This work was supported in whole by funding from the NIH through the R21 grant, "Mechanisms of Perineural Invasion in Head and Neck Cancer" and the NCI T32 training grant, "Post-Doctoral Research Training in Head and Neck Oncology (2T32CA060397-21)." Thank you to Richard Steiman, MD, PhD and lab staff.

1.培養培地の調製と料理（10分） <ol><li> 100μLを96ウェルV底プレートのウェルに、10％ウシ胎児血清（FBS）を有するダルベッコ変法イーグル培地（DMEM）を加えます。 </li><li> 20℃の冷凍庫から半固体マトリックスの約100μLの前等分し、バイアルを取り外し、氷の上に直接配置します。 注：半固体マトリックスは、その後の工程のために必要である、氷の上に液体状態に達するのに約30分かかりますので、この前後根神経節に（DRG）の収穫を行います。すべての回で、氷上で半固体マトリックスを維持するために失敗すると、低品質のマトリックス液滴を発生します。 </li><li>ラベルグラスボトム永久インクでの培養プレート、プレートの下面に四隅のそれぞれに一意の識別子を作成します。プレート表面を冷却するために氷の上にプレートが右サイドを置きます。 注：各マウス利回り32から40 DRGので、1から40にラベルを付けそれはプレチルパイする必要はありませんチルドのヒントは、潜在的に、マトリックス液滴を配置する行列の一貫性の違いを導入するコースを渡って温めますよう、ヒントをペット。 </li></ol>マウスDRGの2解剖（45分） <ol><li>例えばCO 2室と開胸の使用を通じてなど、特定の研究室プロトコル、あたりなどの無胸腺ヌードマウスを安楽死させます。 </li><li>解剖エリアと顕微鏡を設定します。 15-または50-mLコニカルチューブ用ポリスチレン容器の、きれいな滅菌し、平らな底面を使用してください。 注：また、彼らは、使い捨て、安価であるため、解剖面として使用するのに適しており、ピンまたは針を使用して脊柱の固定を可能にします。唯一の滅菌機器や針を使用しています。 </li><li>自然なビジョンの下では、直線状または曲線状の微細なハサミを持つマウスの頭に尾の付け根から背骨に沿って縦切開を行います。 </li><li> SACRの下に横方向に分割する同じハサミを使用してくださいアル背骨。背骨の両側にハサミを使用して頭蓋底までのすべての方法を分析。この時点で、頸椎が頭蓋できるだけハサミで横に切開されていることを確認してください。 </li><li>低電力で手術用顕微鏡下で脊柱の子宮頸終了を確認します。白い脊髄は傍脊柱筋群および軟組織の可変量に囲まれている骨リングの真ん中には明らかです。 </li><li>微視的な春のはさみで3椎体 - 背側または第2の優れた面を分割します。ハサミのバネ作用を使用して、椎体の解剖は正中線で発生したことを確実にするために、この初期の骨のカットを開きます。仙骨に向かって少しずつ続行し、次に椎体の腹側または下面に同一のカットを完了することによって、背骨を二分します。 注：下のDRG収率をもたらす骨脊柱の正中切開を確保するために失敗しました。 </li><li> THIでS点は、脊柱を二等分に分割されています。代わりに脊髄で、脇1ヘミ背骨を置き、滅菌プレートに下向きに。 注：DRGを脊髄に深いような場所に脊髄を残して、乾燥からのDRGを防ぐことができます。 </li><li>ポリスチレン解剖プラットフォーム上の2つの18ゲージの針を他のヘミ背骨の両端を固定します。子宮頸終わりに出発し、穏やか約4椎レベルから脊髄をバック剥離、（背骨が狭いので明らかである、DRGは互いに接近している、とリブ挿入があります）。 </li><li> DRGを接続する感覚神経（通常は2）を確認します。神経がDRGに挿入する領域で、静かに微細な鉗子で周囲の筋膜を把握します。 DRGを解放するために微細な春のはさみでこの筋膜およびその他の神経組織を解剖し、トリム。穏やかな後退は、骨、脊椎内のその位置からDRGをもたらすでしょう。 注：増加この段階で、顕微鏡力が有益です。 DRGへの圧挫損傷は大幅に神経突起の成長を制限します。これは、直接DRGを把握することはありませんすることにより、むしろ周囲の筋膜を把握することにより回避されます。 </li><li> DRGを解放するために微細な春のはさみで（DRGは一般的に解剖ビューに深い相対的なものである1末梢神経を持っている）、末梢神経をカット。 注：それは神経が終了し、DRGはので、この時点でのDRGのできるだけ近くに、このカットを作り、緊張にしながら、始まりが理想的である把握するのが最も簡単です。この遠位枝が切断されると、近位の枝も同様にトリミングされています。 </li><li>微視的な春のハサミでの浮遊神経線維や筋膜の添付ファイルのDRGをトリミング。適切な単離後、96ウェルV底プレート内の室温の培地にDRGを配置します。 