この作品はリーグ Genevoise contre le 癌によって支えられた財団博士デュボワ Ferriere ディヌ ・ リパッティ、OCS-02260-08-2008、2914-02-2012 KPS OCS Oncosuisse スイス国立科学財団・ グラント 31003A_156760 および 310030 153456。

ヒトの血液サンプルは、ローカル倫理とジュネーブ大学病院の人体実験委員会によって承認された手続きに従って使用されました。
動物の手術は、機関倫理委員会動物ケアのジュネーブ、スイス連邦共和国のカントンの獣医のオフィスに従って (承認番号: GE/26/15)。
1. 主な末梢血単核球 (PBMCs) 密度勾配分離の準備
注: 3-5 mL 末梢血は白血病段階で MCL を呈する患者から得られました。診断は、染色体転座 t(11;14) の存在とサイクリン D1 の発現によってその後確認およびフローサイトメトリー (CD5 +、CD23−、CD200 +、単クローン性 B 細胞人口) の標準的な診断基準によると設立されました。すべてのケースで単クローン性 B 細胞を構成する > 合計 B 細胞の人口の 90%。残りのセルは、単球および T 細胞に主にいた。
<ol><li>ロズウェル パーク記念研究所メディア 1640 (RPMI) (図 1 a) の 5 mL と MCL 血液サンプルの (1:1) 5 mL を希釈します。</li>
	<li>徹底的な混合を使用する前に数回の密度勾配メディア (資材表) を反転します。</li>
	<li>ピペット吸引を使用して 15 mL 遠心管に 5 mL の密度勾配のメディアを追加します。</li>
	<li>軽くアスピレーター (血液密度勾配メディア比を 2:1、例えば、10 mL を密度勾配メディアの 5 mL の血液のはず) を使用して 15 mL 遠心管の密度勾配メディアの希釈血液サンプルをレイヤーします。(図 1 b、C) 2 つのレイヤーを混在させるに注意をすべき。</li>
	<li>室温で 40 分間 400 x g でサンプルを遠心分離機、遠心ブレーキがオフになっています。</li>
	<li>中間層 (白っぽい、薄いリング (図 1, E) の矢印でマーク) を優しく吸引滅菌パスツール ピペットを使用して単核細胞を含む、クリーン チューブにそれらを転送します。</li>
	<li>単核細胞の洗浄<ol>
<li>転送細胞懸濁液の容積を推定し、それに追加 1% ウシ血清アルブミン (BSA) とリン酸緩衝生理食塩水 (PBS) x 滅菌 1 の 3 つの容積。</li>
		<li>上下に数回ピペッティングによる懸濁液を混ぜます。</li>
		<li>室温で 10-15 分のための 400-500 × g の懸濁液を遠心します。</li>
		<li>細胞ペレットを取得、洗浄ステップを繰り返しますして上澄みを廃棄します。</li>
		<li>上澄みを除去し、希望の音量、例えば、1 または 2 mL の PBS で細胞を再懸濁します。</li>
		<li>自動化された細胞カウンターまたは検定 (商工会議所 · ノイバウアー) を使用して手動でセルの数をカウントします。</li>
	</ol>
</li>
</ol>2. B 細胞の負の淘汰による B 細胞の濃縮
<ol><li>最終濃度 50 x 106セル/mL の PBS のセルを希釈します。</li>
	<li>セルを下部ポリスチレン チューブ ラウンド 5 mL に転送します。細胞懸濁液 2 mL に B 細胞濃縮キットから抗体カクテルの 100 μ L を追加し、(図 2-1) 上下ピペットで穏やかに混合します。</li>
	<li>10 分間室温でセルを孵化させなさい。</li>
	<li>簡単に渦磁性粒子を提供 B 細胞濃縮キット 30 s (図 2-2)。サンプル 2 mL に磁性粒子の 150 μ L を追加します。</li>
	<li>室温で 5 分間別サンプルをインキュベートします。</li>
	<li>1x PBS 2% 牛胎児血清 (FCS) と 1 mM エチレンジアミン四酢酸 (EDTA) を含むと、2.5 mL にサンプル ボリュームをもたらします。磁石でチューブを置き、別の 3-5 分 (図 2-3) のチューブを孵化させなさい。</li>
	<li>管場所で、1 つの連続的な動きとマグネット ピックアップ、チューブをデカントした新鮮な管 (図 2-4) で濃縮細胞懸濁液を収集します。自動化された細胞カウンターまたは B 細胞の総収率を取得する診断を使用して手動でのセルをカウントします。 注: 通常、利回りは通常 B 細胞と MCL の 10 の6セル/mL x 5-50 〜 105 B 細胞/ml です。</li>
	<li>豊かな B 細胞の純度を確認します。<ol>
<li>取る ~ 50,000 細胞 CD19/抗 CD20 抗体で細胞をインキュベートし、(1:20 希釈) 異なる蛍光色素と結合した抗 CD45 抗体と (1:10 希釈) 15-30 分の室温で。</li>
		<li>1 × PBS + 1 %bsa の 1 mL で洗い、400-500 × g で 5 分で細胞を遠心し、〜 200 μ L の PBS で細胞を再懸濁します。</li>
		<li>フローサイトメトリーによる細胞サンプルを取得し、B 細胞の純度の分析します。純度はほとんど > 90% このメソッド (図 3)。</li>
	</ol>
</li>
</ol>3. 異種移植腫瘍モデルマウスの開発
<ol><li>1 × PBS (マウスあたりを注入するために必要なボリューム) の 150-200 μ L の 10 の6セル × 40 〜 60 を中断します。</li>
	<li>細胞懸濁液の 150 μ L を静脈内注射 (静注) 6-7 週齢 (図 4 a) 性別に関わらずうなずいて scid ガンマ (NSG) マウスのそれぞれの尾静脈に。</li>
	<li>〜 10 週間の人間のリンパ腫を開発するマウスを許可します。 注: プライマリ ヒトリンパ腫細胞人間のリンパ腫細胞株 (~ 3 週間) と比較して腫瘍を開発する時間がかかります。</li>
	<li>動物は、減量のような致命的な病気の症状を表示する開始、波立たせられた髪、活性低下、後肢麻痺、等、犠牲によって CO2窒息心臓心臓穿刺血を取得に続いてにマウス。</li>
	<li>収集さまざまな器官マウスの解剖によって骨髄、脾臓と肝臓のように、腫瘍生着分析のための犠牲の時に心臓から血液を描画することによって末梢血を収集 (図 4 b -F)。</li>
</ol>4. 腫瘍細胞がさまざまな臓器の中にしみ込んでキメリズム解析
<ol><li>さまざまな器官を収集した後、次の赤血球 (RBC) 換散血、骨髄、脾臓の 500 μ L と肝臓の 2 mL の塩化アンモニウム カリウム バッファー x 1 の 200 μ L を使用して単一細胞懸濁液を生成する機械の破壊によってそれらを処理します。.</li>
	<li>カウントし、ヒト B 細胞抗体すなわち抗 CD19、抗 CD20、抗 CD45 の細胞を染色します。 注: NSG マウスの B 細胞を欠いている、したがって分析のため細胞はヒトの特定の B 細胞マーカーとステンド グラスします。</li>
	<li>フローサイトメトリーによる各器官由来 B 細胞 (cd19、cd20、CD45 +) のゲートによって細胞を分析します。</li>
	<li>4.3 (図 4) の手順で示されているように、ゲートのセルに基づいて MCL 注入マウスから派生したさまざまな器官に明らかな移植腫瘍細胞を定量化します。 注: 詳細については材料の表を参照してください。</li>
</ol>

