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実験手順や動物の倫理的な治療は、ピッツバーグ大学IACUC（プロトコル＃1104674）によって承認されました。 G6PDの染色用混合物の調製注：次のように各試薬の最終濃度であることを確認してください。 5mMのグルコース-6-リン酸（G6P）。 2mMのNADP、5mMのMgCl 2、0.35 mMの1-メトキシ-5-メチルフェナジニウムメチルサルフェート（MMPMS）及び0.8mMのNBTの初期濃度は40 mlの最終容積のために導出された各試薬については1,8,9。溶液を、新たに調製し、毎日使用しました。 <ol><li> 50mlのチューブに10月培地35 mlに200mMのpH7.4のリン酸緩衝液（PB）の2ミリリットルを加え、激しく振ります。ソリューションは、pH約7.4であるpH試験紙で確認してください。そうでない場合は、解決策を捨てます。 </li><li> 61ミリグラムG6Pが200mm PBの0.94ミリリットルに溶かし追加、61ミリグラムNADPは、200mMのPBの0.94ミリリットルに溶解し、41mgの塩化マグネシウムは 0.96ミリリットルの100mMのPBに溶解しました。 pHがあることを再確認〜7.4ウィット時間pH試験紙。ない〜7.4場合は、ソリューションを破棄します。 </li><li> 200mMのPB（pH7.4）での0.25ミリリットルに溶かしMMPMS 5mgを追加します。精力的に透明な暗赤色のホモジネートを生成するために、得られた反応混合物を振ります。解決策が濁っている場合、混合物は捨ててください。反応混合物を含むチューブは、光への暴露を最小限に抑えるためにホイルで包まれています。最後のコンポーネント、NBTは、用意されている間、室温で反応混合物を保管してください。 </li><li> Oリングと蓋2 mlチューブにNBTの164 mgの0.4 mlの無水ジメチルホルムアミド（DMF）と0.4 mlの無水エタノールを添加することにより、別々に5mMのNBT溶液を調製します。 loosely蓋を閉じます（しっかりと密封されていない）と、NBTが溶液になるまでオイルバスで活発な沸騰の解決策をもたらす。8 </li><li>可溶化されたNBTを室温まで冷却した後、10月の反応混合物へのソリューションのすべてを追加し、透明な溶液を得るために、積極的に振るようにします。暗赤色またはパープに最初の橙赤色から溶液の色の変化を観察経時ル色。これは正常です。 </li><li>使用前に1時間37℃の暖かい部屋で、最終的なG6PD染色混合物を置きます。 </li></ol> 2.動物の治療や組織採取<ol><li> DNA損傷剤 、例えばで6週齢のC57BL / 6雄マウスを治療、腹腔内注射により200μlのPBSの容量で10mg / kgのアゾキシメタン（AOM）。腹腔内注射により200μlのPBS対照マウスを扱います。 </li><li> IACUCプロトコルによって承認され、90日には、頸椎脱臼に続いてCO 2窒息によりマウスを安楽死させます。 </li><li>外科的胸骨のベースにちょうど肛門上から腹腔を開きます。体腔から小腸に移動し、それを切除しないでください。ちょうど直腸に盲腸以下の低い腸を切除。10 </li><li>慎重にマイクロ解剖ハサミを使用して、コロンの一辺に沿ってスライスし、きれいな滅菌PBSで洗浄することによって開かれたコロンから糞便を削除します。物品税スイスロールDの外側に向け腔側とコロン、および組織型内の場所。10 </li><li>完全に10月の媒体にコロンを浸すと、液体N 2を含むデュワーに金型を配置します。プラスチックベースはちょうど10月の媒体が固体凍結されるまで液体N 2と接触するように金型を持ちます。 </li><li>凍結切片を切断するまで-80で凍結組織ブロックを格納します。 G6PD酵素の不活性化をもたらす任意の固定液を、使用しないでください。 </li></ol>凍結切片の調製<ol><li> -25℃でクライオスタットを用いて、7ミクロンの厚さで凍結組織切片をカットします。 4連続した7μmのセクションをカットし、同じスライド（ 図1）上でそれらのうちの2つを配置します。コロン（ 図1）の1つのレベルを表すために2スライド、または4つの連続した7μmの切片を作製。 </li><li> PRの最後のセクションから&gt;50μmの最小距離と次のレベルのために繰り返しeviousレベル。この距離は、各レベルについて評価陰窩が互いに重複していないことを確実にしました。 </li><li>よく切断中に蓄積する10月の残留物を除去するために、100％エタノールでクライオスタットブレードを拭きます。切片が再開される前に、乾燥したブレードは、蓄積された静電荷を消散させる帯電防止ドライヤーシートで拭きます。 C. O -80で凍結切片と店舗スライド</li></ol>凍結切片の組織の4染色<ol><li>湿度の高い37℃の暖かい部屋で、酵素組織化学反応を実行します。 注：インキュベーションオーブン内またはスライドウォーマー上の酵素反応をしようとは、貧しい人々や一貫性のないG6PD染色につながりました。 </li><li>シリコーングリース（ 図1）を使用して一緒に結合された2つの1.5センチメートルのx 0.15センチメートル鋼ワッシャー（内径×高さ）を使用して、組織染色用の井戸を構築します。中心とウェルのベースに合わせてパラフィルムをカット切り取り、井戸の場所スライド上の組織切片の上にD。 注：洗濯機の底部にパラフィルムは、スライドし、組織切片上の粘性G6PD染色混合物を維持して座金との間にシールを作成します。研究では、各組織切片は、G6PD反応染色混合物の同じ体積を受信するように、0.5ミリリットル容量 - この構築物は0.4とよくを与えます。 </li><li> 10分間、暖かい部屋で37°Cの時に凍結した組織スライドをインキュベートします。 37℃で、45〜50分間、各組織切片ウェル（ス​​ライドあたり2つのセクション）にG6PD染色反応混合物を追加します。暖かい部屋からスライドを取り外し、慎重にスライドオフステンレス製の井戸を持ち上げます。 </li><li>反応媒体を排出できるようにするために彼らの長いエッジでスライドを設定します。組織染色を乱すことなく組織からG6PD染色反応混合物を除去するために30〜60分間、100mMのPB（pH7.4）中置きスライド。 PB塩を除去するために5〜10分間蒸留水中でスライドを沈めます</li><li>追加することによって、組織をシール組織上にスポイトからフルオロゲルをると、それが乾燥するために〜30分を待っています。変異した陰窩の識別を混乱させるできる気泡を避けてください。 注：気泡が形成した場合は、手順は、蒸留水に浸漬してフルオロゲルを除去した後に繰り返すことができます。彼らはトラップ気泡に傾向があるように、カバースリップを使用しないでください。 </li></ol> 5. G6PD変異陰窩の決意と変異頻度<ol><li>光学顕微鏡下でG6PD変異陰窩のためのスライドを分析します。 注：完全に変異クリプトを識別するために使用される基準は、（ⅰ）全体陰窩は青色を欠いていました。 （ii）の陰窩の外部構造（iii）は、変異体のcryptは、（iv）の二人の観察者は、変異体と同じ地下室を識別しなければならなかった、隣接する7μmの切片で観察した、無傷でした。隣接7μmの切片で観察された変異体は、1つの変異体としてカウントされます。 </li><li>画像各G6PD変異40倍の倍率でcryptと5.0メガピクセルのカメラとを使用して画像をカタログイメージングソフトウェア。 </li><li> 10倍の倍率（図1C）で最小の表面積とイメージを持つレベルから各マウスについての代表的な部分を選択してマウスで調べた陰窩の合計数を定量化します。 注：レベルは、その近接にしばしば複数のセクションに陰窩変異SHOW 4つの連続7μmの切片（ 図1D）を表します。レベルは、コロンの異なる領域から陰窩を可視化するためにミクロン50&gt;で分離されています。 </li><li>各画像ファイルを開いて、視覚的に（ 図1Cにボックスとして示されている）ファイルを解析することにより、レベルに陰窩の数を数えます。画像ファイルは、一つのファイルに20〜300&gt;に陰窩ような低いから観察陰窩の数が異なります。 </li><li>各コロンのためのG6PD変異陰窩についてスクリーニングレベルの数によって各マウスのために選択されたレベル（ 図1C）から陰窩の合計数を掛けます。例えば、の総数変異陰窩についてスクリーニング8レベルを乗じ選択されたレベルごとに1,250陰窩10,000陰窩の合計数に等しいです。統計分析のために各コロン10,000陰窩の最小値を評価します。 </li><li>各治療のため、MFを計算するためにスクリーニング陰窩の総数ですべての動物で観察された変異体の数を組み合わせます。未処理マウスにおける背景幹細胞MFレベルが&lt;0.1x10 -4以前にC57BL / 6マウスで報告されているものに近い（ 表1）、。11であります</li></ol>

