我们感谢实验室成员的有益讨论和支持。我们对乳腺癌干细胞和肿瘤微环境的研究由加拿大癌症研究学会研究所和美国陆军国防部乳腺癌计划（赠款#BC160912）资助。V.B.由西部博士后奖学金（西部大学）支持，A.L.A.和V.B.都得到加拿腺癌协会的支持。C.L.得到了加拿大政府颁发的Vanier加拿大研究生奖学金的支持。

作者没有什么可透露的。

乳腺癌转移和对治疗的抵抗已成为全世界妇女死亡的主要原因。乳腺癌干细胞（BCSC）亚群的存在有助于增强转移26，43，44，45，46和治疗耐药性21，47，48。因此，未来治疗的重点应旨在根除BCSC以达到更好?...

了解人类乳腺癌干细胞（BCSC）的细胞和分子机制对于解决乳腺癌治疗中遇到的挑战至关重要。BCSC概念的出现可以追溯到21世纪初，当时发现少量CD44 + CD24-/低乳腺癌细胞能够在小鼠中产生异质肿瘤1，2。随后，观察到具有高酶活性的醛脱氢酶（ALDH高）的人乳腺癌细胞也表现出类似的干细胞样性质3。这些BCSC代表能够自我更新和分化的细胞群，有助于本体肿瘤的异质性1，2，3。越来越多的证据表明，进化保守信号通路的改变推动了BCSC的存活和维持4，5，6，7，8，9，10，11，12，13，14.此外，细胞外在微环境已被证明在决定不同的BCSC功能中起着关键作用15，16，17。这些分子途径和调节BCSC功能的外部因素有助于乳腺癌复发，转移18和对治疗耐药性的发展19，20，21，治疗后BCSC的残留存在对乳腺癌患者的总体生存构成重大挑战22，23.因此，这些因素的临床前评估对于确定BCSC靶向疗法非常重要，这些疗法可能有利于实现更好的治疗结果和提高乳腺癌患者的总生存期。
几种体外人乳腺癌细胞系模型和体内人异种移植模型已被用于表征BCSCs24，25，26，27，28，29。细胞系在每次连续传代后不断重新填充的能力使其成为进行基于组学和药物基因组学研究的理想模型系统。然而，细胞系通常无法概括在患者样本中观察到的异质性。因此，用患者来源的样本补充细胞系数据非常重要。以最纯净的形式分离BCSC对于实现BCSC的详细表征非常重要。 实现这种纯度取决于BCSC特有的表型标记的选择。 目前，ALDH高CD44+CD24- 细胞表型最常用于使用荧光活化细胞分选 （FACS） 从大量乳腺癌细胞群中区分和分离人 BCSC，以获得最大纯度1，3，26.此外，可以使用体外和体内技术评估分离的BCSCs的特性，例如自我更新，增殖和分化。
例如，体外集落形成测定可用于评估单个细胞在不同治疗条件下自我更新以形成50个或更多细胞集落的能力30。乳腺球测定也可用于评估乳腺癌细胞在锚定非依赖条件下的自我更新潜力。该测定法测量单细胞在无血清非贴壁培养条件下每次连续传代时以球体（BCSC 和非 BCSC 的混合物）生成和生长的能力31。此外，三维（3D）培养模型可用于评估BCSC功能，包括细胞-细胞和细胞-基质相互作用，这些相互作用密切概括了体内微环境，并允许研究潜在的BCSC靶向疗法的活性32。尽管体外模型的应用多种多样，但仅使用体外测定很难模拟体内条件的复杂性。这一挑战可以通过使用小鼠异种移植模型来评估体内BCSC行为来克服。特别是，这些模型可作为评估乳腺癌转移33，研究疾病进展期间与微环境的相互作用34，体内成像35以及预测患者特异性毒性和抗肿瘤药物功效的理想系统34。
该协议详细描述了从大量异质乳腺癌细胞群中以最大纯度分离人ALDH高CD44 + CD24-BCSC。我们还详细介绍了三种体外技术（集落形成测定、乳腺球测定和 3D 培养模型）和可用于评估 BCSC 不同功能的体内肿瘤异种移植测定。这些方法适合于有兴趣从人类乳腺癌细胞系或原发性患者来源的乳腺癌细胞和肿瘤组织中分离和表征BCSC的研究人员使用，以了解BCSC生物学和/或研究新的BCSC靶向疗法。