<ol>
	<li>Baggiolini, M., Dewald, B., Moser, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chemokines:+an+update.">Human chemokines: an update.</a> Annu Rev Immunol. 15, 675-705 (1997).</li><li>Baggiolini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+and+leukocyte+traffic.">Chemokines and leukocyte traffic.</a> Nature. 392, 565-568 (1998).</li><li>Janeway, C. A., Travers, P., Walport, M., Shlomchik, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunobiology: The Immune System in Health and Disease. , (2001).</li><li>De Silva, N. S., Klein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+B+cells+in+germinal+centres.">Dynamics of B cells in germinal centres.</a> Nat Rev Immunol. 15 (3), 137-148 (2015).</li><li>Cohen, P. L., Kurtin, P. J., Donovan, K. A., Hanson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+and+peripheral+blood+involvement+in+mantle+cell+lymphoma.">Bone marrow and peripheral blood involvement in mantle cell lymphoma.</a> Br. J. Haematol. 101, 302-310 (1998).</li><li>Meusers, P., Hense, J., Brittinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mantle+cell+lymphoma:+diagnostic+criteria,+clinical+aspects+and+therapeutic+problems.">Mantle cell lymphoma: diagnostic criteria, clinical aspects and therapeutic problems.</a> Leukemia (Baltimore). 11 (Suppl 2), S60-S64 (1997).</li><li>Herrmann, 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=Improvement+of+overall+survival+in+advanced+stage+mantle+cell+lymphoma.">Improvement of overall survival in advanced stage mantle cell lymphoma.</a> J Clin Oncol. 27 (4), 511-518 (2008).</li><li>Martin, P., 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=Intensive+treatment+strategies+may+not+provide+superior+outcomes+in+mantle+cell+lymphoma:+overall+survival+exceeding+7+years+with+standard+therapies.">Intensive treatment strategies may not provide superior outcomes in mantle cell lymphoma: overall survival exceeding 7 years with standard therapies.</a> Ann Oncol. 19 (7), 1327-1330 (2008).</li><li>Iyengar, 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=Characteristics+of+human+primary+mantle+cell+lymphoma+engraftment+in+NSG+mice.">Characteristics of human primary mantle cell lymphoma engraftment in NSG mice.</a> Br J Haematol. 173 (1), 165-169 (2016).</li><li>Klco, J. M., 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=Functional+heterogeneity+of+genetically+defined+subclones+in+acute+myeloid+leukemia.">Functional heterogeneity of genetically defined subclones in acute myeloid leukemia.</a> Cancer cell. 25 (3), 379-392 (2014).</li><li>Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+tumor+xenografts+as+predictive+preclinical+models+for+anticancer+drug+activity+in+humans:+better+than+commonly+perceived-but+they+can+be+improved.">Human tumor xenografts as predictive preclinical models for anticancer drug activity in humans: better than commonly perceived-but they can be improved.</a> Cancer Biol Ther. 2 (4 Suppl 1), S134-S139 (2003).</li><li>Johnson, J. I., 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=Relationships+between+drug+activity+in+NCI+preclinical+in+vitro+and+in+vivo+models+and+early+clinical+trials.">Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials.</a> Br. J. Cancer. 84, 1424-1431 (2001).</li><li>Scholz, C. C., Berger, D. P., Winterhalter, B. R., Henss, H., Fiebig, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+drug+response+in+patients+and+in+the+clonogenic+assay+with+solid+human+tumour+xenografts.">Correlation of drug response in patients and in the clonogenic assay with solid human tumour xenografts.</a> Eur. J. Cancer. 26, 901-905 (1990).</li><li>Do&#241;ate, C., Vijaya Kumar, A., Imhof, B. A., Matthes, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frontline+Science:+Anti-JAM-C+therapy+eliminates+tumor+engraftment+in+a+xenograft+model+of+mantle+cell+lymphoma.">Frontline Science: Anti-JAM-C therapy eliminates tumor engraftment in a xenograft model of mantle cell lymphoma.</a> J Leukoc Biol. 100 (5), 843-853 (2016).</li></ol>

