Name: enrichment and characterization of the tumor immune and non-immune microenvironments in established subcutaneous murine tumors
Uploaded: 2018-06-07T14:45:00.000+00:00
Duration: 8 min 32 s
Description: Watch this Scientific Journal Video about Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors at JoVE.com

によりは、国立衛生研究所の両方の一般的な医学の国立研究所 (T32GM088129) と国立歯科・頭蓋顔面研究 (F31DE026682) からの財政支援を認めています。内容は著者の責任と国立衛生研究所の公式見解を必ずしも表さない。このプロジェクトは、また NIH (P30 AI036211、P30 CA125123 S10 RR024574) とジョエル ・ m ・ Sederstrom の専門家の助力からの資金とフローサイトメトリーおよび薬の Baylor の大学で細胞選別の中心によって支えられました。

ここで説明したすべてのメソッドは、機関動物ケアおよび使用委員会 (IACUC) の薬の Baylor の大学によって承認されています。
1. 腫瘍の収穫と消化
注: 収穫に要する時間は 3 ~ 5 分/腫瘍と処理に必要な時間は ~ 1 + 2-3 min/腫瘍。
<ol><li>炭酸ガス吸入によるマウスを安楽死させるし、髪の汚染を防ぐために収穫前に各マウスをダウン 70% エタノールをスプレーします。マウス皮下腫瘍を削除し、腫瘍が任意の汚染組織(すなわち髪、肌、腹膜等)の無料手術。24 ウェル プレートでのウシ胎児血清 (FBS) なし基本 RPMI 1640 メディアの 1.8 mL の各腫瘍を配置します。大きな収穫の細胞を維持するために氷の寒さのプレートを維持します。 注: 不妊が必要なすべての腫瘍の解剖必要がありますバイオ セーフティ キャビネット (すなわちハサミ、鉗子、等)、生殖不能の外科ツールとして次のステップで実行します。0.5 - 1 cm 最長の直径が付いている腫瘍が最適とより大きいまたは小さい腫瘍試薬のスケーリングが必要になります。</li>
	<li>各ウェル内の3個セット 1 mm 以下に腫瘍をカットするのにはさみを使用します。</li>
	<li>コラゲナーゼを溶解することにより消化カクテル x 10 の準備で 10 mg/mL、コラゲナーゼ IV 2,500 U/mL と DNase 私 200 U/mL 基本 RPMI 1640 メディア (FBS) なしで私。 注: カクテルの消化日を前もって準備して格納できます-20 ° c;ただし、酵素は一度解散時間をかけて活動を失うので、長期の保管はお勧めしません。</li>
	<li>私、コラゲナーゼ IV、DNase コラゲナーゼを含む解離カクテル × 10 の 200 μ l 添加 1 mm の3個の各ウェルに。</li>
	<li>軌道プレート シェーカーを使用しての 60-100 rpm で軽く振りながら 37 ° C で 1 時間のダイジェスト サンプルを許可します。 注: 酵素消化中に 37 ° C までの温度の上昇は、免疫細胞の活性化を引き起こす可能性があります。</li>
