このプロトコルは、検査の結果、博士リチャードハインズ（MIT）の研究室で最初に最適化されていました。資金調達はNYSTEMアイデア賞（LXへ）とルースL.キルシュシュタイン国立リサーチサービス賞（LXへの）によって提供されます。

1。サンプル準備<ol><li>成長因子やFBS（例えば、MC - 1細胞​​を10％FBS含有DMEM）で、その特定のメディアで〜70％コンフルエントに細胞を成長させる。プレートから培地を吸引除去し、穏やかに1 × PBS（塩化ナトリウム8gの/ L、塩化カリウム0.2 g / Lの、1.15のNa 2 HPOのG / L 4 · 7H 2 O、KH 2 POを0.2g / Lで数回洗浄4、pHは7.3）。 </li><li>吸引PBSとヴェルセンの0.05％トリプシン（フェノールレッドおよび1 × PBS、pH7.2中EDTA - Naは0.2 g / Lの0.014グラム/ L）2 mLを加える。静かにプレートから細胞の剥離を容易にするためにプレートを揺らし。顕微鏡下で細胞を観察。それは、通常、切断するセル2-5分かかります。 </li><li>トリプシン活性をクエンチし、50mLのファルコンチューブに細胞を収集するメディアを含むFBS（または大豆トリプシン阻害剤）の十分なボリュームを追加します。血球計を用いて細胞を数える。きれいな血球計算盤の上に細胞懸濁液の10μLをロード。 mL当たりの細胞数は、10 4を掛けた5つの正方形のそれぞれの＃セルの平均値に等しくなります。 </li><li> 3分間ベンチトップ遠心機で1000 RPM（または〜200 × g）で細胞を遠心分離します。 </li><li>慎重に細胞ペレットを乱すことなくメディアを削除します。 〜5 × 10 6細胞/ mlの最終濃度に到達するためのハンクス平衡緩衝液（HBSS）の適切な量で細胞を再懸濁します。 </li><li>ファルコン70μmのセルストレーナーを介してフィルタ細胞は大きな細胞凝集体を除外する。ラベル付け5mLのファルコンの培養チューブオーバーフィルター上に直接ピペットの先端を置きます。迅速に培養チューブにフィルターを介してサスペンションを取り出します。 </li><li>血球計を使用して、再度セルをカウントし、2.5 × 10 6 / mlの最終濃度に希釈する。氷上で細胞を維持する。 </li><li>細胞の生存率を決定する。トリパンブルーで一部の細胞を混合し、血球計算盤を用いて全細胞を介してデッド（青）細胞の割合を測定する。細胞の生存率は、注入前≥90％以上にする必要があります。 </li></ol> 2。静脈注射<ol><li>手袋を変更します。ゆっくりと免疫不全マウス（ヌード、NOD - SCID、またはNSG、ジャクソンの研究所）の尾をつかんで制止する人の後ろに小さな開口部から突き出てスリットとその尾に対して、その後ろで、マウスの制止する人にそれを引く。 </li><li>リングは、マウスの口をキャッチした後、ゆっくりスリットに沿って内側のリングをスライドさせて所定の位置にロックします。マウスは、自由に動くことができないようにする必要がありますが、呼吸の正常な速度を持つ必要があります。 </li><li>主要な尾静脈を見つける。四主要な血管は、マウスの尾部に存在しています。尾の背側と腹側の側面上の血管は動脈です。静脈は尾の側面にあります。 </li><li> 1mLの注射器に準備した細胞の200以上μLを描く。 30 G1 / 2インチの針を取り付け、存在する可能性のある気泡を押し出す。シリンジ内の細胞の最終的な容積は200μL（すなわち、合計で5 × 10 5細胞）でなければなりません。 </li><li>尾静脈に細胞を注入する。 <ol class="lalpha"><li>尾の先端から開始、第一審は失敗した場合、尾部のより近位の領域は、第2のtryを使用することができる。 </li><li> 70％エタノールで尾を拭きます。まっすぐ尾を引いて。親指と尾の先端を押さえ、人差し指で注入するためのポイントをサポートしています。 </li><li>静脈に針を挿入し、細胞を注入する。針と注射器は注射時の静脈に平行な、そうでなければ針が血管壁を通って突くと、隣接する尾の組織に細胞を注入することを確認してください。 </li><li>注射後に針を撤回。注入が正常に行った場合、血液が注射部位から多量のはずです。凝固促進し、循環に静脈内の任意の残留サンプルをプッシュするように尾を上を触診する注射部位での紙タオルや綿棒のきれいな部分を押してください。 </li><li>制止する人からマウスを解放し、ケージに戻す。実験ノートに（例えば、どのように多くの試験では、細胞を注入するために取り、どの程度の細胞を注入した）注入プロセスを記録します。 </li><li>として試料調製のセクションで説明されている注射後の細胞の生存率、、ステップ9を決定します。このステップでは、細胞が注入プロセスを通して生き続けるという安心感を提供します。注入実験の終了時に、細胞の生存率は低下するが、80％以上でなければなりません。 </li><li> （省略可能）残りの細胞をスピンダウンし、PBSで1回ペレットを洗う。今後の分析（例えば、遺伝子発現やノックダウンを確認するためにウエスタンブロット）を-80℃でペレットを凍結する。 </li></ol></li><li>通常、1つまたは2カ月後、マウスを解剖され、転移の場所がひどく決定されます。それは細胞が彼らの後に遭遇する最初の毛細血管床を含んでいるので、肺は、転移のプライマリサイトです。循環を入力してください。 </li><li> PBSで洗浄後、肺（または転移を含む他の組織）の各葉は、解剖顕微鏡（図1）で観察される。肺の両側に検出可能な転移の数がカウントされ、すべての4つの葉で肺転移の番号は、肺転移の合計数として加算されます。肺はホルマリン一晩で修正される場合は、1肺転移がより容易に検出されています、転移は隣接する暗褐色の肺組織とは対照的に白っぽい斑点としてapppearなりますので。 </li></ol> 3。肺転移からの細胞培養<ol><li>肺転移は、注入されたマウスから分離されています。それぞれが別々に培養する必要があります。 </li><li>それぞれの転移は、70μmのセルストレーナーで針のキャップ（無菌）の終わりまでに刻まれている。 </li><li>培地と培養皿でフィルタを通過回収した細胞のいくつかのMLSとセルストレーナーを洗浄します。 </li><li>煩わされることなく、少なくとも4日間、37℃で細胞をインキュベートする。 </li><li> PBSでシャーレに血液や組織の破片を洗い流す。 </li><li>新鮮な培地を追加。初めに、癌細胞と線維芽細胞の両方は、プレート上に成長するが、徐々に、線維芽細胞が死に絶えるだろうと癌細胞に置き換えられます。がん細胞は、任意の薬剤耐性遺伝子を運ぶ場合は、対応する抗生物質で細胞を選択する。 </li><li>由来細胞の純度は、ヒト特異的蛋白質に対する抗体を用いた免疫染色によって評価される。我々は、抗ヒトビメンチン抗体（NCL - VIM - V9、Novocastra）を使用します。我々が由来する細胞は、さらに薬剤の選択がない場合には、ヒト細胞の99％以上、典型的に含まれています。新たに由来する細胞は、上述したように、それらの転移能力をテストするための免疫不全マウスに再注入することができます。 </li></ol> 4。代表的な結果<ol><li>非常に少数の肺転移が不完全に転移性癌細胞株から来る間、このアッセイの終わりには、高転移性癌細胞株は、通常、多くの肺転移（図1）を生じさせる1。 </li><li>このメソッドを使用して派生した細胞は通常、彼らは再びこのアッセイを使用してテストしている親の行、より多くの転移を生じさせる。1-3 </li></ol><img src="/files/ftp_upload/1942/1942fig1.jpg" alt="図1" /> がん細胞の尾静脈注射後のマウスの肺の図1。代表画像。転移性ヒトメラノーマ細胞株、SM細胞、4の5 × 10 5は、免疫不全マウスの尾静脈に注射した。 2ヵ月後、肺を単離し、そして転移は通常の肺組織に分散発見された。