著者は、彼らは競合する金銭的な利益があることを宣言します。

臨床試験、医薬品開発の先進的な段階で、創には使用できません。初期段階の薬をテストするためにひと細胞の異種移植の受信者を動物を開発する努力を追求されている長い間。人間拒絶反応を回避し、MCL など、人間の病気のためのモデルを確立することができます動物のモデルをご紹介します。これは現時点ではひと腫瘍生着と腫瘍成長のメカニズムを研究する最先端の異種移植モデルで...

自然にリンパ球が免疫監視に大きな役割を果たすし、抗原特異免疫1,2をマウントの重要なステップは、リンパ球が人身売買します。このプロセスには、血流に胸腺から、二次リンパ器官、リンパ節、パイエル板、脾臓など同種抗原を満たす彼らそこからナイーブ T リンパ球の移行が含まれます。B リンパ球は骨髄で分化し、二次リンパ器官3包にナイーブ細胞として移行します。これらの B 細胞の一部は受容器の抗原をバインドし、特定の T 細胞によってアクティブ化されます。これらの B 細胞の増殖と分化は、卵胞のマントル層に非活性化、ナイーブ B 細胞をプッシュします。活性化細胞が、パトロール、体や免疫グロブリン分泌形質細胞骨髄4に移行するに成熟細胞のメモリ B に分化します。
MCL は、マントル層でナイーブ B リンパ球が腫瘍に変換するときに発生します。これらのリンパ腫細胞は、リンパ器官の微小環境に存在し、特定の T リンパ球のコントロールとは独立して増殖します。ただし、密度のある特定の段階で彼らはこのニッチから脱出し、他の臓器でのニッチを求めて血流に循環させます。接着分子の複雑さとケモカインとその受容体の混乱を考えるこの細胞人身売買体内のメカニズムはよくわかっていない、したがって治療を妨げます。新しい微小に達することからリンパ腫 B 細胞を防ぐためには、この移行プロセスを効果的にブロックする手法が必要です。
MCL は、B 細胞の悪性腫瘍を治療するために最も困難の一つです。MCL の腫瘍の表現型の開発は、ユニークな生物学的特性の獲得によって特徴付けられる多段カスケードの結果です。診断時に、ほとんどの患者 (70%) 既に存在脾臓、骨髄、および/または消化管5,6舌癌の関与を示す場合の大半の播種性疾患。にもかかわらず、従来の化学療法短期7,8で高い寛解率を誘導する治療を受けた患者の耐性腫瘍によって数年以内の再発は、一般的です。MCL の普及とその基盤となる生物学の理解を助けることができる新しい疾患モデルの紹介: 我々 は患者の腫瘍細胞に由来するひと MCL 異種移植マウス モデルを確立します。我々 は、このモデルは MCL 普及に対する治療戦略を開発し、おそらく再発患者の最適な診断と治療の新しい臨床的視点を提供を助けることを望みます。

