研究室のメンバーの有益な議論とサポートに感謝します。乳がん幹細胞と腫瘍微小環境に関する私たちの研究は、カナダがん研究協会研究所および米国陸軍国防総省乳がんプログラム(Grant # BC160912)からの助成金によって資金提供されています。V.B.はウエスタン・ポスドク・フェローシップ(ウエスタン大学)の支援を受けており、A.L.A.とV.B.はどちらもカナダ乳がん協会の支援を受けています。CLは、カナダ政府からのバニエカナダ大学院奨学金によってサポートされています。

著者は開示するものは何もありません。

乳がんの転移と治療に対する抵抗性は、世界中の女性の死亡の主な原因となっています。乳がん幹細胞(BCSC)の亜集団の存在は、転移の増強に寄与する26、43、44、45、46および治療抵抗性21、47、48に寄与する。<sup class="xre...

ヒト乳がん幹細胞(BCSC)の細胞的および分子的メカニズムを理解することは、乳がん治療で直面する課題に取り組むために重要です。BCSCの概念の出現は21世紀初頭にさかのぼり、CD44 + CD24-/低乳がん細胞の小さな集団がマウスで不均一な腫瘍を生成できることが判明しました1,2。その後、アルデヒドデヒドロゲナーゼの酵素活性が高い(ALDHhigh)ヒト乳がん細胞も同様の幹細胞様特性を示すことが観察されました3。これらのBCSCは、自己複製および分化が可能な細胞の小さな集団を表し、バルク腫瘍の不均一な性質に寄与する1、2、3。進化的に保存されたシグナル伝達経路の変化がBCSCの生存と維持を促進することを示唆する証拠の蓄積4,5,6,7,8,9,10,11,12,13,14 .さらに、細胞外因性微小環境は、異なるBCSC機能を指示する上で極めて重要な役割を果たすことが示されている15、16、17。これらの分子経路とBCSC機能を調節する外的要因は、乳がんの再発、転移18、および治療に対する耐性の発達に寄与しており19,20,21、治療後のBCSCの残存存在は、乳がん患者の全生存に大きな課題をもたらします22,23。.したがって、これらの因子の前臨床評価は、乳がん患者のより良い治療結果と全生存期間の改善を達成するために有益である可能性のあるBCSC標的療法を特定するために非常に重要です。
いくつかのインビトロヒト乳癌細胞株モデルおよびインビボヒト異種移植片モデルが、BCSCを特徴付けるために使用されています24、25、26、27、28、29。連続継代のたびに細胞株が継続的に再増殖する能力により、これらはオミクスベースおよび薬理ゲノム研究を実施するための理想的なモデルシステムになります。しかし、細胞株は、患者サンプルで観察された不均一性を再現できないことがよくあります。したがって、細胞株データを患者由来のサンプルで補完することが重要です。BCSCを最も純粋な形で単離することは、BCSCの詳細な特性評価を可能にするために重要です。 この純度を達成することは、BCSCに特異的な表現型マーカーの選択に依存します。 現在、ALDH高CD44 + CD24-細胞表現型は、最大純度の蛍光活性化細胞選別(FACS)を使用して、バルク乳がん細胞集団からヒトBCSCを区別および分離するために最も一般的に使用されています1、3,26。さらに、自己複製、増殖、分化などの単離されたBCSCの特性は、in vitroおよびin vivo技術を使用して評価できます。
例えば、インビトロコロニー形成アッセイは、異なる処理条件30の存在下で50個以上のコロニーを形成するために単一細胞が自己複製する能力を評価するために使用することができる。マンモスフェアアッセイは、足場非依存条件下での乳がん細胞の自己複製の可能性を評価するためにも使用できます。このアッセイは、無血清非接着培養条件における各連続継代において、単一細胞がスフェア(BCSCと非BCSCの混合物)として生成および増殖する能力を測定する31。さらに、3次元(3D)培養モデルを使用して、 in vivo 微小環境を厳密に再現し、潜在的なBCSC標的療法の活性の調査を可能にする細胞-細胞間および細胞-マトリックス相互作用を含むBCSC機能を評価できます32。in vitroモデルの多様なアプリケーションにもかかわらず、in vitroアッセイのみを使用して in vivo 条件の複雑さをモデル化することは困難です。この課題は、マウス異種移植片モデルを使用して in vivoでのBCSC挙動を評価することで克服できます。特に、このようなモデルは、乳癌転移33を評価し、疾患進行中の微小環境との相互作用を調査し34、 in vivo イメージング35、および抗腫瘍剤34の患者特異的毒性および有効性を予測するための理想的なシステムとして役立つ。
このプロトコルは、不均一な乳がん細胞のバルク集団から最大純度でヒトALDH高CD44 + CD24-BCSCを単離するための詳細な説明を提供します。また、3つのin vitro技術(コロニー形成アッセイ、マンモスフェアアッセイ、および3D培養モデル)と、BCSCのさまざまな機能を評価するために使用できるin vivo腫瘍異種移植アッセイの詳細な説明も提供します。これらの方法は、BCSC生物学の理解および/または新規BCSC標的療法の調査を目的として、ヒト乳がん細胞株または初代患者由来の乳がん細胞および腫瘍組織からのBCSCの単離および特性評価に関心のある研究者による使用に適しています。