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</ol>

The authors have nothing to disclose.

多くの場合、化合物の遺伝毒性作用はDNAを改変する能力によって決定されます。これは通常、組織を単離し、DNA付加体の全体的なレベルを測定することによって行われます。 AOMの場合、これはOを結腸6 -メチルグアニン付加物を定量伴います。そのような幹細胞ニッチのような特定の細胞型内の損傷に対するこのアプローチの情報を使用して、失われます。付加体の小さなサブセット...

大腸癌は、DNAに損傷を与えることができ、環境エージェントと食物成分への曝露を伴い、（ 例えば 、APC）の腫瘍抑制遺伝子に変異を癌遺伝子に体細胞変異を活性化（例えば、β-カテニン）または不活性化生成すると考えられています。これは、これらの重要な突然変異は結腸幹細胞において生じることが想定されます。なぜなら結腸上皮の固有の暗号アーキテクチャのために、それは、結腸発癌に関連する化学物質に動物を曝露した後に、結腸中の幹細胞の変異を測定することができます。突然変異がクリプト内の全ての細胞に存在することになるなど、いくつかのX連鎖酵素は、幹細胞で起こる突然変異のための指標としての役割を果たすことができます。 以前は、手順は、マウスにおいて結腸腫瘍を誘発する化学物質はまた、X連鎖グルコース-6-リン酸デヒドロゲナーゼ（G6PD）遺伝子の突然変異の分析によって結腸における体性幹細胞の突然変異を生成することを実証掲載されました。1-3この方法は、任意選択圧なしで結腸幹細胞におけるランダムな体細胞変異の発生率を定量化します。手順は、処理されたマウスおよび対照マウスからの未固定凍結された大腸セクションの生産と機能G6PD活性を産生する細胞を欠いている陰窩の同定を含みます。 、白く見えるこれらの変異した陰窩は、変異はまた、変異したG6PD遺伝子を有する子孫を生じさせた幹細胞で発生したことを示しています。酵素アッセイでは、G6PD欠損変異体の陰窩は、ニトロブルーテトラゾリウム（NBT）の還元のために必要とされるグルコース-6-リン酸を酸化することができません。 G6PD酵素が機能している場合には、染色用混合物中のNBT青色酵素及び「染色」は、細胞の位置に蓄積し、不溶性のホルマザン沈殿物に還元されます。 G6PDの変異体である細胞は青く染色された野生型陰窩とは対照的に白っぽいまま。この方法は、「ヌル」表現型を有する変異を測定します。 enため、zymes、例えば、G6PDとして、切片を水溶液中に置かれた場合に非固定組織切片上で細胞外に拡散することができ、それは分析されるべき組織切片中の酵素を安定化するために必要であり、図4中の酵素の安定化組織は、G6PD酵素反応に必要な試薬の小分子の拡散を妨害してはなりません。 私たちは、元のプロシージャに大幅な変更の数を行いました。重要な酵素染色培地は、pH制御およびアッセイにおいて使用される成分の可溶化を容易に最適切削温度化合物（OCT）にポリビニルアルコールから変更されています。それぞれの組織切片は、G6PD反応混合物の同じ量を受信するようにスチールワッシャからなる0.5 mlのウェル - 染色は、0.4を用いて行われます。手順がなくても、結腸組織の大規模なサンプリングを提供して撮像されたセクション内の陰窩の数を推定するために開発されましたmanually&gt;各コロンのための10 5陰窩をカウントします。隣接7μmの組織切片の分析は、潜在的な染色アーチファクトを低減する複数のスライド上の変異陰窩の視覚化を提供します。これらの変更は、手順がより効率的かつ再現可能にします。 この修正されたプロトコルを使用して、我々は、げっ歯類においてよく知られている結腸の発癌物質のアゾキシメタンをC57BL / 6マウスの曝露後の結腸幹細胞の突然変異頻度を定量化している。5-7