	<li>1 時間後の各ウェルに 5 %fbs と 2 mM EDTA 添加 RPMI 1640 メディアの 1 つの mL を追加することによって反応を中和します。</li>
	<li>50 mL の遠心管の上に座っている 40 μ m セル ストレーナーに腫瘍消化、残りの腫瘍部分をピペットします。 注: は、ストレーナーに腫瘍消化を容易に転送する 1 mL ピペット チップの先端をカットします。</li>
	<li>こし器を通して腫瘍部分を機械的に細かく分けた 10 mL シリンジのプランジャーを使用します。定期的に 2 ml の補われた RPMI 1640 メディアのストレーナーを洗浄 (5% を含む FBS) セル ストレーナーが腫瘍のクリアされるまでを繰り返します。 注: メディア総数の約 4-10 mL は十分に腫瘍のストレーナーを洗浄するための十分なはずです。 注: は、細胞生存率を維持するためにすべてのサンプルのこのステップを完了するまで氷にサンプルを配置します。</li>
	<li>補足 RPMI 1640 メディアを使用して同じボリュームに各 50 mL 遠心管を調整します。 注: ボリュームの調整は、遠心力のバランスをとるため重要です。遠心分離機の損傷やけがこのステップをスキップ可能性があります。</li>
	<li>(5 分、805 × g、4 ° C) 細胞懸濁液を遠心分離、培養上清を破棄し、再補足 RPMI 1640 メディアの 2 mL で中断します。 注: セルの集計は次の再懸濁しますにくい遠心分離フォームがあります。続行する前にそれを破るため急速に 5 mL の血清ピペットとミックスを使用します。</li>
</ol>2. 免疫と腫瘍細胞分画の分離
注: この手順の所要時間は約 1 時間です。
<ol><li>密度勾配媒体の 3 mL を 15 mL 遠心管の下部に追加します。準備し、ラベルを各腫瘍に対する十分なチューブ。</li>
	<li>層の 2 つの層の混合を防ぐために管の側をゆっくりとピペッティングによる密度勾配媒体上に腫瘍細胞懸濁液 2 mL。</li>
	<li>慎重に遠心分離機で 805 x g は、20 ° C、ないブレーキで 20 分間スピン多層チューブを配置します。 注: 適切なバランスを確認し、遠心分離中にブレーキを使用しないことを非常に重要です。このプロセスを中断する層の混合になります、サンプルの完全回復が困難になります。</li>
	<li>(ゴマ) 層と転送ピペットを使用して新しい 15 mL チューブにトップ メディア層白血球浸潤腫瘍を慎重に転送します。すべての残りの密度勾配媒体を破棄し、補われた RPMI 1640 の 2 mL でチューブの下部に腫瘍ペレットを再懸濁します。 注: 遠心分離後がありますそれぞれに対応する下に上から 4 つの可視レイヤー: 腫瘍細胞ペレット密度勾配媒体、ティルズ メディア。図 2さまざまな層の例についてはを参照してください。</li>
	<li>両方のティルを遠心し、腫瘍のサンプル チューブ (5 分、805 × g、4 ° C) を分離し、上澄みを廃棄します。</li>
	<li>200 μ L のティル サンプルと補われた RPMI 1640 メディアの 2 mL で腫瘍ペレットを再懸濁します。生存率を維持するために氷の上を保ちます。</li>
</ol>3. めっきと染色
注: めっきに必要な時間は 30 s/腫瘍;表面の汚損のための所要時間は 1 時間です。細胞内染色に要する時間は 40 分です。フローサイトメトリー解析は 1 - を必要な時間 4 分/腫瘍。
