Name: establishment of cancer stem cell cultures from human conventional osteosarcoma
Uploaded: 2016-10-14T13:00:00.000+00:00
Duration: 9 min 25 s
Description: Watch this Scientific Journal Video about Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma at JoVE.com

This study was supported by ITT (Istituto Toscano Tumori) Grant Proposal 2010.

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The authors declare that they have no competing financial interest.

CSCが腫瘍バルク中のこの特定の細胞サブセットの同定を可能にするいくつかのプロパティを持っています。そのようなATP結合カセットの多剤排出トランスポーターの過剰発現28細胞毒性化学療法剤に対する獲得耐性のようなこれらの特性に基づいて、32、33、またはそのような発現のためのALDH 32として解毒酵素の発現のアップレギュレーションのためにCSCを単離するためのこのようなCD133、CD44、CD34、CD90、および他の30、34、35、41、いくつかの異なる方法として、特定の表面マーカーの42-44が開発されてきました。これらの技術の一つは、非付着条件下で増殖するためのCSCの能力に基づいて球体形成アッセイです。 球体を形成するための組織幹細胞とのCSCの能力は、最初のレイノルズらによる神経幹細胞の同定の研究に記載された。37。その後、ギブスら38たちこれらの研究edは骨肉腫から、特に、固形腫瘍からのCSCを単離することを開始します。私たちは、ギブスらによって示されている球形成アッセイ法を使用することを決定した。従来のOSの生検から得られたOSA細胞株からのCSCを隔離します。我々は、このアッセイの結果を向上させ、他の癌細胞株のための再現性を容易にするための独自の方法を適合しました。球形成アッセイの確立を参照すると、我々は、40,000細胞/ウェルをプレーティングすることが確認され、アッセイの開始時に分離して細胞を維持するための良い方法です。このトリックは、球状コロニーが細胞凝集からではなく、非付着条件下で増殖し、球状のコロニーを形成するための単一のCSCの特定と排他的な能力に由来する可能性を回避するために非常に重要です。この能力は、このアッセイの特に重要な点です。 また、球形成の良好な速度を得るためにそれを認定します、元の方法に記載のように増殖因子のアリコートごとに3次元ではなく、毎日更新するのに十分です。本研究では、我々はまた、確立され、広範囲に非接着条件下で培養したときに形成される球状のコロニーを単離するための良い方法を説明しました。それらを損傷することなく、各ウェル内に形成されている可能な限り球のような多くを隔離しようとすることは非常に重要であるため、この手順は、このアッセイにおいて重要です。唯一の球ではなく、アッセイの期間中、懸濁液中に残っている可能性があり、単一の細胞を単離することも重要です。これらの重要な点を克服するために、我々は、CSCの単離のための良好な結果が得られ、上記のように、特定の分離法を開発しました。明らかに、そこに形成された全ての球を回収することができない可能性があるが、損失率は非常に低いです。実際に、我々はまた、（フォーム大きくなった後に球を単離するために40μmの孔を有するフィルターを使用する可能性を有していますEDは約100から200細胞）。 この分離は、球形成を停止しますが、単一細胞、メチルセルロース残基の一部、および最小の球をフィルタリングすることができます。プロトコールに記載されるように、この除去は、完全な濾過により行われます。 また、最小の球状コロニーの必然的な損失と40μmのメッシュを介して最大の球状コロニーの選択は1つが球状コロニーを形成する最大容量で、より大きな幹細胞性とのCSCを選択することができます。これらの修飾の全ては、アッセイを改善し、理解し、球体形成アッセイの元の方法の最も重要なステップを再現するためのCSCを研究する研究者を支援するために行きました。 CSCを単離するためのin vitro法に関する研究の中で、これは球形成アッセイを適応方法を示すことを目的としたこの研究からのCSCを単離するための良い方法かもしれませんOSAの細胞株。オリジナルの方法と詳細な分離技術への適応は、その有効性を改善します。短時間でのCSCのかなりの数を得ることができ、いくつかの実験のために使用されます。したがって、迅速にOS-のCSCを特徴付ける二幹様の表現型を研究するために、特に、幹様の表現型を確認し、することが可能です。したがって、この修正されたアッセイは、CSCをを単離し、それらの生物学を研究するための優れた手法である可能性があります。将来的には、この方法は、追加の適応でも希少な固形腫瘍の生検により得られた他の有限の癌細胞株からのCSCを単離するために使用されてもよいです。 OSなど希少な固形腫瘍からのCSCを単離する可能性は、この特定の癌に関する研究の改善を可能にするだけでなく、それらの単離のためのより良い方法を開発する異なるタイプの癌の研究にとの生物学の今後の研究にも及ぶだけでなく、この重要な細胞サブセット。したがって、我々として本研究で行っている、おそらくのために責任があるこの特定の細胞サブセットに対して向けの非常に特定の抗癌治療を、分子標的を発見し、開発の最終目標で、CSC生物学の研究を通じてCSC分離の方法を向上させることが重要です原発腫瘍の維持、再発の開発、およびいくつかの器官における転移の起源。 CSC生物学の研究でも、このようなネオアジュバント治療後の生存率は非常に貧しいままであるためにOS、などの癌の治療法に鋭いかもしれない治療法を見出すために重要です。