<ol>
	<li>Xu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+changes+in+an+animal+melanoma+model+correlate+with+aggressiveness+of+human+melanoma+metastases.">Gene expression changes in an animal melanoma model correlate with aggressiveness of human melanoma metastases.</a> Mol Cancer Res. 6 (5), 760-760 (2008).</li><li>Clark, E. A., Golub, T. R., Lander, E. S., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+metastasis+reveals+an+essential+role+for+RhoC.">Genomic analysis of metastasis reveals an essential role for RhoC.</a> Nature. 406 (6795), 532-532 (2000).</li><li>Xu, L., Begum, S., Hearn, J. D., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPR56,+an+atypical+G+protein-coupled+receptor,+binds+tissue+transglutaminase,+TG2,+and+inhibits+melanoma+tumor+growth+and+metastasis.">GPR56, an atypical G protein-coupled receptor, binds tissue transglutaminase, TG2, and inhibits melanoma tumor growth and metastasis.</a> Proc Natl Acad Sci U S A. 103 (24), 9023-9023 (2006).</li><li>Kozlowski, J. M., Hart, I. R., Fidler, I. J., Hanna, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+melanoma+line+heterogeneous+with+respect+to+metastatic+capacity+in+athymic+nude+mice.">A human melanoma line heterogeneous with respect to metastatic capacity in athymic nude mice.</a> J Natl Cancer Inst. 72 (4), 913-913 (1984).</li><li>Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multigenic+program+mediating+breast+cancer+metastasis+to+bone.">A multigenic program mediating breast cancer metastasis to bone.</a> Cancer Cell. 3 (6), 537-537 (2003).</li></ol>