<ol><li>準備し、各免疫染色パネル、セルの追加プレート用ラベル 96 ウェルの U 底プレートをカウントします。 注: 通常、3枚のプレートは、合計で用意をしています: リンパ球に焦点を当てたパネル プレート、骨髄性に焦点を当てたパネル プレート、セル数プレート。</li>
	<li>分注染色のそれぞれに単一細胞懸濁液パネル プレートとセットのボリューム カウント板に。 メモ: カウント板により正確なセル人口それぞれの染色パネルに追加されるセルの合計数を計算するため使用されます。96 ウェル プレート サンプリングおよび容積測定分析機能を備えたフローサイトは各サンプルの μ L あたりのセル数を提供し、したがって、各染め色のパネルに追加されるセルの数を計算できます。数板は 4 ° C、FACs バッファーで洗浄で一晩固定後次の日を得ることができる (リン酸緩衝生理食塩水 (PBS) 2 %fbs)、FACs バッファーの設定ボリュームで再と。</li>
	<li>(5 min, 805 × g、4 ° C) を遠心分離し、任意の無料の FBS を削除する各リンス間清を破棄してサンプルあたりダルベッコ リン酸緩衝生理食塩水 (DPBS) 200 μ L で 2 回細胞を洗ってください。</li>
	<li>Fc ブロックの 50 μ L マルチ チャンネル ピペットを使用してサンプルをミックスし、4 ° C で 20 分間インキュベートを追加します。</li>
	<li>2 倍の 50 μ L 濃縮細胞ターゲティング抗体と修正可能な生存汚れの混合物、マルチ チャンネル ピペットを使用してミックス、RT または 4 ° C で暗闇の中で 30 分間インキュベートを追加します。 注: 抗体の正確な染色条件は、製造元によって異なります。すべての染色希釈は、修正可能性色素の飽和を防ぐためにない追加の FBS の DPBS でなければなりません。この原稿で使用されている染色パネルの最適な希釈液のためのテーブルの材料/機器を参照してください。Fc ブロックに追加される染色容量 100 μ L の濃度を染色 × 最終的な 1 を提供するために 2 倍の濃度では、抗体と修正可能性染色液を用意しています。</li>
	<li>(5 分、805 × g、4 ° C) を遠心分離し、各リンス間培養上清を破棄して FACs バッファーで 2 回サンプルを洗います。</li>
	<li>1 x 固定/透過液 200 μ L で再懸濁します、光から保護 4 ° C で一晩インキュベートします。 注: 実験を一時停止できる一晩この時点: 光から保護 4 ° c のサンプルを保ちます。</li>
	<li>次の日は (5 分、805 × g、4 ° C) プレートを遠心し、上澄みを廃棄します。</li>
	<li>濯ぎ 1 回 x 透過バッファー、遠心分離機 (5 分、805 × g、4 ° C)、1 の 200 μ L で、上澄みを廃棄します。</li>
	<li>1 x 透過バッファーの準備 1 x 集中細胞抗体染色パネルの 100 μ L を追加し、RT または 4 ° C (製造元から染色の推奨によって異なります) で暗闇の中で 30 分間インキュベートします。</li>
	<li>1 x 透過とバッファー (5 分、805 × g、4 ° C) は、上澄みを廃棄したらすすぎとリンス FACs バッファーと 2 回目 (PBS 2 %fbs)。</li>
	<li>FACs バッファーの 300 μ L のサンプルを再懸濁します (PBS 2 %fbs)、流れの cytometry チューブにサンプルを転送およびフロー フローサイトメトリー分析を実施します。</li>
</ol>