<table>
	<tbody>
		<tr>
			<td>7-Aminoactinomycin D (7AAD)</td>
			<td>BD</td>
			<td>51-68981E</td>
			<td>suggested: 0.25 &#181;g/1x106 cells</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher</td>
			<td>A18-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Aldehyde dehydrogenase (ALDH) substrate</td>
			<td>Stemcell Technologies</td>
			<td>1700</td>
			<td>Sold commerically as part of the ALDEFLOUR Assay kit; follow manufacturer&#39;s instructions for ALDH substrate preparation</td>
		</tr>
		<tr>
			<td>Basement membrane extract (BME)</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Sold under the commercial name Matrigel</td>
		</tr>
		<tr>
			<td>Cell culture plates: 6 well</td>
			<td>Corning</td>
			<td>877218</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates: 60mm</td>
			<td>Corning</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates: 96-well ultra low attachment</td>
			<td>Corning</td>
			<td>3474</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer: 40 micron</td>
			<td>BD</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen</td>
			<td>Stemcell Technologies</td>
			<td>7001</td>
			<td>Prepare 1:30 dilution of 3 mg/mL collagen in PBS</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>11088807001</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Conical tubes: 50 mL</td>
			<td>Fisher scientific</td>
			<td>05-539-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal violet</td>
			<td>Sigma</td>
			<td>C6158</td>
			<td>Use 0.05% crystal violet solution in water for staining</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Stemcell Technologies</td>
			<td>7913</td>
			<td>5U/mL</td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td>1x, With L-glutamine and 15 mM HEPES</td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma</td>
			<td>D5052</td>
			<td>0.1 mg/mL final concentration</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Avantor Seradigm Lifescience</td>
			<td>97068-085</td>
			<td>&#160;</td>
		</tr>
		<tr>
			<td>Flow tubes: 5ml</td>
			<td>BD</td>
			<td>352063</td>
			<td>Polypropylene round-bottom tubes</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>84124</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-Human CD24 antibody</td>
			<td>BD</td>
			<td>561646</td>
			<td>R-phycoerythrin and Cyanine dye conjugated Clone: ML5</td>
		</tr>
		<tr>
			<td>mouse anti-Human CD44 antibody</td>
			<td>BD</td>
			<td>555479</td>
			<td>R-phycoerythrin conjugated, Clone: G44-26</td>
		</tr>
		<tr>
			<td>N,N-diethylaminobenzaldehyde (DEAB)</td>
			<td>Stemcell Technologies</td>
			<td>1700</td>
			<td>Sold commerically as part of the ALDEFLOUR Assay kit; follow manufacturer&#39;s instructions DEAB preparation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Wisent Inc</td>
			<td>311-425-CL</td>
			<td>1x, Without calcium and magnesium</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mammosphere Media Composition</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504-44</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Sigma</td>
			<td>F2006</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Bioshop</td>
			<td>ALB003</td>
			<td>04%</td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td>1x, With L-glutamine and 15 mM HEPES</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma</td>
			<td>E9644</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>16634</td>
			<td>5 &#181;g/mL</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3D Organoid Media Composition</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A8301</td>
			<td>Tocris</td>
			<td>2939</td>
			<td>500 nM</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504-44</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td>1x, With L-glutamine and 15 mM HEPES</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma</td>
			<td>E9644</td>
			<td>5 ng/mL</td>
		</tr>
		<tr>
			<td>FGF10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>FGF7</td>
			<td>Peprotech</td>
			<td>100-19</td>
			<td>5 ng/mL</td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>Sigma</td>
			<td>A9165</td>
			<td>1.25 mM</td>
		</tr>
		<tr>
			<td>Neuregulin &#946;1</td>
			<td>Peprotech</td>
			<td>100-03</td>
			<td>5 nM</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>Peprotech</td>
			<td>120-10C</td>
			<td>100 ng/mL</td>
		</tr>
		<tr>
			<td>R-spondin3</td>
			<td>R&#38;D</td>
			<td>3500</td>
			<td>250 ng/mL</td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma</td>
			<td>S7067</td>
			<td>500 nM</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>5 &#181;M</td>
		</tr>
	</tbody>
</table>

isolation and functional assessment of human breast cancer stem cells from cell and tissue samples