転移は、がん患者における死亡の主要な原因である。彼らの主要な遺伝子座、循環への参入（管内）、循環（血管外漏出）から、これらの終了、および遠隔臓器の生存と成長から癌細胞の剥離：これは、4つの主要な手順が含まれます。人間の転移はゆっくりとしたプロセスとみなされ、多くの場合、待ち時間の年後に明示​​される。タイムリーにその進行を勉強するために、上記の比較的...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10313-021</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30088.03</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14175-095</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S9888</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Mallinckrodt Chemicals</td>
<td>6838-04</td>
<td>&nbsp;</td>
<tr>
<td>Na2 HPO4.7H2O</td>
<td>&nbsp;</td>
<td>Mallinckrodt Chemicals</td>
<td>7896-04</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO4</td>
<td>&nbsp;</td>
<td>Mallinckrodt Chemicals</td>
<td>7100-12</td>
<td>&nbsp;</td>
<tr>
<td>Phenol red</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P3532</td>
<td>&nbsp;</td>
<tr>
<td>EDTA-Na</td>
<td>&nbsp;</td>
<td>EMD Biosciences</td>
<td>4010</td>
<td>&nbsp;</td>
<tr>
<td>Trypan Blue</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15250-061</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin 2.5%</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15090-046</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Ultrapure</td>
<td>200-CSGP</td>
<td>&nbsp;</td>
<tr>
<td>50 ml Falcon Tube</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89039-656</td>
<td>&nbsp;</td>
<tr>
<td>Microcentrifuge Tubes</td>
<td>&nbsp;</td>
<td>Axygen Scientific</td>
<td>MCT-175-C</td>
<td>&nbsp;</td>
<tr>
<td>Falcon 70&#181;m Cell Strainer</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>5ml Falcon Culture Tube</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>60818-576</td>
<td>&nbsp;</td>
<tr>
<td>Hemacytometer</td>
<td>&nbsp;</td>
<td>Hausser Scientific</td>
<td>15170-208</td>
<td>&nbsp;</td>
<tr>
<td>1 ml syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>329650</td>
<td>&nbsp;</td>
<tr>
<td>30 &frac12; gauge needle</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>305106</td>
<td>&nbsp;</td>
<tr>
<td>Mouse restrainer</td>
<td>&nbsp;</td>
<td>Plas Labs Inc.</td>
<td>551-BSRR</td>
<td>&nbsp;</td>
<tr>
<td>Surgical scissors</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-940</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-902</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>SMZ1500</td>
<td>&nbsp;</td>
<tr>
<td>Benchtop Centrifuge</td>
<td>&nbsp;</td>
<td>Thermoscientific</td>
<td>75003491</td>
<td>&nbsp;</td>		</tbody>
		</table>
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experimental metastasis assay

転移は、がん患者における死亡の主要な原因である。転移のメカニズムを理解するために、実験的転移アッセイは、免疫​​不全マウスを用いて設立されました。この記事では、サンプル調製、静脈内注射、および肺転移から培養細胞を含むこの検定に関わる手順を、群を区別。簡単に言えば、ヒト癌細胞の予め決められた数を調製した<em&gt; in vitroで</em&gt;とは直接彼らの尾静脈を介して免疫不全マウスの循環に注入。細胞の数が少ない循環の乱後も存続し、肺などの臓器、の転移として成長する。注入されたマウスは、一定期間後に解剖されています。転移の組織分布は、解剖顕微鏡下で決定されます。特定の組織への転移の数がカウントされ、それが直接注入された癌細胞の転移能と相関する。生じた転移を単離し、培養する<em&gt; in vitroで</em&gt;のような免疫不全マウスに再び注入すると、しばしば親系統よりも強化された転移性の能力を示す細胞株、。これらの転移性の高いデリバティブ取引は、転移性の進行を調節する遺伝子や分子経路を同定するために有用なツールとなる。

Watch this Scientific Journal Video about Experimental Metastasis Assay at JoVE.com

Experimental Metastasis Assay

この記事では、ヒト癌細胞株の転移能を決定するために使用される実験的転移アッセイの手順を説明します。

実験的転移アッセイ

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Research

JoVE Journal

Medicine

20.2K ビュー.  University of Rochester Medical Center. この記事では、ヒト癌細胞株の転移能を決定するために使用される実験的転移アッセイの手順を説明します。

ビデオ: Experimental Metastasis Assay

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

鳥類胚のモデルを使用して、がんの転移の定量的解析

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

医学

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

in vitroで中皮クリアランスアッセイでそのモデルの卵巣がんの転移の初期段階を

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

骨転移モデル

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

実験的な人間の肺炎球菌キャリッジ

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

ザ<em&gt;卵で</em肉腫のためXenograftmodelとして&gt; CAMアッセイ

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

ヒト前立腺癌転移の同所マウスモデル

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

原発腫瘍と肺転移を研究するためのMEF細胞の単離

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

テストのためのひよこ心臓浸潤アッセイ癌細胞の浸潤性および潜在的に抗浸潤化合物の活性

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

肝細胞癌転移の「患者様」同所同系マウスモデル

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

腹腔内卵巣癌転移の定量

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...