Name: lung tumor cell recruitment assay
Uploaded: 2019-02-26T12:30:00
Description: Watch this Scientific Journal Video about Lung Tumor Cell Recruitment Assay at JoVE.com

マウスで実行されたすべてのプロシージャは、動物研究委員会の東京女子医科大学によって承認されました。
1. ルイス肺癌細胞 (LLC) の尾静脈注射
<ol><li>加湿 5%、10% の FCS を DMEM に LLC 細胞を維持 37 ° C で CO2インキュベーターセルは、蛍光タンパク質の発現システム (例えばGFP) を含める必要があります。</li>
	<li>培養皿からセルを削除するには、非酵素細...

<ol>
	<li>Valastyan, S., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+metastasis:+molecular+insights+and+evolving+paradigms.">Tumor metastasis: molecular insights and evolving paradigms.</a> Cell. 147 (2), 275-292 (2011).</li><li>Kaplan, R. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGFR1-positive+haematopoietic+bone+marrow+progenitors+initiate+the+pre-metastatic+niche.">VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.</a> Nature. 438 (7069), 820-827 (2005).</li><li>Hiratsuka, S., Watanabe, A., Aburatani, H., Maru, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-mediated+upregulation+of+chemoattractants+and+recruitment+of+myeloid+cells+predetermines+lung+metastasis.">Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis.</a> Nature Cell Biology. 8 (12), 1369-1375 (2006).</li><li>Hiratsuka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+S100A8-serum+amyloid+A3-TLR4+paracrine+cascade+establishes+a+pre-metastatic+phase.">The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase.</a> Nature Cell Biology. 10 (11), 1349-1355 (2008).</li><li>Tomita, T., Sakurai, Y., Ishibashi, S., Maru, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imbalance+of+Clara+cell-mediated+homeostatic+inflammation+is+involved+in+lung+metastasis.">Imbalance of Clara cell-mediated homeostatic inflammation is involved in lung metastasis.</a> Oncogene. 30 (31), 3429-3439 (2011).</li><li>Deguchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+amyloid+A3+binds+MD-2+to+activate+p38+and+NF-&#954;B+pathways+in+a+MyD88-dependent+manner.">Serum amyloid A3 binds MD-2 to activate p38 and NF-&#954;B pathways in a MyD88-dependent manner.</a> Journal of Immunology. 191 (4), 1856-1864 (2013).</li><li>Erler, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-induced+lysyl+oxidase+is+a+critical+mediator+of+bone+marrow+cell+recruitment+to+form+the+premetastatic+niche.">Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche.</a> Cancer Cell. 15, 35-44 (2009).</li><li>Huang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+vascular+destabilization+in+the+premetastatic+phase+facilitates+lung+metastasis.">Pulmonary vascular destabilization in the premetastatic phase facilitates lung metastasis.</a> Cancer Research. 69 (19), 7292-7237 (2009).</li><li>Sceneay, J., Smyth, M. J., M&#246;ller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pre-metastatic+niche:+finding+common+ground.">The pre-metastatic niche: finding common ground.</a> Cancer Metastasis Reviews. 32 (3-4), 449-464 (2013).</li><li>Castle, J., Shaker, H., Morris, K., Tugwood, J. D., Kirwan, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+circulating+tumor+cells+in+breast+cancer:+A+review.">The significance of circulating tumor cells in breast cancer: A review.</a> The Breast. 23, 552-560 (2014).</li><li>Wolf, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+CCR2+signaling+induced+by+colon+carcinoma+cells+enables+extravasation+via+the+JAK2-Stat5+and+p38MAPK+pathway.">Endothelial CCR2 signaling induced by colon carcinoma cells enables extravasation via the JAK2-Stat5 and p38MAPK pathway.</a> Cancer Cell. 22 (1), 91-105 (2012).</li><li>Hiratsuka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+tumours+modulate+innate+immune+signalling+to+create+pre-metastatic+vascular+hyperpermeability+foci.">Primary tumours modulate innate immune signalling to create pre-metastatic vascular hyperpermeability foci.</a> Nature Communications. 4, 1853 (2013).</li><li>Deguchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eritoran+inhibits+S100A8-mediated+TLR4/MD-2+activation+and+tumor+growth+by+changing+the+immune+microenvironment.">Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment.</a> Oncogene. 35 (19), 1445-1456 (2016).</li><li>Ieguchi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ADAM12-cleaved+ephrin-A1+contributes+to+lung+metastasis.">ADAM12-cleaved ephrin-A1 contributes to lung metastasis.</a> Oncogene. 33 (17), 2179-2190 (2014).</li></ol>