注：プレートの下に暗い背景を配置すると、簡単にサブミリメートルホワイトのDRGを可視化することができます。唯一のEAC内の1つのDRGを置きます時間だけでなく、このように、それらは後の工程でより容易に説明することができます。 </li><li>すべての方法1ヘミ背筋がこのプロセスを繰り返し、その後、他の。 40から32を合計20のDRG、 - それぞれの側は、16が得られます。 注：これより少ないたDRGがある場合、脊椎の解剖は、さらに頭または尾を行う必要があります。 DRGを1が尾側に進行するよう、ますますあまり明確になります。 </li></ol>半固体マトリックス液滴の調製（プレートあたり&lt;1分） <ol><li>氷から一方のガラスだけでなく、底板を外し、手術用顕微鏡下に氷のブロックの上に置きます。マトリックスのアリコートは、すべての回で氷の上に残っていることを確認します。 </li><li>よくガラスの端からの液滴自体と少なくとも同じ偉大な距離を残して、2-μLまたは10μLのマイクロピペッターでガラス底板の四隅に行列の1.5-μLの液滴を配置します。 <ol><li>上に直接ピペットの先端を置き45度の角度でガラス。ゆっくりと行列をピペット。行列が板に従事した後、ゆっくりとガラス底部から離れます。 注：行列とガラスとの間の表面張力が完璧な半球を毎回作成する必要があります。 </li><li>行列の液滴への空気の不慮の注入は、行列の液滴の直径がはるかに大きくなり、除去するのが困難であるためである。先端が、空になる直前にピペッティングを停止 注：ピペッティング手を安定させるために秒針を使用して、より正確な配置を容易にします。 </li></ol></li></ol>半固体マトリックス液滴にDRGの4挿入（&lt;プレートあたり2分） <ol><li>簡単に、室温（〜1分）でマトリックス滴でプレートを残します。これはわずかにDRGを正確に配置するのにそれが容易になり、マトリックスを補強します。 </li><li>スクープ（把握していない）DRGそっと左手に閉じられた微細なピンセットで。ここでも、小さなに対して暗い背景、白DRGは、可視化を容易にします。右手に21ゲージの針の先端にDRGを転送します。この転送は鉗子上の残留メディアを残します。 </li><li>静かに21ゲージの針（ 図1）を使用して、マトリックス液滴の中央にDRGを挿入します。ほとんどの時間、DRGを容易にマトリックスに放出され、その後針を中央に配置することができます。 <ol><li> DRGが針に付着した場合、針のオフとマトリックス液滴にDRGをプッシュするために微細な鉗子を使用しています。ラボでの微視的鉗子から余分なメディアをワイプ吸い取ります。 注：行列への紹介メディアは、アッセイの直径、体積、および一貫性が変更されます。色や色の書き込みと氷のブロックは、この繊細なプロセスの可視化を容易に背景のコントラストを、提供します。 </li></ol></li><li>プレートはすべて4のDRGを持っていた後、DRGは、行列滴の中心にあることを確実にするために、最終的な検査を行います。調整を行います21ゲージの針を用いて、必要に応じて。 </li><li> 37℃のインキュベーターに完成したプレートを転送します。これは、半固体マトリックスを固化した位置にDRGを修正します。 </li><li> DRGのすべてについて、この手順を繰り返します。陰性対照としてDRGずにブランクマトリックス液滴を配置します。 </li><li>すべてのプレートを完了し、少なくとも3分間37℃のインキュベーターにそれらを露出させた後、各ガラス底プレートに10％FBS培地を含むDMEMの4 mLを加え。わずかな角度でプレートを置き、ゆっくりと、それは徐々にマトリックス-DRGユニットと接触するように、メディアを追加します。 注：媒体が速すぎるか、積極的に追加された場合、マトリックスおよび/またはDRGは追い出さになることができます。 </li><li> 72時間 - 次の48を37℃のインキュベーター中でアッセイを保管してください。 注：アッセイの定期検査では、行列のエッジ（ 図2）に向けて神経突起の周方向の伸長が表示されます。神経突起は、マトリックスへの道の4分の3よりも大きい場合には、それを細胞をプレーすることが適切です。 </li></ol>頭頸部癌細胞の5準備注：頭頸部扁平上皮癌細胞以外の細胞株は、この実験の設計に使用することができます。 <ol><li> 37℃のインキュベーター中で好ましいフラスコまたは培養皿に10％FBSを含むDMEM中に扁平上皮癌細胞株を維持します。すべての培地培養フラスコまたはディッシュから実験、吸引用の細胞を調製し、PBSで2回それを洗浄します。 10％FBSを含むDMEMに続いて、5分間、0.025％トリプシンを適量添加することにより細胞を懸濁。 6 mLの培養皿への細胞および培地混合物のピペット4ミリリットル。 注：6 mLの培養プレートは十分な細胞でも、50％未満コンフルエント以上のものを提供します。 </li><li>染色およびDRGアッセイにメッキの24時間前に異なる条件にHNSCC細胞株を公開します。