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

注:此研究是通过适当的伦理委员会。 人肺成纤维细胞1.原代培养<ol><li>收集肺组织: <ol><li>得到直接从外科手术室人肺组织样本。收集来自癌性和非癌肺组织约1cm 3块，以收集作为远离肿瘤尽可能的非癌性样品。 </li><li>暂停在Dulbecco改良的Eagle培养基（DMEM）中补充有100单位/ ml青霉素，100微克/毫升链霉素和0.25微克/毫升两性霉素B（无血清培养基）的样品。保持在无菌50ml试管中悬浮的样品并转移到实验室。 注:成纤维细胞是高度洄游和增殖与其它类型的细胞，如上皮细胞，内皮细胞和平滑肌细胞。该生长方法利用了成纤维细胞的这些特征，并且outgro从组织切片翼细胞丰富的成纤维细胞。经过多次传代细胞，其他细胞类型不能存活和繁殖的DMEM，用10％胎牛血清（FBS），导致纯化的成纤维细胞培养物。所述细胞可用于3-7通道主培养以下。 </li></ol></li><li>植文化和调节细胞生长: <ol><li>执行使用以前报道的协议进行了修改5成纤维细胞原代培养。 </li><li>在实验室中，放置在无培养基10cm的组织培养皿的组织样品切成小切片〜2-3毫米大小用无菌镊子和手术刀。在此过程中，应避免使部分干燥，否则细胞生长的能力受到损害。 </li></ol></li><li>分离上皮细胞层和结缔组织的: <ol><li>泡切的组织切片在含2000的PU /毫升分散酶I培养基和培养16小时，在4℃下&lt;/ li&gt; <li>的组织切片转移到培养皿，并且从相邻的结缔组织分离上皮细胞层。处理在清洁台上的部分，以避免微生物的污染。 注意:如果需要上皮细胞原代培养，执行步骤1.3。如果只成纤维细胞将被分离，则省略步骤1.3，因为在随后的外植体培养和细胞传代是不利的上皮细胞，其产生纯化的成纤维细胞种群。 </li></ol></li><li>附件组织切片: <ol><li>剁碎组织成使用两个手术刀1毫米件。如果这两块不足够小，它们分离更容​​易地从皿中，细胞将无法长大于餐具表面。 </li><li>放置小肺组织切片彼此分开，以维持周围的每一块的空白区域。使用手术刀刀片，以促进附着的组织切片的组织培养皿的表面上的划痕。 注意:抓似乎也促进细胞生长，提供轨道沿着细胞可以迁移。 <ol><li>或者，将组织糜片段单独成6孔板的每个孔中，并用盖玻片覆盖它们。附上盖玻片使用硅脂该板的表面上。 </li><li>放置硅脂在两个点相隔1.5厘米中使用无菌针每个孔中。组织碎片覆盖增强细胞生长和细胞繁殖以下几个段落的疗效。 </li></ol></li></ol></li><li>随后的外植体文化: <ol><li>轻轻培养基添加到培养皿之前组织切片干燥，作为有效的细胞生长失去大部分如果样品变干。不要将媒体过快的组织切片会脱落。 </li><li>培养的细胞在DMEM含10％FBS。使用足够的培养基刚好盖住组织，但不会太大，它允许浮动，作为额外的浮力促进支队从菜。 </li></ol></li><li>细胞传代: <ol><li>小心轻放培养基在任何时候都为培养皿的重复动作可导致组织切片的脱离。 </li><li>将培养皿在培养箱中在37℃下5-7天。刷新媒体隔日，并在媒体变化最小处理培养皿。长大成纤维细胞从组织切片，在未来几周内的边缘。 注:〜2-3周后，outgrowing成纤维细胞几乎达到汇合，这取决于表面积的量。 </li><li>用1×PBS洗涤细胞，并加入胰蛋白酶1毫升我到板上。 </li><li>胰蛋白酶消化后，分离使用9毫升DMEM中的细胞在补充有10％FBS中。与组织切片收集细胞悬浮在10毫升管的介质，在一起。离心以形成细胞沉淀，重悬所得的颗粒在新的平台。 </li><li>种子的细胞和组织切片到一个新的小路TURE盘，在该点的组织中没有附着到培养皿的同时将细胞附着和生长。次日，通过抽吸除去浮动组织片，并改变介质。连接成纤维细胞繁殖与后续培养。 </li></ol></li></ol>人肺成纤维细胞和癌细胞2.三维共培养<ol><li>制备细胞和试剂: <ol><li>大约用2×10 5的上皮细胞和每孔2.5×10 5个成纤维细胞。制备型胶原IA（3毫克/毫升，pH为3），FBS（100％），重建缓冲液（50mM的NaOH，260毫的NaHCO 3，200mM的HEPES），5×DMEM，DMEM补充有10％FBS的成纤维细胞培养，和三维共培养培养基（1:1的混合物的成纤维细胞的培养基和培养基的癌细胞）。 </li><li>培养的A549肺腺癌细胞，成纤维细胞和A549三维共培养在DMEM补充有10％FBS中。 </li></ol></li><li>胶原凝胶˚Formation: <ol><li>洗的成纤维细胞上10cm皿用1×PBS中培养，并添加胰蛋白酶1毫升我到板上。胰蛋白酶消化为〜5分钟，在37℃后，分离使用9毫升DMEM补充有10％FBS的细胞。收集的细胞悬浮在10毫升管的培养基中，离心它们在200-300×g离心以形成细胞小球。 </li><li>重悬在100％FBS的所得粒料在5×10 5 / ml的密度。 </li><li>准备上冰的胶原凝胶。冷却该反应物，移液器和管子在冰箱中之前执行以下步骤。甚至在冰上，在固化到某种程度，总体积小于所有组分的总和。 </li><li>在每个孔中，通过混合0.5毫升成纤维细胞悬浮液（2.5×10 5个细胞）中的FBS的，2.3毫升IA型胶原，670微升5×DMEM，和330微升重建缓冲液制备胶原凝胶。允许凝胶容易地设定在室温。 <ol><li>大力吸管，以确保均匀的英里夹具。避免在移液过程中产生气泡。 </li></ol></li><li>该混合物（3毫升）添加至6孔板的每个孔中，并允许在孵化器在37℃凝胶化，而不扰动30-60分钟。 </li></ol></li><li>在胶原凝胶癌细胞培养: <ol><li>使用先前报道的协议进行了修改6进行三维共培养。 </li><li>重悬的癌细胞在三维共培养培养基（或在DMEM与A549细胞的情况下，10％FBS）以1×10 5个/ ml的密度。 </li><li>倾2毫升再悬浮细胞溶液（2×10 5个细胞）在每个凝胶的表面上。修改癌细胞或成纤维细胞的细胞数目在共培养取决于实验条件。 </li></ol></li><li>凝胶收缩: <ol><li>过夜孵育凝胶在37℃下这样的癌细胞可以附着在胶原凝胶。分离的每个凝胶，并生成每个"流动文化"以及Øf显示6孔板。 注意:通过使用一个浮培养，在凝胶尺寸的各种变化都受到机械张力和胶原降解，其通过共同培养的癌细胞和成纤维细胞之间的细胞相互作用介导的诱导。 </li><li>使用成角度的21g的针或小抹刀将每个凝胶从井的边缘分开。每2-3天，刷新井用2ml三维共培养培养基。每天测量每个凝胶的尺寸为5天。 </li></ol></li><li>分析胶原凝胶: <ol><li>测量使用胶原定量方法，如Sircol测试每个凝胶的胶原含量。从凝胶5收集的RNA后进行转录分析或定量RT-PCR。 </li></ol></li></ol> 3.气 - 液界面文化入侵检测<ol><li>在37℃下5天培养的胶原凝胶后，通过将它们放置在一个网格（70微米孔径）在新的6-暴露收缩凝胶到空气孔板中。使从用于流式细胞市售的细胞滤网的网格。 </li><li>轻轻地用11毫升三维共培养培养基填充每个孔中。 </li><li>凝胶放置到使用无菌勺网格，并从凝胶中除去任何剩余的介质。在此条件下，浸入在介质中的凝胶，而露出的凝胶，以空气的上表面。定义该培养条件如空气 - 液体界面。 注:后5-7天保持在37℃的空气 - 液体界面培养在培养箱的，侵入性癌症细胞迁移到凝胶。根据细胞类型和作为细胞相互作用的结果，可变度在癌细胞中细胞形态的变化是由空气 - 液体界面培养诱导。 </li><li>用于组织学评价，固定在福尔马林溶液中的凝胶在室温下过夜，并嵌入石蜡中。染色垂直切片（4微米）与HE染色（H＆E）。进行免疫组化分析Òn中的第4。 </li></ol>