<table><tbody><tr><td>reagents</td><td></td><td></td><td></td></tr><tr><td>nitroblue tetrazolium</td><td></td><td></td><td></td></tr><tr><td>NADP</td><td></td><td></td><td></td></tr><tr><td>glucose-6-phosphate</td><td></td><td></td><td></td></tr><tr><td>1-methoxy-5-methylphenazinium methyl sulfate</td><td></td><td></td><td></td></tr><tr><td>dimethyl formamide</td><td></td><td></td><td></td></tr><tr><td>phosphate buffer (pH 7.4</td><td></td><td></td><td></td></tr><tr><td>Optimal Cutting Temperature Compound (OCT)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>light microscope equipped with 5 megapixel camera</td><td></td><td></td><td></td></tr><tr><td>cryostat</td><td></td><td></td><td></td></tr><tr><td>warm room&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

quantification of  colonic stem cell mutations

幹細胞の変異を測定する能力は、化学物質が潜在的に癌を引き起こす突然変異を誘発することができた場合、どの程度に重要な細胞集団に定量化するための強力なツールです。 X連鎖グルコース-6-リン酸デヒドロゲナーゼ遺伝子で幹細胞変異を定量化するための酵素アッセイの使用は、以前に報告されている。1この方法は、得られる反応混合物と切片の組織の凍結切片とのインキュベーションの準備が必要青色細胞は、機能的なグルコース-6-リン酸デヒドロゲナーゼ（G6PD）酵素を生成した場合。そうでない場合、細胞が白っぽく見えます。我々は、ポリビニルアルコールの代わりに、最適切削温度化合物（OCT）培地を用いて反応混合物に変更しました。これは、pH測定を容易にG6PD染色成分の可溶化を増加させ、G6PD酵素の拡散を制限します。突然変異は、幹細胞で生じたことを実証するために、全体の陰窩はG6PD酵素を欠いている必要があります活動。幹細胞が抱いている場合のみ、表現型G6PD変異は陰窩における子孫のすべては、G6PD酵素活性を欠くであろう。幹細胞変異を有する陰窩を識別するために、4つの連続した​​隣接する凍結切片（レベル）が7μmの厚さで切断しました。隣接するカットを作るこのアプローチは、同じ変異した地下室は、隣​​接するセクションで観察されるので、地下室が完全に変異させたコンホメーションを提供します。 50μmより離れた組織サンプルとスライドは、マウスあたり&gt; 10 4陰窩の合計を評価するために調製しました。突然変異頻度は、治療群では、野生型（青）陰窩の数で÷観測された変異した（白）陰窩の数です。

動物に結腸幹細胞変異を測定する能力は、癌誘発に変異を相関させるユニークな方法を提供します。通常、これは、発癌における重要な段階は、癌遺伝子における突然変異を活性化および/または腫瘍抑制遺伝子における変異を不活性化することを含むものとします。私たちは、200μlのPBSまたはPBS200μl中10mg / kgのAOMとC57BL / 6マウスに注射。 AOMは、既知の大腸発がん性物質である。コロンは、?...