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著者が明らかに何もありません。

時間は、多様で複雑な細胞成分と分子で構成されます。最近の証拠は、この環境の正確な特性評価が治療の成功または障害のよりよい理解を提供できる治療抵抗性のメカニズムを特定するを助けてもいいこと示唆しています。たとえば、エフェクター免疫反応を軽減することができ、免疫療法の効果を破棄する腫瘍様々 な免疫細胞 (すなわちMDSCs、T 細胞など) のレベルを上げま?...

抑制時間は、最近、癌の特徴として認識されているし、開発、進行固形腫瘍癌の保護に重要な役割を果たすだけでなく、免疫療法1感受性を緩和する知られています。時間は、多数の細胞サブセットと腫瘍の免疫学的状態に重要な洞察力を提供するすべての表現型で構成されます。これらの免疫のサブセットは、一緒に自然免疫と適応免疫応答2,3の大半を構成するリンパ球や骨髄由来の細胞にさらに層化できます。がん免疫療法の分野の最近の進歩は免疫療法戦略(すなわち、免疫チェックポイント阻害剤、キメラ抗原受容体の T 細胞等)に耐久性のある癌を誘発する可能性があることを示しています。リグレッションただし、固形腫瘍の癌4,5で比較的効果が残ります。多数のグループでは、時間治療成功6、7、遊ぶことができる、したがって、特に中心前臨床設定の新しい免疫療法を正確に評価する必要があるいる、重要なハードルを示されている、変調または8時間を克服する機能。
通常時間を特徴付ける現在の努力はどちらか顕微鏡を利用またはフローサイトメトリー抗体の免疫細胞サブセットと9,10代の機能を識別するための戦略を分類と共に。これらの 2 つの戦略顕微鏡により細胞サブセットの空間感謝されフローサイトメトリー高スループット、携帯電話の変更のより広範な定量化一意に異なる情報を提供します。今まで 7 つのパラメーターをサポートできる、スペクトル イメージング システムの最適化多重免疫組織染色の最近の改善にもかかわらず限定パネル サイズ広範なレベル プロファイリング免疫困難は、したがってこの手法はしばしば予約詳細解析に焦点を当てた。その結果、フローサイトメトリーはプロファイリング技術最も広く使われている免疫の一つであります。時間評価技術の広範な使用、にもかかわらず処理と染色に使用される方法は非常に変数です。多くの場合プロトコル利用酵素の分離腫瘍 (すなわちコラゲナーゼ、DNase、等) と単一細胞懸濁液を達成するために手動の解離メソッドの後に抗体染色と分析7。それぞれの方法の利点があるにもかかわらず多数のグループはこれらテクニック11まで誘導することができます大規模な変動を示しています。これにより、同じマウス腫瘍モデルを評価する際にも、腫瘍微小環境のプロファイリングのクロス研究比較が非常に困難になります。さらに、これらのメソッドは、腫瘍細胞を評価する限られた可能性を提供し、腫瘍免疫侵入、独立していないコンポーネント消化後両方のコンポーネントが点在するので。サンプル量は、マルチパネル染色とまれな免疫学的サブセットを特徴付けるように試みとが主要な問題の分析を制限 (すなわち、腫瘍特定の T 細胞)12。質量 cytometry など (CyTOF)、飛行時間によってフローサイトメトリー最新技術は、42 の独立したパラメーター13以上のパネル デザインをサポートいくつかのシステムと細胞サブセットの高次元の表現型解析を許可します。CyTOF 免疫プロファイリング技術の驚異的なパワー、にもかかわらず、それは費用、解析の専門知識、および機器へのアクセスのため限られています。さらに、CyTOF プロトコルの多くは、信号対雑音比の14を改善するために免疫のサブセットの浄化をお勧めします、したがって私たちの濃縮方法を使用できることを提案 CyTOF 分析データの品質を向上させるための上流。
本腫瘍微小環境消化と腫瘍免疫を組み込む方法は侵入分離解析について述べる。このメソッドの目的は、腫瘍免疫浸潤、腫瘍微小環境の広範な評価の腫瘍細胞の一部分の独立した高スループット プロファイルを許可することです。このメソッドを使用すると、さらに示す全腫瘍の分析の重要性と皮下腫瘍モデル発見された重要な免疫不.全体的にみて、このメソッドより正確かつ一貫して比較できますサンプル間腫瘍内免疫細胞サブセットの豊か、独立した腫瘍細胞と腫瘍の免疫分数のプロファイリングが可能になります。

