这项工作是具有特别提到希拉里工艺和道格拉斯·迈尔斯爵士支持帝国BRC，国家卫生研究院的研究，并采取行动战胜癌症。

程序如下一直道德经伦敦帝国学院。 1.原发性代微球体从人乳腺癌细胞系注:执行下无菌培养罩以下步骤。 <ol><li>制备微球体媒体​​含有DMEM / F12补充了2mM L-谷氨酰胺，100U / ml青霉素，100U / ml链霉素。 ，10纳克/ ml重组人碱性成纤维细胞生长因子（bFGF; R＆D系统）和1x B27添加剂;通过加入20纳克/ ml重组人表皮生长因子（Sigma公司，EGF）在使用前立即制备完整的媒体。 </li><li> （或您选择的乳腺癌细胞系; 70​​-80％汇合）从瓶中含有粘附MCF-7或MDA-MB-231细胞吸媒体报道，在1X PBS洗两次，trypsinize细胞。 </li><li>离心细胞，在200×g离心，在室温下放置5分钟。倒出上清和悬浮细胞在1-5毫升的微球体介质。移液器上下，如有必要，使用一个40微米的细胞滤网过滤帽，以获得单细胞悬浮液。使用血球计数器，以确保单细胞悬浮液已形成（如果不是，则使用将25g针注射器，所述细胞悬液最多3次）。 </li><li>如果通过FACS必要分离CD44 + CD24-细胞的子集使用抗CD24藻红蛋白（PE）和抗CD44异硫氰酸荧光素（FITC）的单克隆抗体9。可替代地，使用磁激活细胞分选（MACS）系统用抗CD44和抗CD24的生物素合成微珠按制造商的说明分离这样的人口。用LD柱进行正选择使用LS列，并阴性选择和流确认所有分离细胞的表型流式细胞仪17。 </li><li>计算使用台盼蓝每毫升的活细胞的数目。 注:细胞活力被计算为活细胞通过细胞的总数的网格内的hemocy划分的数目垢仪。这样会占用台盼蓝细胞被认为是非可行的。下面的过程是用来精确地确定活细胞的比例。 <ol><li>制备在PBS中的台盼蓝的0.4％的溶液。加入0.1毫升锥虫蓝原液到1ml细胞。加载血球和显微镜低倍率下的立即检查。计数总细胞数和蓝染色的细胞的数目。 活菌= [1.00 - （蓝色细胞数÷总细胞数）]×100。 </li><li>计算每毫升培养物的活细胞的数量，使用下面的公式。记以校正稀释因子。 活细胞数×10 4×1.1 =细胞/ ml培养</li></ol></li><li>悬浮预先确定的细胞量在2毫升完全微球体媒体​​在6孔超低附着板的各孔中。接种密度通常为500-4,000个细胞/厘米每孔2细胞。可选择使用LO瓦特附件24孔板而在0.5ml的培养基接种的细胞在相同的密度。 注:我们推荐接种密度和文化的时间优化每个感兴趣的细胞系。 </li><li>而不干扰所述板孵育超低附件6孔板，在37℃和5％的CO 2为5-10天（取决于细胞系和乳腺球的大小），特别是在第5天的生长期。 </li></ol> 2.原代微球体从人乳腺癌的临床样本注意:人乳腺癌组织中可从接受手术切除乳腺肿瘤的患者中获得。在冰上商店组织长达24小时的50毫升无菌试管中的培养基含有100U / ml青霉素和100U / ml链霉素。执行下一个无菌培养罩以下步骤。 <ol><li>将样品转移到一个100mm组织培养皿用小体积的介质。祛瘀e。使用无菌剪刀，scapel和镊子的脂肪组织。 </li><li>添加2-3毫升的DMEM / F12和剁碎的样品成1mm 3枚无菌scapel或刀片，直到没有大块依然存在。 </li><li>重悬的组织片在预热10毫升的DMEM含有蛋白水解酶（3000单位/毫升胶原酶和1000 U / ml的透明质酸酶），并在旋转振荡器孵育在37℃，直到所有的组织碎片被消化。通常完全消化需要1-3小时。根据肿瘤的病理特征调整消化时间。 （例如乳腺癌通常是难以消化相比，粘液癌是比较脆弱的，需要更短的消化时间）。通过观察下血球20微升悬架评估每一个消化半小时的程度。 </li><li>允许片段沉降5分钟，然后在200×g离心，在室温统转移上清液在一个15毫升的锥形聚丙烯管中并离心10分钟perature。小心倒出上清，悬浮细胞在1 - 5毫升微球体介质。 </li><li>按照上面描述的用于细胞系，以获得和板单细胞悬浮液中的步骤1.1-1.4。穿过25 G细胞如果需要的最大的2倍注射器限制破坏细胞。 </li></ol> 3.微球体形成率（％）计算<ol><li>培养期结束后，算上微球体（大于40微米）的直径在显微镜下放大40倍。数字图像5无规字段使用数码相机上的光显微镜并使用采集软件确定的微球体的大小。使用任何图像分析软件。 </li><li>使用以下等式计算乳腺球形成率（MFE％）: MFE（％）=（＃每孔微球体）/（＃细胞每孔接种）×100 </li></ol>微球体4.自我更新的评估连续传代<ol><li&gt;移液器含有来自每个井的微球体入15ml管中的介质。用PBS洗涤各孔，并加入到收集介质。离心在115×g离心，在室温下10分钟。 </li><li>弃上清，悬浮颗粒在500微升预热的0.5％胰蛋白酶/ 0.2％EDTA的。孵育2-3分钟。 </li><li>加入500微升的FCS以中和胰蛋白酶。离心，在500×g离心5分钟。弃上清，重新悬浮颗粒在100微升微球体介质，移液，上下分列球。 </li><li>计数细胞用血细胞计数器，并确定是否细胞是在单细胞悬浮液。如果不通过将25g注射器最多3次，以获得单细胞。种子的细胞进入一个新的超低附件6孔板在在初级代中使用的相同的密度。 </li><li>经过5-10天，算球&gt; 40微米，使用前报道的公式计算球体形成效率。 </li></ol>