乳腺癌干细胞（BCSC）是具有遗传或获得性干细胞样特征的癌细胞。尽管它们的频率很低，但它们是乳腺癌发生、复发、转移和治疗耐药性的主要贡献者。了解乳腺癌干细胞的生物学必须确定治疗乳腺癌的新治疗靶点。乳腺癌干细胞是根据独特的细胞表面标志物（如CD44、CD24）和醛脱氢酶（ALDH）的酶活性）的表达进行分离和表征的。这些 ALDH高CD44+CD24- 细胞构成了 BCSC 群体，可通过荧光激活细胞分选 （FACS） 进行下游功能研究。根据科学问题，可以使用不同的 体外 和 体内 方法来评估BCSC的功能特征。在这里，我们提供了一个详细的实验方案，用于从乳腺癌细胞的异质群体以及从乳腺癌患者获得的原发性肿瘤组织中分离人类BCSC。此外，我们还重点介绍了下游体 外 和 体内 功能测定，包括集落形成测定、乳腺球测定、3D 培养模型和可用于评估 BCSC 功能的肿瘤异种移植测定。

所描述的方案允许从异质的乳腺癌细胞群中分离人BCSC，无论是来自细胞系还是从解离的肿瘤组织中。对于任何给定的细胞系或组织样品，产生均匀的单细胞悬液以最大纯度分离BCSC至关重要，因为污染的非BCSC群体可能导致不同的细胞反应，特别是如果研究目的是评估靶向BCSC的治疗剂的功效。 应用严格的分选策略将最大限度地减少污染非BCSC的存在，并导致收集乳腺癌细胞比例的能力具有干细胞样?...

Watch this Scientific Journal Video about Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples at JoVE.com

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

该实验方案描述了从乳腺癌细胞和组织样品中分离BCSC以及可用于评估BCSC表型和功能的 体外 和 体内 测定。

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 ...

isolation-functional-assessment-human-breast-cancer-stem-cells-from

Research

JoVE Journal

Cancer Research

4.2K 次观看.  London Regional Cancer Program. 该实验方案描述了从乳腺癌细胞和组织样品中分离BCSC以及可用于评估BCSC表型和功能的 体外 和 体内 测定。

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

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

巨蟹座的建立源于人类传统的骨肉瘤细胞培养

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

科研

癌症研究

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&#954;B Activity and Breast Cancer Stem Cells

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an anti-inflammatory drug to standard cancer regiments may improve patient outcome. One such drug, aspirin (acetylsalicylic acid, ASA), has been explored for cancer chemoprevention and anti-tumor activity. Besides inhibiting the cyclooxygenase 2-prostaglandin axis, ASA&#39;s anti-cancer activities have also been attributed to nuclear factor &#312;B (NF&#312;B) inhibition. Because prolonged ASA use may cause gastrointestinal toxicity, a prodrug strategy has been implemented successfully. In this prodrug design the carboxylic acid of ASA is masked and additional pharmacophores are incorporated.
This protocol describes how we synthesized an aspirin-fumarate prodrug, GTCpFE, and characterized its inhibition of the NF&#312;B pathway in breast cancer cells and attenuation of the cancer stem-like properties, an important NF&#312;B-dependent phenotype. GTCpFE effectively inhibits the NF&#312;B pathway in breast cancer cell lines whereas ASA lacks any inhibitory activity, indicating that adding fumarate to ASA structure significantly contributes to its activity. In addition, GTCpFE shows significant anti-cancer stem cell activity by blocking mammosphere formation and attenuating the cancer stem cell associated CD44+CD24- immunophenotype. These results establish a viable strategy to develop improved anti-inflammatory drugs for chemoprevention and cancer therapy.

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&amp;#954;B Activity and Breast Cancer Stem Cells

This procedure will demonstrate how we synthesized and characterized the anti-NF&#954;B and anti-cancer stem cell activity of an aspirin-fumarate prodrug.

合成与阿司匹林富马酸酯前药的表征抑制NFκB活动和乳腺癌干细胞

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an ...

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

在斑马鱼胚胎人乳腺癌细胞的侵袭行为

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

细胞凋亡逆转后新形成乳腺癌干细胞样细胞的流式细胞仪检测

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

从妇科和乳腺癌肿瘤获得癌症干细胞球

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

从3维球体培养中分离出类似干细胞的细胞

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C&#62;T, c.154C&#62;T, c.3847C&#62;T, c.5056C&#62;T, and c.4986+5G&#62;A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

People with BRCA1 mutations have a higher risk of developing cancer, which warrants accurate evaluation of the function of BRCA1 variants. Herein, we described a protocol for functional assessment of BRCA1 variants using CRISPR-mediated cytosine base editors that enable targeted C:G to T:A conversion in living cells.

使用 CRISPR 介质基础编辑器对 BRCA1 变体进行功能评估

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter ...

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 ...