<table><tbody><tr><td>6 to 12 week old male C57BL/6 mice</td><td>Jackson Laboratory&amp;nbsp;</td><td>N/A</td><td>Mice used in our tumor studies</td></tr><tr><td>MEER Murine Syngeneic Cancer Cell line</td><td>Acquired from collaborator&amp;nbsp;</td><td>N/A</td><td>E6/E7 HPV antigen expressing murine tonsillar epithelial cancer cell line&amp;nbsp;</td></tr><tr><td>70% isopropyl alcohol&amp;nbsp;</td><td>EMD Milipore&amp;nbsp;</td><td>PX1840</td><td></td></tr><tr><td>Murine surgical tool kit</td><td>World Precision Instruments</td><td>MOUSEKIT</td><td>Primarily only need scissors, tweezers, and a scalpel.&amp;nbsp;</td></tr><tr><td>24-well plate&amp;nbsp;</td><td>Denville Scientific Inc.</td><td>T1024</td><td>Or any 24-well flat bottom plate.&amp;nbsp;</td></tr><tr><td>RPMI-1640 media</td><td>Sigma-Aldrich</td><td>R0883</td><td></td></tr><tr><td>Collagenase I&amp;nbsp;</td><td>EMD Milipore</td><td>234153</td><td></td></tr><tr><td>Collagenase IV</td><td>Worthington Biochemical Corporation</td><td>LS004189</td><td></td></tr><tr><td>DNase I&amp;nbsp;</td><td>Sigma-Aldrich</td><td>11284932001</td><td></td></tr><tr><td>Low Speed Orbital Shaker</td><td>BioExpress (supplier: Genemate)&amp;nbsp;</td><td>S-3200-LS</td><td>Or any orbital shaker.&amp;nbsp;</td></tr><tr><td>Thermoregulating incubator</td><td>Fisher Scientific</td><td>13-255-27</td><td>Or any other thermo-regulated incubator</td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F8192</td><td></td></tr><tr><td>EDTA</td><td>AMRESCO</td><td>E177</td><td></td></tr><tr><td>1 mL Pipette and tips</td><td>Eppendorf</td><td>13-690-032</td><td>Or any other pipette and tips</td></tr><tr><td>40 um Cell Strainers</td><td>Fisher Scientific</td><td>22363547</td><td></td></tr><tr><td>50 mL Centrifuge Tubes</td><td>Denville Scientific Inc.</td><td>C1062-P</td><td></td></tr><tr><td>15 mL Conical Tubes</td><td>Denville Scientific Inc.</td><td>C1012</td><td></td></tr><tr><td>10 mL Luer-Lok Syringe without needles</td><td>BD</td><td>309604</td><td></td></tr><tr><td>Lymphoprep Density Gradient Medium</td><td>STEMCELL Technologies</td><td>7811</td><td></td></tr><tr><td>Transfer Pipettes</td><td>Denville Scientific Inc.</td><td>P7212</td><td></td></tr><tr><td>96-well U-bottom plates</td><td>Denville Scientific Inc.</td><td></td><td></td></tr><tr><td>DPBS</td><td>Sigma Aldrich&amp;nbsp;</td><td>D8573</td><td></td></tr><tr><td>FoxP3 Transcription Factor Staining Buffer Set&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>00-5523-00</td><td>Fix/Permeabilization Buffer and Permeabilization Buffer</td></tr><tr><td>Thermoregulated Centrifuge</td><td>Eppendorf&amp;nbsp;</td><td>5810 R</td><td></td></tr><tr><td>Attune NxT Flow Cytometer</td><td>ThermoFisher Scientific</td><td>N/A</td><td>For 96-well cell volumetric counting&amp;nbsp;</td></tr><tr><td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)&amp;nbsp;Clone 2.4G2&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>553141</td><td>Fc Block</td></tr><tr><td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td><td>ThermoFisher Scientific</td><td>L34962</td><td>Fixable viability stain</td></tr><tr><td>CD45 Monoclonal Antibody (30-F11), APC-eFluor 780</td><td>ThermoFisher Scientific</td><td>47-0451-80</td><td></td></tr><tr><td>TCR beta Monoclonal Antibody (H57-597), APC</td><td>ThermoFisher Scientific</td><td>17-5961-82</td><td></td></tr><tr><td>CD8-&amp;alpha; Antibody (KT15), PE</td><td>Santa Cruz Biotechnology</td><td>sc-53473&amp;nbsp;</td><td></td></tr><tr><td>CD4 Monoclonal Antibody (GK1.5), PE-Cyanine5</td><td>ThermoFisher Scientific</td><td>15-0041-81</td><td></td></tr><tr><td>MHC Class II (I-A/I-E) Monoclonal Antibody (M5/114.15.2), Alexa Fluor 700</td><td>ThermoFisher Scientific</td><td>56-5321-82</td><td></td></tr><tr><td>CD11c Monoclonal Antibody (N418), PE-Cyanine5</td><td>ThermoFisher Scientific</td><td>15-0114-82</td><td></td></tr><tr><td>F4/80 Monoclonal Antibody (BM8), eFluor 450</td><td>ThermoFisher Scientific</td><td>48-4801-80</td><td></td></tr><tr><td>CD11b Monoclonal Antibody (M1/70), APC</td><td>ThermoFisher Scientific</td><td>17-0112-81</td><td></td></tr><tr><td>Ly-6G (Gr-1) Monoclonal Antibody (RB6-8C5), PE</td><td>ThermoFisher Scientific</td><td>12-5931-82</td><td></td></tr><tr><td>CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH5), PE-Cyanine7</td><td>ThermoFisher Scientific</td><td>25-5982-80</td><td></td></tr><tr><td>CD273 (B7-DC) Monoclonal Antibody (122), PerCP-eFluor 710</td><td>ThermoFisher Scientific</td><td>46-9972-82</td><td></td></tr><tr><td>Ki-67 Monoclonal Antibody (B56), BV711</td><td>BD Biosciences</td><td>563755</td><td></td></tr></tbody></table>

