.  The University of Tokyo. Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

ビデオ: Three-dimensional Co-culture Model for Tumor-stromal Interaction

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

腫瘍の4DライブセルイメージングのためのMAMEモデル：微小環境の相互作用に影響を与える悪性進行

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

医学

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

肺扁平上皮癌進行をモデル化する多次元共培養システム

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

がん研究

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

腫瘍間質相互作用と創薬を研究するための新規ストロマ線維芽細胞変調3D腫瘍球状モデル

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

3D培養におけるがん細胞浸潤とT細胞毒性のモニタリング

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

肝細胞癌における腫瘍間質相互作用を調べる三次元スフェロイドモデル

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

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

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 ...

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 ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

三次元混合細胞スフェロイド共培養から乳腺上皮細胞の分離

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

生物学

3D Co-culture of Lung Cancer Cells with CAFs: An In Vitro Model System to Study Tumor Progression

Source: Chen, S., et al. <a href="https://www.jove.com/v/60644/multidimensional-coculture-system-to-model-lung-squamous-carcinoma">Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression.</a>&#160;J. Vis. Exp.&#160;(2020)
This video describes the two invitro model systems to capture the tissue architectural changes during lung squamous carcinoma progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts. This system provides a unique platform to investigate the role of intrinsic and extrinsic changes that affect the tumor phenotype.

腫瘍間質相互作用のための三次元共培養モデル

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