<table>
	<tbody>
		<tr>
			<td>Ficoll-paque media</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td>for separation of mononuclear cells</td>
		</tr>
		<tr>
			<td>RPMI Medium 1640</td>
			<td>Gibco-Life technologies</td>
			<td>61870-010</td>
			<td>for dilution of blood sample</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>washing of cells</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A8412</td>
			<td>for preparation of PBS with 1% BSA used in washing cells during isolation</td>
		</tr>
		<tr>
			<td>CD19-APC700</td>
			<td>Beckman Coulter</td>
			<td>B49212</td>
			<td>human pan-B cell marker</td>
		</tr>
		<tr>
			<td>CD20-APC</td>
			<td>Beckman Coulter</td>
			<td>A21693</td>
			<td>human pan-B cell marker</td>
		</tr>
		<tr>
			<td>CD20-ECD</td>
			<td>Beckman Coulter</td>
			<td>IM3607</td>
			<td>human pan-B cell marker</td>
		</tr>
		<tr>
			<td>CD5-PC 5.5</td>
			<td>Beckman Coulter</td>
			<td>PN A70203</td>
			<td>human T cell marker</td>
		</tr>
		<tr>
			<td>CD23-PE</td>
			<td>Pharmingen</td>
			<td>555711</td>
			<td>Cell surface protein typically absent in MCL</td>
		</tr>
		<tr>
			<td>CD45 KO</td>
			<td>Beckman Coulter</td>
			<td>B36294</td>
			<td>Pan-leucocyte marker</td>
		</tr>
		<tr>
			<td>CD200-PE</td>
			<td>Pharmingen</td>
			<td>552475</td>
			<td>Cell surface protein typically absent in MCL</td>
		</tr>
		<tr>
			<td>NOD scid gamma (NSG) mice</td>
			<td>Charles River Laboratories</td>
			<td>5557</td>
			<td>used to develop MCL xenografts in this study</td>
		</tr>
		<tr>
			<td>Easy sep Human B cell enrichment kit</td>
			<td>Stem cell technologies</td>
			<td>19054</td>
			<td>used to enrich B cells to obtain pure cells for injecting into mice</td>
		</tr>
		<tr>
			<td>FACS</td>
			<td>Beckman Coulter</td>
			<td>Navios</td>
			<td>used to characterize MCL sample and to study the organs for MCL engraftment</td>
		</tr>
		<tr>
			<td>1X ammonium chloride potassium buffer</td>
			<td></td>
			<td></td>
			<td>red blood lysis buffer (NH4Cl 8,024 mg/l; KHCO3 1,001 mg/l; EDTA.Na2&#183;2H2O 3.722 mg/l )</td>
		</tr>
	</tbody>
</table>

tumor engraftment in a xenograft mouse model of human mantle cell lymphoma

B リンパ球は主に家に免疫細胞の循環と彼らのキー ・ プレイヤー、リンパ器官に存在します。正常 B 細胞は、T リンパ球が刺激されるときのみ増殖、発癌性 B 細胞生き残るため、未定義のオルガンのニッチの自律的展開します。マントル細胞リンパ腫 (MCL) はそのような 1 つの B 細胞障害患者の生存率が 4-5 年。これは効果的なメカニズムが、ホーミングとこれらの細胞の生着は生存率を高めるためにブロックの必要性と患者の長寿のために呼び出します。したがって、体内ホーミング機構ブロックによる MCL 治療の有効性を検討する異種移植マウス モデルを開発するための努力は非常に重要です。ひと細胞の異種移植の初期段階の薬をテストするための受信者動物の開発は長い間、関連するマウスの前臨床モデルに欠かせない画面の新しい治療薬として追求されています。この動物モデルの開発人間の移植片拒絶反応を回避し、ひと疾患モデルの確立し、の候補薬の臨床テストを実行する別のリンパ腫型の病気の進行を研究する非常に便利なツールがあります。造血器腫瘍、MCL のよう。私たちは研究し、MCL の新規治療法を開発するための優秀な資源となる異種移植マウス モデルを確立しました。

原稿では、MCL 細胞の生着の異種移植腫瘍モデルマウスの開発に成功のための最適化されたプロトコルについて説明します。(このケース MCL 細胞で)、純粋な細胞集団の準備は成功した MCL 異種移植片を開発する非常に重要です。図 1は、密度勾配分離法による MCL 患者の血液から単核のセル隔離のため分取の手順を表します。単核細胞は、さらに?...