マウス抗体、薬、高脂肪の食事療法または腫瘍細胞の注入前に腫瘍エアコン中の前処理が可能です。この試金で最も困難なステップは、尾静脈注射です。不完全な注入は決定的なデータを結果します。C57bl/6 の尾静脈は、失敗した注射の結果を識別し、特に困難です。役立ちます加熱パッド (37 ° C) にマウスを配置する尾静脈注入が容易になるようにを拡張させます。
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腫瘍の転移は癌関連疾患の高い死亡率を占めています。分子レベルで調査の観点から転移が複数のステップに分けることができます: 原発腫瘍部位、腫瘍の成長と周囲の組織、intravasation、血管の循環に侵入で腫瘍開始、遠隔臓器や腫瘍の再増殖で血管外漏出。各ステップには、異なる分子/信号の1のセットが含まれます。
研究は、腫瘍細胞が転移前フェーズ2,3,4,5は別名、血の循環を開始する前に、遠く離れた臓器に発生するイベントに注目しました 6。マウス モデル研究では、転移前の相が肺のと低レベルの長期的な炎症性応答を作成することによって肺転移を促進を明らかにしました。転移前の相は themicrovasculature の血管、骨髄由来細胞 (腎不全) の採用、S100A8 と SAA33,4を含むサイトカイン/ケモカインのような分子の発現によって特徴付けられる.そのリジルオキシダーゼ変更黒谷7を募集する肺の細胞外マトリックスといわれています。別のレポートは前転移相8Angpt2、MMP3、および MMP10 の重要性を明らかにしました。つまり、原発性腫瘍、骨髄、およびリモート臓器間の複雑な相互作用を理解し正しく (例えば、によってこれらの相互作用に関与する蛋白質を目標とする薬剤を使用して) 変調必要があります9 今後の転移を防ぐために.転移前の相を調整するため、それする必要があります最初可視化し、診断や治療介入の評価。ここでは、肺の転移前の相の研究を可能にする肺腫瘍細胞募集試験を報告します。
マウスの血管に直接注入する腫瘍細胞は、培養細胞の違いはありますが、癌患者の腫瘍細胞を循環し、循環腫瘍細胞に似ています。原発腫瘍から循環に入るときいくつかの特徴的な変化を受ける循環腫瘍細胞自身。それにもかかわらず、培養細胞は、免疫不全マウスの尾静脈に注入されるとき、腫瘍を形成できます。さらに、マウスの系における蛍光腫瘍細胞を標識使用できます体内でこれらの細胞の追跡を許可します。循環腫瘍細胞の挙動はがん患者10で究極の結果を決定するために重要です。血液は腫瘍細胞2の生存のための良い場所ではないです。彼らは宿主の免疫系による攻撃からの脱出、物理的なサポートの不足のためのアポトーシスを防ぐために抗アポトーシス シグナルを必要とする必要があります。転移巣の腫瘍開始のより高い能力を持つ腫瘍細胞より多くの腫瘍結節を生成します。腫瘍細胞は、特定の臓器親和性を示す、それらの特定の臓器に転移が守らなければ。この分析では、転移の翼列 (原発腫瘍の増殖および浸潤) の最初の手順は含まれていません、循環腫瘍細胞と間質細胞 (内皮細胞、上皮細胞、血液細胞) 間の相互作用にフォーカスがあるので。従来腫瘍細胞注入分析の研究者は腫瘍結節の転移を検出するための具体的なサイズになるまで待たなければなりません。この場合、血管に腫瘍細胞の正確な数を定量化することはできません。さらに、このプロセスには、腫瘍細胞の他の要因が結果に影響を与える可能性がありますを示す腫瘍の再生の段階が含まれています。
カウント標識蛍光顕微鏡下で腫瘍細胞は単純なプロセスです。個人差は、全肺をスキャンすることができますように、広い視野を提供します。フローサイトメトリーは、また即座に組織に腫瘍細胞の正確な数を与える便利なツールです。共焦点顕微鏡下組織に蛍光セルをカウントも可能です、転移性腫瘍細胞の最も正確な画像が表示されますが、それは時間がかかり、選択した領域がカウントされる場合にのみ偏りのある結果を与える可能性があります。この試金の究極の目標は、腫瘍細胞が肺に蓄積される方法を簡単に観察することです。既に報告されている3,4, 肺前転移段階第一次腫瘍から炎症性シグナル伝達のための場合、注入された腫瘍細胞はこうして肺転移の高い可能性を示唆、肺に蓄積する傾向があります。(慢性関節リウマチ、喘息、等) 他の条件とその治療法 (抗 tnf α) などの抗炎症剤は肺転移を減衰させる可能性があります。したがって、この試金は肺転移の可能性を評価するための強力なツールであることが分かった。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C6885</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>DN-25</td>
			<td>trace amount</td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>BD</td>
			<td>352340</td>
			<td>40 micrometer mesh</td>
		</tr>
		<tr>
			<td>Fluorescence labeling kit</td>
			<td>Sigma</td>
			<td>MIN26-KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m syringe filter</td>
			<td>sartorius</td>
			<td>17597K</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde Phosphate Buffer Solution</td>
			<td>Wako</td>
			<td>163-20145</td>
			<td></td>
		</tr>
	</tbody>
</table>