肉腫は胚の中胚葉1から主に由来するまれな悪性の結合組織腫瘍の異種群です。異なるタイプの骨肉腫および軟部組織肉腫を含みます。骨肉腫は、比較的まれ原発腫瘍のグループは、骨肉腫（OS）を含むいくつかのサブタイプ、で構成されています。 OS、骨の最も一般的な原発腫瘍の一つは、大規模な臨床、組織学的、および分子不均質2、3を示す間葉系悪性腫瘍である。残念ながら、OSは5、子供におよび若年成人4で主に発生し、60％を占めます小児期における骨肉腫の一般的な組織学的サブタイプは6、7。OSは通常影響を与える急速な骨の成長によって特徴付けられる骨格領域、（ 例えば 、長骨の骨幹）。 OS、従来のOSの組織学的に異なるサブタイプの中でも、また髄または中央OSと呼ばれる、悪性度の高いグレードとクォータ社を持っています80％8の再。この80％、60％、従来の骨芽細胞のOS、10％軟骨芽OS、および10％の線維芽OS 6、8-10から構成されています。他のOSのサブタイプは、未分化、毛細血管拡張、巨細胞が豊富で小細胞OSが含まれています。 OS管理で組み合わせ、手術と化学療法の進歩にもかかわらず、結果は転移11、12のない患者では65から70パーセントの長期生存率は、貧しいまま。遠い再発が頻繁に転移として、それほど頻繁に、肺転移として発生したり、遠くの骨や地元の再発13へ。転移は、多くの場合、従来の治療に抵抗性です。この抵抗は、10年の無病生存率は診断14、15に転移性疾患を有する患者では約30％である理由です。 正常組織と同様に、癌組織は、細胞型の異種コレクションから構成されています。腫瘍内の細胞は、発生の異なる段階に対応するように見えます。任意のn個以内ormal組織は、このように組織の恒常性のための前駆細胞および成熟細胞を提供し、selfrenewする能力を有する細胞の亜集団を常駐します。同様に、癌が増殖および腫瘍形成能の程度が異なると、発生の異なる段階における細胞の同様の不均一な集団で構成されています。これらの癌細胞のサブセットは、がん幹細胞（CSCを）と呼ばれる、このようにして腫瘍塊16を構成する種々の細胞系統を生成する、腫瘍の悪性度をselfrenewし維持する排他的な能力を有する細胞をselfsustainingのリザーバを構成しています。 1990年代には、急性骨髄性白血病の研究は、CSCの亜集団17、18の存在のための最初の説得力のある証拠を提供した。CSCがあるため、それによって癌研究の中で最も研究テーマの一つになって、固形腫瘍19の多数から単離されています。 CSCが実際に正常を作る遺伝子の変異により正常な幹細胞から生じる可能性があります20-23癌幹細胞。微小環境を持つ複数の転換の突然変異と相互作用ものCSCを代表するselfrenewal容量と不死を取得し、健康な前駆細胞および成熟細胞に寄与し得ます。この変換に関するいくつかの仮説があります。健康な前駆細胞、健康な成熟細胞、癌細胞は、selfrenewal関連遺伝子24-28を活性化することにより幹様表現型を得る、幹細胞に脱分化することができます。