Watch this Scientific Journal Video about Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma at JoVE.com

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

マントル細胞リンパ腫 (MCL) は B 細胞の障害を治療することは困難と同様に研究および治療法を開発するプライマリ MCL の異種移植マウス モデルを確立することは困難です。ここでは、その基盤となる生物学を理解するためにマウスの MCL を異種移植片の成功確立について述べる。

人間マントル細胞リンパ腫の異種移植マウス モデルにおける腫瘍生着

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

tumor-engraftment-xenograft-mouse-model-human-mantle-cell

Research

JoVE Journal

Cancer Research

11.1K ビュー.  University of Geneva. マントル細胞リンパ腫 (MCL) は B 細胞の障害を治療することは困難と同様に研究および治療法を開発するプライマリ MCL の異種移植マウス モデルを確立することは困難です。ここでは、その基盤となる生物学を理解するためにマウスの MCL を異種移植片の成功確立について述べる。

ビデオ: Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

前臨床患者由来の腫瘍新規抗がん療法の調査のための異種移植モデルの開発とメンテナンス

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

がん研究

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

結腸直腸癌モデルマウスにおける循環腫瘍細胞の単離

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

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 ...

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

同系マウス B 細胞リンパ腫モデル CD19 車 T 細胞の前臨床評価

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

ヒト尿路上皮細胞癌および大腸癌腫瘍増殖および自発性転移に対する患者由来の眼科医異所性Xenograft移植片モデル

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

黒色腫患者由来異種移植片モデル

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

ヒト末梢血単核細胞(PBMC)移植型ヒト型異物移植モデル翻訳免疫腫瘍学(I-O)研究

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

胃癌患者由来異種移植片モデルおよび原発細胞株の確立

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

腫瘍増殖における癌関連線維芽細胞の役割を調べるためのマウスモデル

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

レプトメニンゲレス病の直接標的療法を研究するマウス・オンマヤ異種移植片モデル

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...

人間マントル細胞リンパ腫の異種移植マウスモデルにおける腫瘍生着

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この動画の章

前臨床患者由来の腫瘍新規抗がん療法の調査のための異種移植モデルの開発とメンテナンス

結腸直腸癌モデルマウスにおける循環腫瘍細胞の単離

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

同系マウス B 細胞リンパ腫モデル CD19 車 T 細胞の前臨床評価

ヒト尿路上皮細胞癌および大腸癌腫瘍増殖および自発性転移に対する患者由来の眼科医異所性Xenograft移植片モデル

黒色腫患者由来異種移植片モデル

ヒト末梢血単核細胞(PBMC)移植型ヒト型異物移植モデル翻訳免疫腫瘍学(I-O)研究

胃癌患者由来異種移植片モデルおよび原発細胞株の確立

腫瘍増殖における癌関連線維芽細胞の役割を調べるためのマウスモデル

レプトメニンゲレス病の直接標的療法を研究するマウス・オンマヤ異種移植片モデル

人間マントル細胞リンパ腫の異種移植マウス モデルにおける腫瘍生着

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さらに動画を探す

この動画の章

前臨床患者由来の腫瘍新規抗がん療法の調査のための異種移植モデルの開発とメンテナンス

結腸直腸癌モデルマウスにおける循環腫瘍細胞の単離

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

同系マウス B 細胞リンパ腫モデル CD19 車 T 細胞の前臨床評価

ヒト尿路上皮細胞癌および大腸癌腫瘍増殖および自発性転移に対する患者由来の眼科医異所性Xenograft移植片モデル

黒色腫患者由来異種移植片モデル

ヒト末梢血単核細胞(PBMC)移植型ヒト型異物移植モデル翻訳免疫腫瘍学(I-O)研究

胃癌患者由来異種移植片モデルおよび原発細胞株の確立

腫瘍増殖における癌関連線維芽細胞の役割を調べるためのマウスモデル

レプトメニンゲレス病の直接標的療法を研究するマウス・オンマヤ異種移植片モデル

人間マントル細胞リンパ腫の異種移植マウスモデルにおける腫瘍生着

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