lung tumor cell recruitment assay

腫瘍の転移を支配する分子メカニズム解明のため、モデル動物としてマウスを使用してさまざまな試金が提案されています。ここでは、腫瘍細胞の浸潤や微小転移を評価する簡易測定法を示す.この分析では、腫瘍細胞を尾静脈投与と短い期間後肺解剖し、蓄積されたラベル付けされた腫瘍のセルの個数を消化します。このアッセイの血管に原発腫瘍の侵入の最初のステップをスキップして、遠隔臓器転移が発生するイベントの検討が容易になります。転移の限られた数を観察するための血管に注入された細胞の数を最適化できます。それは、遠隔の臓器の間質細胞が転移に貢献することが報告されています。したがって、このアッセイは、潜在的な治療薬や腫瘍転移予防のためのデバイスを探索する便利なツールかもしれない。

肺は、注入 (図 1 a) 後多くの GFP LLC 細胞 2 h を提示します。また赤のフィルターで検出された蛍光スポットがセル数から除外すべきことに留意。LLC 細胞の大半は肺 24 h から注入 (図 1) 後に消えます。
肺の GFP LLC の数を確認するには、フローサイトメトリーによる解析 (<strong class...

Watch this Scientific Journal Video about Lung Tumor Cell Recruitment Assay at JoVE.com