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The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this article.

中小学微球体的成功评估依赖于细胞被镀在足够低的密度，从单一的克隆微球体的形式，以最小的领域聚集。然而，在密度过低，过少的微球体可能形成区分疗效的统计。播种密度应为使用，因为他们可以有很大的不同在他们的领域形成效率每个细胞系进行优化（那些表达低E-cadherin的可能形成不太稳定，短期微球体培养18）。这种优化阶段应确保从单细胞的克隆生长最初级和次级球形成?...

通过干细胞样肿瘤干细胞为首的肿瘤细胞系的存在，极大地增加我们的肿瘤异质性的理解。而在一些肿瘤的遗传多样性并从基因不同克隆的克隆产物出现，大幅组件出现从后生差异导致:癌细胞可以通过激活特定基因或抑制干，祖和分化状态之间转换（有时可逆）表达程序1 - 3。这可能反映了细胞的内在或外在的因素，这反映当前正从邻近癌，间质或免疫细胞递送调节因子表达与在与旁分泌信号结合其所得自分泌信号传导的细胞中的基因表达程序，microenviromental条件如度缺氧2,4,5。 虽然创新的谱系追踪的方法在前进我们研究推定的癌干细胞在它们的体内利基6能力-如图8所示 ，球形成测定法仍估计乳腺癌细胞的潜力，以行为像干细胞，至少在所用的测定条件一种流行和方便的方法。它经常被用来一起回顾性方法用于净化癌症干细胞，通过对已提出的膜标记物CD44和CD24 9，和酶乙醛脱氢酶（醛脱氢酶）的活性水平10，标志物的表达，以对应于更mesenchymal-和上皮分别为11样癌干细胞。球体的形成方法首先被开发作为神经球测定法，使得能够从单个克隆在非粘附，无血清条件，增加了上皮生长因子（EGF）12推定的干细胞的生长，后来被有效地应用于正常和癌乳腺组织。 骨真正的干细胞，认为休息G0期，将不会遇到的因素将有利于激活体内的精确组合。所述微球体测定代替使细胞要么蓄势有丝分裂或已经除以13的生长。这些祖细胞，虽然不是一个真正的静态细胞，可能是细胞阶段，与在测定中使用的表皮生长因子和碱性成纤维细胞生长因子（bFGF）促分裂原增殖。然而，它们包含了一系列激活干细胞相关的信令的通路14。另外，它们的形成的速率涉及当通过在有限稀释测定法效力在小鼠异种移植2,15,16测定它们取自组织的致瘤&lt;/ SUP&gt;。 在这里，我们提供详细的协议，以分离单个细胞，并产生来自人乳腺癌细胞系和乳腺肿瘤的临床样品初级乳腺球。我们还描述了如何执行主微球体的连续传代，以评估自我更新，以及如何计算球体形成效率，允许在不同的接种密度比较（见方案在图1中）。

<table><tbody><tr><td>DMEM/F12</td><td>Lonza</td><td>CC-3151</td><td></td></tr><tr><td>2 mM L-glutamine</td><td>Sigma Aldrich</td><td>G8540</td><td></td></tr><tr><td>100 U/ml Penicillin &amp;amp; Streptomycin</td><td>Sigma Aldrich</td><td>P4083</td><td></td></tr><tr><td>20 ng/ml Recombinant human epidermal growth factor (EGF)</td><td>Sigma Aldrich</td><td>E9644</td><td></td></tr><tr><td>20 ng/ml Recombinant human basic fibroblast growth factor (bFGF)</td><td>R&amp;amp;D systems</td><td>233-FB-025</td><td></td></tr><tr><td>1x B27 supplement</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Thermo Scientific</td><td>12399902</td><td></td></tr><tr><td>0.5% trypsin/0.2% EDTA</td><td>Sigma Aldrich</td><td>59418C</td><td></td></tr><tr><td>Fetal calf serum</td><td>First Link UK</td><td>02-00-850</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma Aldrich</td><td>93595</td><td></td></tr><tr><td>Low attachment 6-well plates</td><td>Corning</td><td>CLS3814</td><td></td></tr><tr><td>Collagenase type 1A</td><td>Sigma Aldrich</td><td>C9891</td><td></td></tr><tr><td>Hyaluronidase</td><td>Sigma Aldrich</td><td>H3506</td><td></td></tr><tr><td>Sterile razor blades</td><td>Fisher Scientific</td><td>12443170</td><td></td></tr><tr><td>Sterile scalpel</td><td>Fisher Scientific</td><td>11758353</td><td></td></tr><tr><td>Sterile micro-dissecting scissors</td><td>Sigma Aldrich</td><td>S3146</td><td></td></tr></tbody></table>