Watch this Scientific Journal Video about Quantification of  Colonic Stem Cell Mutations at JoVE.com

Quantification of  Colonic Stem Cell Mutations

We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

結腸幹細胞変異の定量

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce ...

quantification-of-colonic-stem-cell-mutations

Research

JoVE Journal

Developmental Biology

6.6K ビュー.  University of Pittsburgh. We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

ビデオ: Quantification of  Colonic Stem Cell Mutations

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

タッチ捺印細胞診を用いた臨床病理標本から質の高い腫瘍 DNA を取得する簡易法

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

がん研究

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single &#x2018;marker&#x2019; has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. 
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. 
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6. 
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

抗体マイクロアレイと蛍光多重を用いた大腸癌の細胞表面タンパク質のプロファイリング

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

免疫・感染学

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.

The presented diagnostic FL-DNA kit is a time-saving and user-friendly method to determine the reliable probability of the presence of colorectal cancer lesions.

便DNA完全性検出による大腸癌リスクと罹患率の評価

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the initiation of cancer stemness properties. Since gene expression in the formation of colorectal cancer-derived tumorspheres is unclear, it takes time to discover the mechanisms working on one gene at a time. This study demonstrates a method to quickly discover the driver genes involved in the survival of the colorectal cancer stem-like cells in vitro. Colorectal HT29 cancer cells that express the LGR5 when cultured as spheroids and accompany an increase CD133 stemness markers were selected and used in this study. The protocol presented is used to perform RNAseq with available bioinformatics to quickly uncover the overexpressed driver genes in the formation of colorectal HT29-derived stem-like tumorspheres. The methodology can quickly screen and discover potential driver genes in other disease models.

Presented here is a protocol to discover the overexpressed driver genes maintaining the established cancer stem-like cells derived from colorectal HT29 cells. RNAseq with available bioinformatics was performed to investigate and screen gene expression networks for elucidating a potential mechanism involved in the survival of targeted tumor cells.

HT29由来癌幹細胞様腫瘍球におけるドライバ遺伝子の発見

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

精製造血幹細胞/前駆細胞のDNAの変異を特定する

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

大腸癌幹細胞を研究するスフェロイドの3次元モデル

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

目標シーケンシングアプローチによる比較病変分析

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

行動学

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

大腸癌細胞の3次元モデルにおける侵襲性腫瘍微小環境の分子プロファイリングと<em&gt;生体外</em&gt;線維芽細胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

医学

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.
Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.
DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.
We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

大腸癌への進行中に視野欠損欠損PMS2、ERCC1、Ku86、CcOI

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other ...

生物学

MLH1 Gene Expression in Peripheral Blood in Colon Cancer Patients and Their Association with Colon Cancer

MutL homolog 1 (MLH1) is a component of the heterodimeric complex MutL&#945; that detects and fixes base-base mismatches and insertion/deletion loops caused by nucleotide misincorporation. In the absence of MLH1 protein, the frequency of non-repaired mismatches increases, resulting in organ cancer. The current study sought to quantify MLH1 gene expression and its relationship with tumor invasion (T) and lymph node invasion (N) in blood samples from patients with colorectal cancer (CRC). Blood samples were obtained from 36 CRC patients. RNA was extracted, and cDNA was synthesized using a kit. The primers were built using the exon-exon junction approach, and MLH1 and &#946;-actin genes were tested 3x using real-time polymerase chain reaction (Real-Time PCR). Gene expression analysis software was used to analyze the data, and a t-test was used to examine the expression of MLH1 and its connection with T and N variables. In this study, 36 patients with colorectal cancer, including 15 (41.6%) women and 21 (58.4%) men, with a mean age of 57.35 &plusmn; 4.22 years and in the age range of 26-87 years, were included. The results showed that the ratio of MLH1 gene expression in patients decreased compared to that in healthy individuals, and the decrease in gene expression at different stages of the disease was significant. The results of this study showed that the reduction of MLH1 gene expression has an effective role in the development of CRC.

This study examines the association between MLH1 gene expression in peripheral blood and colon cancer, utilizing a case-control approach to compare expression levels in patients and matched healthy controls.

MLH1 の大腸がん患者における末梢血中の遺伝子発現と大腸がんとの関連

MutL homolog 1 (MLH1) is a component of the heterodimeric complex MutL&#945; that detects and fixes base-base mismatches and insertion/deletion loops ...