いくつかの最近の研究にもかかわらず、CSCをの起源はまだ発見されなければなりません。 CSCの特定の特性は、その容量は異なる薬剤との併用手術と化学療法で構成されたマルチ治療アプローチを、抵抗することです。最近の研究でのCSCはまた、細胞毒性化学療法剤に対する耐性を獲得することができることを示しています。この抵抗のための可能な説明は、ATP結合カセット（ABC）多剤輸送体（ すなわち、の過剰発現を含みますMDR1およびBCRP1）、アルデヒドデヒドロゲナーゼ1（ALDH1）、および/または細胞周期動態30-33の変化として化学療法代謝酵素の過剰発現。これまでに記載されているすべてのこれらの概念の直接的な結果は、CSCの亜集団が完全に排除された場合にのみ、1つでもCSCが生き残った場合は局所再発や遠隔転移が発生する可能性がありながら、癌治療は、効率的であるということです。 それは多くの治療が最初に有効であると思われる理由として可能性のある説明が、患者後に再発していますので、人間の肉腫34、特にOS 35で、または任意の他の骨と軟部組織のがんでのCSCの発見は、大きな臨床的重要性を持っています。そのため、従来のOSに対する今後の戦いのための希望は、このサブの分子特性にOS-のCSCのおかげに向けた革新的な医薬品の開発に基づいて、新しいと特定の標的療法を見つけることです人口とCSC生物学の研究に。 1992年には、幹細胞のサブセットは、成人哺乳類の脳に存在したかどうかを調査したレイノルズらは、幹細胞様細胞36、37であることが疑われる細胞を分離する方法を開発した。この方法は、特定の能力に基づいています非付着条件下で増殖するとき、これらの細胞は球状のコロニーを形成します。同様の技術は、骨肉腫38中の幹細胞様細胞の亜集団を研究するために2005年にギブスらによって採用されました。従来のOSの異なるタイプの初代細胞培養物から、OSのCSCを単離し、特徴づけるために、我々は、OS細胞株は、この技術を適応することを決定しました。 ここでは、球体形成アッセイのこの適合方法を説​​明し、従来のOSのヒト生検に由来する有限の初代細胞株からOS-のCSCを単離することができる「sarcosphereアッセイ」と呼ばれます。我々はまた、すべての技術のuを記述しますこのアッセイにより単離された細胞株の幹様CSC表現型を検証するためのsed：多能性胚性幹細胞（ESC）を特徴づける遺伝子とのCSCのマーカーであるCD133遺伝子の発現の1）の評価を、 2）コロニー形成単位（CFU）アッセイ。 3）適切な分化条件下での骨芽細胞と脂肪細胞に分化するために、これらの細胞の能力を評価します。間葉系幹細胞（MSC）の表面マーカーの4）研究（ すなわち、CD44、CD105及びSTRO-1）の免疫蛍光染色により、フローサイトメトリー分析によって、 5）これらの細胞のALDH活性を評価します。