enrichment and characterization of the tumor immune and non-immune microenvironments in established subcutaneous murine tumors

腫瘍免疫微小環境 (時間) は最近特に免疫療法のための固形腫瘍の治療反応の重要な仲介人として認識されています。免疫療法の臨床的進歩は、再現可能な方法で正確かつ完全に特徴付ける腫瘍とその関連付けられた免疫潜入の必要性を強調表示します。腫瘍の酵素消化とフローサイトメトリーによる解析により、多数の免疫細胞サブセットおよび表現型; の広範な評価しかし、分析の深さは多くの場合パネルのデザインと大きな腫瘍のサンプルを取得する必要があります興味のまれな免疫個体を観察する fluorophore 制限によって制限されます。したがって、分離・非免疫腫瘍コンポーネントから腫瘍免疫潜入を豊かに高スループットの効果的な方法を開発しました。記載されている腫瘍消化と遠心密度ベースの分離技術により、腫瘍と腫瘍の個別特性免疫潜入分数と細胞生存率を保持し、したがって、腫瘍の広範な評価を提供します免疫学的状態。このメソッドは、さらに一貫性のある全体の腫瘍免疫学的プロファイリング技術の必要性を示す固形腫瘍広範なの空間的免疫不均一を特徴付けるために使用されました。全体として、このメソッドは固体マウス皮下腫瘍の免疫学的特性に対する効果的かつ適応手法を提供します。よい腫瘍の免疫学的機能を特徴付けるためのこのツールの使用など、新しい免疫療法戦略の前臨床評価。

プロトコルで説明したように、非免疫腫瘍コンポーネントから分離までの大きな利点を示した。また、記載されているメソッドを使用して確立された固形腫瘍の重要な免疫学的不均一性を示す.
多くの腫瘍解離技術の大きな問題として、過酷な消化条件 (すなわち、上昇温度、不十分な栄養供給など) の結果と...

Watch this Scientific Journal Video about Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors at JoVE.com

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

ここで分離し、確立された皮下腫瘍における腫瘍免疫と免疫のない微小環境のコンポーネントを豊かにする方法をについて説明します。この手法は、腫瘍免疫浸潤と非免疫腫瘍腫瘍免疫微小環境の包括的な評価を可能にすることができます独立した分析のためことができます。

濃縮と確立された皮下マウス腫瘍における腫瘍免疫と免疫のない微小のキャラクタリゼーション

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

enrichment-characterization-tumor-immune-non-immune-microenvironments

Research

JoVE Journal

Cancer Research

9.5K ビュー.  Baylor College of Medicine. ここで分離し、確立された皮下腫瘍における腫瘍免疫と免疫のない微小環境のコンポーネントを豊かにする方法をについて説明します。この手法は、腫瘍免疫浸潤と非免疫腫瘍腫瘍免疫微小環境の包括的な評価を可能にすることができます独立した分析のためことができます。

