Name: isolation and functional assessment of human breast cancer stem cells from cell and tissue samples
Uploaded: 2020-10-02T14:00:00.000+00:00
Duration: 7 min 3 s
Description: Watch this Scientific Journal Video about Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples at JoVE.com

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 &amp;micro;g/1x10&lt;sup&gt;6&lt;/sup&gt; 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&#039;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>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</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&#039;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>&lt;strong&gt;Mammosphere Media Composition&lt;/strong&gt;</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 &amp;micro;g/mL</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;3D Organoid Media Composition&lt;/strong&gt;</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 &amp;beta;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&amp;amp;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 &amp;micro;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

4.3K 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

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.

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 ...

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

加工的主要脑肿瘤组织的干细胞检测和流量排序

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

医学

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 ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

评价的人卵巢癌细胞干细胞特性使用多和单细胞为基础的化验球

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

人胰腺癌移植瘤的干细胞的分离

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

生物学

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

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 self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

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 ...