<table><tbody><tr><td>Dulbecco&#039;s Phosphate Buffered Saline with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; (DPBS)</td><td>LONZA</td><td>BE17-513F</td><td>_</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline&amp;nbsp; without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; (DPBS)</td><td>LONZA</td><td>BE17-512F</td><td>_</td></tr><tr><td>Porcine Trypsin 1:250</td><td>BD Difco</td><td>215310</td><td>Solvent: DPBS. Stock concentration: Powder</td></tr><tr><td>Ethylenediamine tetraacetic acid disodium salt dihydrate(EDTA)</td><td>Sigma-Aldrich</td><td>E4884</td><td>Solvent: DPBS. Stock concentration: Powder</td></tr><tr><td>Collagenase from Clostridium histolyticum</td><td>Sigma-Aldrich</td><td>C0130</td><td>Solvent: Buffer Solution, pH 7.4. Stock concentration: Powder</td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>BDH Chemicals-VWR</td><td>10323</td><td>_</td></tr><tr><td>Nutrient Mixture F-12 Ham</td><td>Sigma-Aldrich</td><td>F6636</td><td>Solvent: Ultrapure dH&lt;sub&gt;2&lt;/sub&gt;O. Stock concentration: Powder</td></tr><tr><td>2-Phospho-L-ascorbic acid trisodium salt</td><td>Sigma-Aldrich</td><td>49752</td><td>Solvent: DPBS. Stock concentration: 5 mg/mL</td></tr><tr><td>&amp;beta;-Glycerol phosphate disodium salt pentahydrate</td><td>Sigma-Aldrich</td><td>50020</td><td>Solvent: DPBS. Stock concentration: 1 M</td></tr><tr><td>Insulin. Human Recombinant</td><td>Sigma-Aldrich</td><td>91077</td><td>Solvent: NaOH 0.1 M. Stock concentration: 10 mM</td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma-Aldrich</td><td>I5879</td><td>Solvent: DMSO. Stock concentration: 500 mM</td></tr><tr><td>Indomethacin</td><td>Sigma-Aldrich</td><td>I7378</td><td>Solvent: DMSO. Stock concentration: 200 mM</td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td>Solvent: DMSO. Stock concentration:&amp;nbsp; 1 mM / 100 &amp;micro;M. Store in liquid nitrogen to maintain the biological activity</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>F7524</td><td>_</td></tr><tr><td>Fetal Bovine Serum South America&amp;nbsp;</td><td>EUROCLONE</td><td>ECS0180L</td><td>_</td></tr><tr><td>Penicillin-Streptomycin (PEN-STREP) 10,000 U/mL</td><td>LONZA</td><td>DE17-602E</td><td>_</td></tr><tr><td>Methyl cellulose</td><td>Sigma-Aldrich</td><td>274429</td><td>Solvent: Ultrapure dH&lt;sub&gt;2&lt;/sub&gt;O. Stock concentration: 2%</td></tr><tr><td>Putresceine dihydrochloride</td><td>Sigma-Aldrich</td><td>P5780</td><td>Solvent: Ultrapure dH&lt;sub&gt;2&lt;/sub&gt;O. Stock concentration: Powder</td></tr><tr><td>apo-Transferrin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T-1147</td><td>Solvent: DPBS. Stock concentration: 25 mg/mL</td></tr><tr><td>Human Epidermal Growth Factor (EFGF)</td><td>Sigma-Aldrich</td><td>E5036</td><td>Solvent: DPBS pH 7.4. Stock concentration: 10 &amp;micro;g/mL</td></tr><tr><td>Fibroblast Growth Factor-Basic Human</td><td>Sigma-Aldrich</td><td>F0291</td><td>Solvent: DPBS + 0.2% BSA. Stock concentration: 25 &amp;micro;g/mL</td></tr><tr><td>Selenous Acid</td><td>Sigma-Aldrich</td><td>211176</td><td>Solvent: DPBS. Stock concentration: 30 mM</td></tr><tr><td>Progesterone</td><td>Sigma-Aldrich</td><td>P8783</td><td>Solvent: ETOH. Stock concentration: 10 mM</td></tr><tr><td>Toluidine Blue O</td><td>Sigma-Aldrich</td><td>198161</td><td>Solvent: Ultrapure dH&lt;sub&gt;2&lt;/sub&gt;O. Stock concentration: Powder</td></tr><tr><td>Oil Red O</td><td>ICN Biochemicals</td><td>155984</td><td>Solvent: 2-Propanol. Stock concentration: Powder</td></tr><tr><td>Naphtol AS-MX Phosphate Disodium Salt</td><td>Sigma-Aldrich</td><td>N5000</td><td>Solvent: DMSO. Stock concentration: Powder</td></tr><tr><td>Fast Blue BB Salt</td><td>Sigma-Aldrich</td><td>F3378</td><td>Solvent: Tris HCL, pH 