再線量細胞は一貫environmeを維持するためにメッキされた後の状態をNT。 注： - マトリックスへのDRGのマウスを解剖し、移植後2日目の細胞培養皿に1抗体、成長因子、サイトカイン、または他の分子を加えます。 </li><li> （ - 3日マトリックスへのDRGの収穫と移植後2）細胞をプレーティングする前に、蛍光細胞の汚れを1時間を追加します。 注：ここで使用される特定の汚れは、細胞膜を自由に通過。しかしながら、細胞内のチオール基と反応した後、染色は、細胞内に留まると娘細胞に受け継がれます。染色は大幅アッセイ内の可視化を容易にします。これらの実験の時間枠である3日、 - 蛍光は2のために存在しているので、これらの一時的な汚れは、これらのアッセイのために理想的です。また、多くの異なる色の使用を可能にし、蛍光タンパク質、トランスフェクトされた細胞株を作成する必要がなくなります。 <ol><li>無血清DMEMの2mLに2μLの10mMストック細胞の汚れを混合することにより、色素溶液の10μMの準備すべてのセルの状態のため。細胞に添加する前に37℃にこれを暖めます。 </li><li>プレートに付着HNSCC細胞を残し、6 mLのプレート内の培地を除去します。 DMEMを各プレートに添加し、リン酸緩衝生理食塩水（PBS）の2 mLを加え、10μM細胞染色/無血清2mLのを削除</li><li> 40分間37℃のインキュベーターに細胞染色で6 mLの培養プレートを返します。 </li><li> 40分後、培地を吸引除去します。 PBSの2ミリリットルを追加し、それを吸引除去します。各プレートに0.025％トリプシンの1 mLを加え、37℃のインキュベーターでそれらを交換してください。 </li><li> 3の後に10％FBSを含むDMEMの2mLのを追加 - 5分および15-mLコニカルチューブに懸濁した細胞を移します。細胞をスピンし、10％FBSを含むDMEMを1mLで再懸濁しました。 </li><li>血球計又は自動化細胞カウンターで細胞をカウントし、その後mLの30万の細胞の最終細胞濃度を作成するために、10％FBSを含むDMEMを加えます。 </li></ol></li></ol> 6.メッキヘッドとNECK癌細胞<ol><li>インキュベーターから行列-DRGアッセイとガラス底プレートを取り外します。 注： - 3日神経突起は、一般的に2の後に起こるマトリックスのエッジへの道の少なくとも75％である場合もやはり、それらが適切です。これは最高の、少なくとも10倍の対物レンズを用いて見られています。 </li><li>ガラス底プレート内の培地を吸引除去します。完全によくマトリックス-DRGユニットを取り外すことなく、ガラス内の培地を吸引除去します。 </li><li> 200-μLのピペットを用いて30万細胞/ mL培地の200μLを描画します。各マトリックス-DRGアッセイを介して細胞の2滴を置きます。一方のプレート上の各セルの状態の4反復を作成し、各マトリックス滴に対してこの手順を実行します。 注：行列の半球形状は、細胞が、アッセイ（ 図3a、図3b）の外周に沿ってリングに定住することができます。滑らかなトリガーとピペットを見つけることは、均一の配置ははるかに簡単に低下させます。 </li><li>同様の細胞数およびジを確認します4X顕微鏡下での様々な細胞条件のstribution。 </li><li>約60分間、37℃のインキュベーターにガラス底プレートを置きます。注：細胞および培地（〜150μL）のわずかなボリュームがありますがインキュベータ時間の数時間が経過するまで、アッセイが乾燥しないでください。 </li><li> 60分後、そっとガラス底板の側壁に沿って10％FBSを含むDMEMの4 mLを加え。 注：時間の期間にわたり、細胞が部分的にプレート底部に付着し、そして培地を添加する方法は、細胞に影響を与えません。異なる細胞型は、ガラス底プレートに付着し始めるために、多かれ少なかれ時間がかかるします。 </li><li>前の濃度レベルを維持するために、この時点で、任意の外因性薬剤または成長因子を交換してください。 </li><li>彼らは顕微鏡で検査されている場合を除いて、37℃のインキュベーターにアッセイを返します。顕微鏡イメージングによってこのアッセイの結果を定量化します。簡単に言うと、神経周囲浸潤の鎖を数えます4X顕微鏡画像を使用して、4つの象限インチ 注：4X目的と蛍光機能を備えた顕微鏡は、光文書化アッセイのために適切です。アッセイの大きさは、神経周囲浸潤の個々の領域をキャプチャするのに十分な詳細である4X対物レンズの視野内にうまく収まります。神経周囲の浸潤は、24時間以内に明らかになったが、48時間を超えて、アッセイの整合性は、腫瘍細胞がマトリックスの周囲に沿って分割して侵入するように、弱体化し始めます。 48時間を超えて、明視野顕微鏡を用いて可視化を不明瞭DRG、からの線維芽細胞の実質的な流出もあります。したがって、このウィンドウの間に少なくとも2回のフォトドキュメント4X顕微鏡での結果と得策である（ 例えば、腫瘍細胞をプレーティング後24および48時間で）。これらは、3次元のアッセイであり、それは完全に画像全体を分析することは困難であることを留意します。ほとんどの神経周囲の浸潤は、プレートの底、トンから飛行機で発生します彼細胞はわずかにプレートの底の上にマトリックス中に埋め込まれています。この平面が水平に近いため、腫瘍細胞と神経突起の大半は4Xでの単一レベルの画像に取り込まれます。 </li></ol>