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The authors declare that they have no competing financial interests.

的CAF形成周围癌细胞ECM的一个重要组成部分，而不是仅提供一个支架的肿瘤，但也积极参与肿瘤发展7。越来越多的证据揭开CAF或与其相关的分子上的最终预后的影响，突出CAF介导的肿瘤进展8的关键作用。 在以前的研究中，我们采用的方法生长隔离肺的CAF 4。在这个实验中，组织切片的粘附维持到餐具表面是关键的。细胞不能从组织切片漂浮在培养基?...

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.

该共培养方法，模仿肿瘤微环境，是一种有用的工具，调查癌细胞和嵌入在胶原凝胶的成纤维细胞之间的相互作用。胶原凝胶收缩，癌细胞的侵袭和形态变化:在以前的研究中，三个参数在这个实验模型进行了评价。还预计使用Ki67的免疫染色4癌细胞增殖。肺组织样品从一个切除肺叶（ 图1A）的癌性和非癌的部分收集。样品切成小片，并在DMEM中培养在补充有10％FBS的（ 图...

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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.

对于肿瘤基质相互作用的三维共培养模式

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

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17.3K 次观看.  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.

视频: 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.

MAME的模型四维活细胞成像肿瘤微环境的相互作用，影响的恶性进展

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

科研

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.

多维共同培养系统模拟肺鳞癌进展

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

癌症研究

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.

用于研究肿瘤-斯特罗马相互作用和药物发现的新型斯特罗姆纤维细胞-模组3D肿瘤球体模型

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.

监测三D培养中的癌细胞入侵和T细胞毒性

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.

一种三维球体模型研究肝细胞癌肿瘤-基质相互作用

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.

制备及分析<em&gt;体外</em&gt;三维乳腺癌代理项

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

生物工程

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.

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

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.

三维培养试验探索肿瘤细胞浸润和卫星肿瘤形成

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.

从三维混合细胞球体共培养的乳腺上皮细胞的隔离

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.

对于肿瘤基质相互作用的三维共培养模式

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