9.0. Stock concentration: Powder</td></tr><tr><td>Fast Red Violet LB Salt</td><td>Sigma-Aldrich</td><td>F3381</td><td>Solvent: Tris HCL, pH 9.1. Stock concentration: Powder</td></tr><tr><td>Bovine Serum Albumin, Fraction V (BSA)</td><td>Sigma-Aldrich</td><td>A-4503</td><td>Solvent: DPBS. Stock concentration: 2%</td></tr><tr><td>Alizarin Red S</td><td>ICN Biochemicals</td><td>100375</td><td>Solvent: Ultrapure dH&lt;sub&gt;2&lt;/sub&gt;O. Stock concentration: Powder</td></tr><tr><td>Formaldehyde solution</td><td>Sigma-Aldrich</td><td>533998</td><td>4%</td></tr><tr><td>Triton-100X</td><td>MERCK</td><td>11869</td><td>Solvent: DPBS. Stock concentration: 0.2%. Danger - Use only under chemical hood</td></tr><tr><td>Calcein</td><td>MERCK</td><td>2315</td><td>Solvent: DPBS . Stock concentration: 200 &amp;micro;g/mL</td></tr><tr><td>2-Propanol</td><td>MERCK</td><td>109634</td><td>Danger - Use only under chemical hood</td></tr><tr><td>Ab-CD105&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Mouse monoclonal [SN6] to CD105 (FITC)</td><td>Abcam</td><td>ab11415</td><td>Liquid. Application:&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Flow Cytometry (Flow Cyt)</td></tr><tr><td>Ab-CD44&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Mouse monoclonal&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; [F10-44-2] to CD44 (PE/Cy7&amp;reg;) )</td><td>Abcam</td><td>ab46793</td><td>Liquid. Application:&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Flow Cytometry (Flow Cyt)</td></tr><tr><td>Ab-CD45&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Mouse monoclonal&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; [MEM-28] to CD45 (PerCP))&amp;nbsp;</td><td>Abcam</td><td>ab65952</td><td>Liquid. Application:&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Flow Cytometry (Flow Cyt)</td></tr><tr><td>Ab-CD105&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Human CD105 Purified Antibody)&amp;nbsp;</td><td>Invitrogen</td><td>MHCD10500</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application: Immunofluorescence staining (IF)</td></tr><tr><td>Ab-CD44&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Anti-CD44 Antibody)&amp;nbsp;</td><td>Abcam</td><td>EPR1013Y(ab51037)</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application: Immunofluorescence staining (IF)</td></tr><tr><td>Ab-Stro-1&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Mouse anti-STRO-1)&amp;nbsp;</td><td>Invitrogen</td><td>398401</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application:&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Flow Cytometry (Flow Cyt)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Immunofluorescence staining (IF)</td></tr><tr><td>Alexa Fluor 488&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Anti-Rabbit IgG (Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L))</td><td>Invitrogen</td><td>A-21206</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application: Immunofluorescence staining (IF)</td></tr><tr><td>FITC Anti-Mouse IG (FITC-Rabbit Anti Mouse IgG (H+L))</td><td>Invitrogen</td><td>61-6511</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application:&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Flow Cytometry (Flow Cyt)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Immunofluorescence staining (IF)</td></tr><tr><td>Alexa Fluor 635 Phalloidin&amp;nbsp;</td><td>Invitrogen</td><td>A34054</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application: Immunofluorescence staining (IF)</td></tr><tr><td>AutoMACS&amp;trade;Running Buffer MACS Separation Buffer</td><td>Miltenyi</td><td>130,091,221</td><td>Liquid. Store at 4 &amp;deg;C</td></tr><tr><td>QIAzol&amp;reg;Lysis Reagent</td><td>QIAGEN</td><td>79306</td><td>Danger - Use only under chemical hood</td></tr><tr><td>QUANTITECT&amp;reg;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Reverse Transcription Kit</td><td>QIAGEN</td><td>205314</td><td>_&amp;nbsp;</td></tr><tr><td>Chlorophorm</td><td>Sigma-Aldrich</td><td>C2432</td><td>Liquid. Danger - Use only under chemical hood</td></tr><tr><td>Laminar flow hood</td><td>GELAIRE</td><td>BSB6A</td><td>_&amp;nbsp;</td></tr><tr><td>Chemical hood</td><td>ARREDI TECNICI&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Villa</td><td>Modello&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; DYNAMICA</td><td>_&amp;nbsp;</td></tr><tr><td>Centrifuge</td><td>EPPENDORF</td><td>5415R</td><td>_&amp;nbsp;</td></tr><tr><td>Laser Scanning Confocal Microscopy LSM 5109 Meta&amp;nbsp;</td><td>ZEISS</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>iCycler PCR Thermalcycler&amp;nbsp;</td><td>BIORAD</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>CyFlow&amp;reg;SPACE&amp;nbsp;</td><td>(PARTEC)</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Inverted Micrposcope Axiovert 200M&amp;nbsp;</td><td>ZEISS</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Freezing container ,</td><td>Nalgene</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Original Pipet-Aid</td><td>pbiBrand</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Micropipettes</td><td>EPPENDORF</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Glass Pasteur Pipette</td><td>SIGMA</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>VICTOR3&amp;trade;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>PERKIN ELMER</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Conical tubes&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (15 and 50 mL)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD FALCON</td><td>352096 (for 15 mL)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 352070 (for 50 mL)</td><td>_&amp;nbsp;</td></tr><tr><td>24-Well Clear Flat Bottom TC-Treated Multiwell-Cell-Culture-Plate</td><td>BD FALCON</td><td>353047</td><td>_&amp;nbsp;</td></tr><tr><td>6-Well Clear Flat Bottom Ultra Low Attachment Multiple-Well-Plates</td><td>CORNING</td><td>3471</td><td>_&amp;nbsp;</td></tr><tr><td>Serological pipettes&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (5 and 10 mL)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD FALCON</td><td>357543 (for 5 mL)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 357551 (for 10mL)</td><td>_&amp;nbsp;</td></tr><tr><td>Syringe (5mL)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>B|BRAUN</td><td>4617053V</td><td>_&amp;nbsp;</td></tr><tr><td>Petri dish 100X20 mm&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD FALCON</td><td>353003</td><td>_&amp;nbsp;</td></tr><tr><td>R&amp;ouml;hren Tubes&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (3.5 mL, 55x12mm, PS)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>SARSTEDT</td><td>55,484</td><td>_&amp;nbsp;</td></tr><tr><td>Petri dish 60X15 mm&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD FALCON</td><td>353004</td><td>_&amp;nbsp;</td></tr><tr><td>Cryovials 1.5 mL&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>NALGENE</td><td>5000-1020</td><td>_&amp;nbsp;</td></tr><tr><td>Cell-Culture Flasks 25 cm&amp;sup2;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD FALCON</td><td>353014</td><td>_&amp;nbsp;</td></tr><tr><td>Nylon Net Filter, Hydrophilic</td><td>MERCK</td><td>NY4104700</td><td>_&amp;nbsp;</td></tr><tr><td>Swinnex Filter Holder</td><td>MERCK</td><td>SX0002500</td><td>_&amp;nbsp;</td></tr><tr><td>Perry tweezer&amp;nbsp;</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Lancet</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Dounce</td><td>_</td><td>_</td><td>_</td></tr></tbody></table>