Lung Tumor Cell Recruitment Assay

肺腫瘍細胞募集試験

本稿では、腫瘍の転移の動物モデルにおける肺腫瘍細胞蓄積を定量化する方法について説明します。

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

この方法は、転移の分子メカニズムに関する腫瘍生物学分野の重要な質問に答えるのに役立ちます。この技術の主な利点は、遠隔器官内の腫瘍細胞の募集を定量化できることである。まず、非酵素細胞解離試薬を使用して、蛍光タンパク質発現ルイス肺癌(LLC)を、標準的なプロトコルに従って培養容器から細胞を除去することから始めます。得られた細胞溶液を遠心分離前に15ミリリットルの円錐管に移し、遠心分離機は1回の洗浄につき5ミリリットルのPBSでペレットを2回洗浄する。2回目の洗浄後、40マイクロメートルの孔細胞ストレーナーを通して細胞を濾過し、1ミリリットル濃度の6細胞に10倍に希釈します。その後、29ゲージの針を備えた1ミリリットルの注射器に細胞をロードし、8〜10週齢のC57-Black-6、オスマウスの尾静脈に100マイクロリットルの細胞を注入します。適切な実験エンドポイントで、はさみを使用して各マウスの腹側表面から皮膚を取り除き、肋骨と横隔膜を切断して胸腔を露出させます。右心室を通してPBSの15ミリリットルを洗い流し、心臓と胸腺を取り除くために25ゲージの翼を持つ注入針とチューブを使用してください。気管を鉗子でつかみ、引き上げ、気管の周りの結合組織を解剖して肺を収穫する。次にPBSで肺を洗浄し、蛍光顕微鏡で蛍光細胞を可視化するために1つのローブを取り外します。肺組織を消化するには、まず尾頭葉をはさみでサイコロにし、肺片を10ミリリットルの注射器に入れる。プランジャーでシリンジを閉じ、5ミリリットルの消化液をシリンジに吸い込み、肺組織が溶液全体に広まるまでプランジャーを注射器シャフトの中と外に移動させる。肺スラリーを50ミリリットルのチューブに分配し、逆シェーカーで37°Cと150rpmで15分間組織を消化します。インキュベーションの終わりに、肺細胞が完全に分散するまで残りの組織を三分化し、さらに30分間シェーカーにチューブを戻す。その後、40マイクロメートルの細孔ストレーナーを通して細胞を濾過し、遠心分離によって細胞を収集する。単離した肺細胞をフローサイトメトリーで解析するには、ペレットを室温で1分間赤血球リシスバッファーの2ミリリットルで再懸濁し、遠心分離により細胞を採取する。次いで、ペレットを5ミリリットルの新鮮なPBSで2回遠心分離し、1回の洗浄につき1%ウシ血清アルブミンを補う。第2遠心分離後、新鮮なPBSとBSAの1ミリリットルでペレットを再懸濁し、フローサイトメトリーによる腫瘍細胞の定量化のために新しい40マイクロメートルの孔ストレーナーを介して細胞を濾過する。肺は注射の2時間後に多くのGFP-LLC細胞を提示し、細胞の大半は24時間以内に肺から消失する。なお、赤色フィルター内で検出された蛍光スポットは、細胞数から除外する必要があります。肺中のGFP-LLCの数を確認するために、示されているようにフローサイトメトリック分析にローブの1つを使用することができる。この手順を試みる間、凍結断面手順は、腫瘍細胞の正確な位置を観察するためのより正確な方法であることを覚えておくことが重要です。

Research

JoVE Journal

Cancer Research

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

単球/マクロファージにおける腫瘍微小環境の採用を検討する癌細胞株における遺伝子発現の条件付きのノックダウン

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

がん研究

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

肺組織に腫瘍のセルの視覚化を循環させるため肺植民地化アッセイの確立

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

無細胞性と細胞の肺転移モデル

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

住セルイメージ投射アッセイ研究神経膠腫脳腫瘍幹細胞の遊走と浸潤に

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

チップ チューブ形式を使用して散発的な家族性大腸腺腫症患者の遺伝的変異のデジタル ポリメラーゼ連鎖反応分析

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

それらの相互作用を研究する歯髄組織と癌細胞との共培養から間葉系幹細胞の分離

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

CD8 による培養腫瘍細胞のアポトーシスのリアルタイム検出+ T 細胞腫瘍浸潤骨髄細胞の免疫抑制機能を勉強するには

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

免疫蛍光による非小細胞肺癌患者からの循環腫瘍細胞の半自動PD-L1特性図と列挙

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

腫瘍マクロファージ相互作用を研究するインビトロアッセイ

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

超音波画像によるマウスにおける肺腫瘍の進行の検出

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...