mammosphere formation assay from human breast cancer tissues and cell lines

类似的健康组织，许多血液和实体恶性肿瘤现在认为是分层组织的，与干细胞样癌细胞自我更新而引起更加分化的后代的一个子集。理解并针对这些癌干细胞在乳腺癌，其可以具有增强化疗和放射抗性相比非干肿瘤散装，已成为一个重要的研究领域。该微球体检测也成为广泛使用的，因为它能够识别追溯sphere-:标志物CD44包括，CD24和乙醛脱氢酶的活性可以用荧光活化细胞分选（FACS）来隔离前瞻性当细胞植入免疫缺陷小鼠显示增强的致瘤性进行评估形成细胞，从单一的干细胞样克隆的发展。在这里，我们列出的微球体从细胞系或小学患者样本，他们的传代，并计算估计范围f显示相应的培养方法orming效率（SFE）。首先我们讨论关键的考虑和陷阱在适当的规划和微球体的实验解释。

不同的样品或那些经受不同处理可以在微球体&gt;40μm的用于接种的初始细胞正火后形成的数目而变化。计算乳腺球形成率（MFE），用于在一式三份培养各处理。这使得能够用不同的接种密度实验以进行比较。数据最好显示在一条形图，理想地与阳性和阴性对照，并显示在整个一式三份孔的标准偏差。始终在60-80％汇合之间粘附细胞转移到非粘附板。小心细胞计数是必不可少的准确定量的治疗效果?...

Watch this Scientific Journal Video about Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines at JoVE.com

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

从人乳腺癌组织及细胞系微球体形成分析

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

mammosphere-formation-assay-from-human-breast-cancer-tissues-cell

Research

JoVE Journal

Medicine

33.2K 次观看.  Imperial College London. Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

视频: Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.

The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

利用微阵列研究微环境对癌症细胞表型的影响

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

科研

癌症研究

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

小鼠乳腺皮下HC11和EpH4细胞的分化

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

医学

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

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.

使用微生理系统模拟人体乳房组织中的乳腺癌

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 ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

小鼠乳腺上皮细胞形成过程中Lactogenic分化的微球体

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

生物学

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Quantification of Self-renewal in Murine Mammosphere Cultures

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. The role of mammary stem cells (MaSCs) is widely studied both in the physiological/developmental context and with regards to breast carcinogenesis. In this aspect, ex vivo studies focused on MaSC properties are highly sought after. Mammosphere cultures represent a surrogate of organ formation and have become a valuable tool for both basic and translational research. Here, we present a detailed protocol for the generation of murine primary mammosphere cultures and the quantitation of MaSC growth properties. The protocol includes mammary gland collection and digestion, isolation of primary mammary epithelial cells (MECs), establishment of primary mammosphere cultures, serial passaging, quantitation of mammosphere growth parameters and interpretation of the results. As an example, we present the effect of low-level constitutive Myc expression on normal MECs leading to increased self-renewal and proliferation.

Here, we describe the implementation and interpretation of the results of an in vitro mammosphere self-renewal quantitative assay.

鼠乳层文化中自我更新的量化

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. ...

发育生物学

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

非酶中，预浸润性乳腺癌病变的无血清组织培养的微球体的自然发生

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

从人乳腺癌组织及细胞系微球体形成分析

摘要

探索更多视频

此视频中的章节

利用微阵列研究微环境对癌症细胞表型的影响

小鼠乳腺皮下HC11和EpH4细胞的分化

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

人类乳腺上皮细胞致癌转化的体外评价

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

使用微生理系统模拟人体乳房组织中的乳腺癌

小鼠乳腺上皮细胞形成过程中Lactogenic分化的微球体

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型

鼠乳层文化中自我更新的量化

非酶中，预浸润性乳腺癌病变的无血清组织培养的微球体的自然发生

从人乳腺癌组织及细胞系微球体形成分析

摘要

探索更多视频

此视频中的章节

利用微阵列研究微环境对癌症细胞表型的影响

小鼠乳腺皮下HC11和EpH4细胞的分化

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

人类乳腺上皮细胞致癌转化的体外评价

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

使用微生理系统模拟人体乳房组织中的乳腺癌

小鼠乳腺上皮细胞形成过程中Lactogenic分化的微球体

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

鼠乳层文化中自我更新的量化

非酶中，预浸润性乳腺癌病变的无血清组织培养的微球体的自然发生

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型