establishment of cancer stem cell cultures from human conventional osteosarcoma

骨肉腫（OS）に対する治療における現在の改善は、癌患者の生命を延長しているが、転移が発生した場合に5年の生存率は悪いまま。癌幹細胞（CSC）理論は、腫瘍を維持し、多剤化学療法に抵抗する能力を含む幹様特性を有する腫瘍内の腫瘍細胞のサブセットが存在することを保持します。したがって、OS生物学および病因論のより良い理解は、この特定のサブセットを根絶し、患者の間で罹患率および死亡率を減少させるために標的治療の開発を進めるために必要とされます。 、のCSCの単離のCSCの細胞培養を確立し、それらの生物学を研究すると、OSの生物学および病因の我々の理解を改善するための重要なステップです。 OSの生検からのヒト由来のOS-のCSCの確立がnonadhere下3次元幹細胞培養物を作成する能力を含むいくつかの方法を用いて可能になりましたNT条件。これらの条件下では、CSCを娘幹細胞によって形成された球状の浮遊コロニーを生成することができます。これらのコロニーは、「細胞球」と呼ばれます。ここでは、OSの生検から得られた従来のOSの初代細胞培養物からのCSCの培養物を確立するための方法を記載しています。我々は明らかにのCSCを分離し、特徴付けるために必要ないくつかの通路を説明します。

Watch this Scientific Journal Video about Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma at JoVE.com

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

骨肉腫における癌幹細胞（CSCを）の存在は、最近、その病因に関連しています。本稿では、非接着性条件下で増殖するのCSCの能力を用いて、従来の骨肉腫（OS）の人間の生検から得られた初代細胞培養物からのCSCの単離を提示します。

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 ...

establishment-cancer-stem-cell-cultures-from-human-conventional

Nanyang Technological University

Research

JoVE Journal

Cancer Research

18.9K ビュー.  University of Florence. 骨肉腫における癌幹細胞（CSCを）の存在は、最近、その病因に関連しています。本稿では、非接着性条件下で増殖するのCSCの能力を用いて、従来の骨肉腫（OS）の人間の生検から得られた初代細胞培養物からのCSCの単離を提示します。 

ビデオ: Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

患者由来の軟部肉腫の主な文化の確立

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

がん研究

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

肉腫患者由来異種移植片からの腫瘍始発細胞の単離と特徴付け

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

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 ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

マウス横紋筋肉腫から単離された原発性腫瘍細胞からの腫瘍球誘導と治療

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

遺伝学

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).

CD133の患者由来の細胞培養および分離<sup&gt; +</supメラノーマから&gt;推定されるがん幹細胞

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

医学

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

<meta charset="utf8"></head><body>ヒト間質を用いた卵巣癌の同所性マウスモデルによる転移促進

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

ヒト間質を用いた卵巣癌の同所性マウスモデルによる転移促進

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

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.

ヒト前立腺癌サンプルからがん幹細胞の単離

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

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

ヒト細胞株から化学療法抵抗性の卵巣がん幹細胞の濃縮

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

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.

がんの確立は、ヒト従来の骨肉腫からの幹細胞培養物

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患者由来の軟部肉腫の主な文化の確立

肉腫患者由来異種移植片からの腫瘍始発細胞の単離と特徴付け

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

マウス横紋筋肉腫から単離された原発性腫瘍細胞からの腫瘍球誘導と治療

CD133の患者由来の細胞培養および分離

ヒト間質を用いた卵巣癌の同所性マウスモデルによる転移促進

ヒト間質を用いた卵巣癌の同所性マウスモデルによる転移促進

ヒト前立腺癌サンプルからがん幹細胞の単離

ヒト細胞株から化学療法抵抗性の卵巣がん幹細胞の濃縮

マルチとシングルセルベース球アッセイを用いてヒト卵巣癌細胞における幹細胞特性の評価