ビデオ: Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

濃縮細胞集団を生成し、細胞間コミュニケーションを調べるための簡単​​な方法：腫瘍微小環境のミミック

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

がん研究

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

腫瘍免疫プロファイル用のマウスおよび治療応答評価における扁平上皮癌細胞の粘膜接種

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

乳癌のマウスモデルにおいて腫瘍微小環境における薬剤応答のイメージングを生きる

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

医学

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

直交トピック膵腫瘍の生成と腫瘍浸潤T細胞細胞傷害のEx生体内特性評価

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

マウスにおける腫瘍浸潤CD8+ T細胞の動態を評価するための腫瘍移植

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

ポリ無水物IL-1αナノ粒子の 生体内 抗腫瘍活性の評価

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, &#34;humanized&#34;) mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2R&#947;C-/- NODSIRP&#945;) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; &#34;hot&#34; tumors exhibit large immune infiltration while &#34;cold&#34; tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

This protocol outlines the generation of human immune system (HIS) mice for immuno-oncology studies. Instructions and considerations in the use of this model for testing human immunotherapeutics on human tumors implanted in this model are presented with an emphasis on characterizing the response of the human immune system to the tumor.

ヒト腫瘍を有するヒト化マウスモデルにおける癌免疫療法の試験

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

皮下腫瘍モデルにおける腫瘍浸潤白血球サブセットの評価

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

原発腫瘍から免疫細胞の分離

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

生物学

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

in vitro腫瘍微小環境における免疫応答を解剖するためのマイクロ流体共培養モデル

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

濃縮と確立された皮下マウス腫瘍における腫瘍免疫と免疫のない微小のキャラクタリゼーション

要約

さらに動画を探す

この動画の章

濃縮細胞集団を生成し、細胞間コミュニケーションを調べるための簡単な方法：腫瘍微小環境のミミック

腫瘍免疫プロファイル用のマウスおよび治療応答評価における扁平上皮癌細胞の粘膜接種

乳癌のマウスモデルにおいて腫瘍微小環境における薬剤応答のイメージングを生きる

直交トピック膵腫瘍の生成と腫瘍浸潤T細胞細胞傷害のEx生体内特性評価

マウスにおける腫瘍浸潤CD8⁺ T細胞の動態を評価するための腫瘍移植

ポリ無水物IL-1αナノ粒子の生体内抗腫瘍活性の評価

ヒト腫瘍を有するヒト化マウスモデルにおける癌免疫療法の試験

皮下腫瘍モデルにおける腫瘍浸潤白血球サブセットの評価

原発腫瘍から免疫細胞の分離

in vitro腫瘍微小環境における免疫応答を解剖するためのマイクロ流体共培養モデル

濃縮と確立された皮下マウス腫瘍における腫瘍免疫と免疫のない微小のキャラクタリゼーション

要約

さらに動画を探す

この動画の章

濃縮細胞集団を生成し、細胞間コミュニケーションを調べるための簡単​​な方法：腫瘍微小環境のミミック

腫瘍免疫プロファイル用のマウスおよび治療応答評価における扁平上皮癌細胞の粘膜接種

乳癌のマウスモデルにおいて腫瘍微小環境における薬剤応答のイメージングを生きる

直交トピック膵腫瘍の生成と腫瘍浸潤T細胞細胞傷害のEx生体内特性評価

マウスにおける腫瘍浸潤CD8+ T細胞の動態を評価するための腫瘍移植

ポリ無水物IL-1αナノ粒子の 生体内 抗腫瘍活性の評価

ヒト腫瘍を有するヒト化マウスモデルにおける癌免疫療法の試験

皮下腫瘍モデルにおける腫瘍浸潤白血球サブセットの評価

原発腫瘍から免疫細胞の分離

in vitro腫瘍微小環境における免疫応答を解剖するためのマイクロ流体共培養モデル

濃縮細胞集団を生成し、細胞間コミュニケーションを調べるための簡単な方法：腫瘍微小環境のミミック

マウスにおける腫瘍浸潤CD8⁺ T細胞の動態を評価するための腫瘍移植

ポリ無水物IL-1αナノ粒子の生体内抗腫瘍活性の評価