<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

乳がん幹細胞(BCSC)は、遺伝性または後天性の幹細胞のような特徴を持つがん細胞です。頻度が低いにもかかわらず、乳がんの開始、再発、転移、治療抵抗性の主な原因です。乳がんを治療するための新しい治療標的を特定するためには、乳がん幹細胞の生物学を理解することが不可欠です。乳がん幹細胞は、CD44、CD24などのユニークな細胞表面マーカーの発現およびアルデヒドデヒドロゲナーゼ(ALDH)の酵素活性に基づいて単離および特徴付けられます。これらのALDH高CD44+CD24- 細胞はBCSC集団を構成し、下流の機能研究のために蛍光活性化セルソーティング(FACS)によって単離することができます。科学的問題に応じて、異なるインビ トロ および インビボ の方法を使用して、BCSCの機能的特性を評価することができます。ここでは、乳がん細胞の不均一な集団と乳がん患者から得られた原発腫瘍組織の両方からヒトBCSCを単離するための詳細な実験プロトコルを提供します。さらに、BCSC機能の評価に使用できるコロニー形成アッセイ、マンモスフェアアッセイ、3D培養モデル、腫瘍異種移植アッセイなど、下流のin vitro および in vivo 機能アッセイを強調しています。

記載されたプロトコルは、細胞株または解離した腫瘍組織のいずれかからの乳癌細胞の不均一な集団からのヒトBCSCの単離を可能にする。特定の細胞株または組織サンプルについて、汚染された非BCSC集団が変動する可能性があるため、BCSCを最大純度で単離するための均一な単一細胞懸濁液を生成することが重要です。 厳格な選別戦略を適用すると、汚染された非BCSCの存在が最小限に抑えら?...

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

この実験プロトコルでは、乳がん細胞および組織サンプルからのBCSCの単離、ならびにBCSCの表現型と機能を評価するために使用できるin vitro および in vivo アッセイについて説明します。

細胞および組織サンプルからのヒト乳がん幹細胞の単離と機能評価

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 ビュー.  London Regional Cancer Program. この実験プロトコルでは、乳がん細胞および組織サンプルからのBCSCの単離、ならびにBCSCの表現型と機能を評価するために使用できるin vitro および in vivo アッセイについて説明します。

ビデオ: 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.

がんの確立は、ヒト従来の骨肉腫からの幹細胞培養物

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

がん研究

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.

癌細胞スフェロイド培養における潜在的に新規薬剤標的を同定するためのスクリーニング機能ゲノミクスを利用

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.

NFκB活性および乳癌幹細胞を阻害するアスピリン、フマル酸プロドラッグの合成とキャラクタリゼーション

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.

胚ゼブラフィッシュにおけるヒト乳癌細胞の浸潤挙動

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.

アポトーシス反転後新しく形成された乳癌幹細胞様細胞の流れフローサイトメトリー法による検出

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.

それらの相互作用を研究する歯髄組織と癌細胞との共培養から間葉系幹細胞の分離

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.

婦人科腫瘍および乳がん腫瘍から癌幹細胞球体を取得する

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

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.

CRISPR媒介ベース・エディタを用いた BRCA1 バリアントの機能評価

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.

微小生理学的システムを用いたヒト乳房組織における乳癌のモデル化

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 ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

患者由来グリオーマ幹細胞を用いたGBMの潜在的併用療法を同定するための迅速スクリーニングワークフロー

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...