<ol>
	<li>Jemal, 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=Cancer+statistics,+2006.">Cancer statistics, 2006.</a> CA Cancer J Clin. 56 (2), 106-130 (2006).</li><li>Hinerman, R. W., 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=Postoperative+irradiation+for+squamous+cell+carcinoma+of+the+oral+cavity:+35-year+experience.">Postoperative irradiation for squamous cell carcinoma of the oral cavity: 35-year experience.</a> Head Neck. 26 (11), 984-994 (2004).</li><li>Rahima, B., Shingaki, S., Nagata, M., Saito, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+significance+of+perineural+invasion+in+oral+and+oropharyngeal+carcinoma.">Prognostic significance of perineural invasion in oral and oropharyngeal carcinoma.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 97 (4), 423-431 (2004).</li><li>Woolgar, J. A., Scott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+cervical+lymph+node+metastasis+in+squamous+cell+carcinoma+of+the+tongue/floor+of+mouth.">Prediction of cervical lymph node metastasis in squamous cell carcinoma of the tongue/floor of mouth.</a> Head Neck. 17 (6), 463-472 (1995).</li><li>Tai, S. 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=Treatment+for+T1-2+oral+squamous+cell+carcinoma+with+or+without+perineural+invasion:+neck+dissection+and+postoperative+adjuvant+therapy.">Treatment for T1-2 oral squamous cell carcinoma with or without perineural invasion: neck dissection and postoperative adjuvant therapy.</a> Ann Surg Oncol. 19 (6), 1995-2002 (2012).</li><li>George, D. L., 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=Nosocomial+sinusitis+in+patients+in+the+medical+intensive+care+unit:+a+prospective+epidemiological+study.">Nosocomial sinusitis in patients in the medical intensive care unit: a prospective epidemiological study.</a> Clin Infect Dis. 27 (3), 463-470 (1998).</li><li>Fagan, J. 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=Perineural+invasion+in+squamous+cell+carcinoma+of+the+head+and+neck.">Perineural invasion in squamous cell carcinoma of the head and neck.</a> Arch Otolaryngol Head Neck Surg. 124 (6), 637-640 (1998).</li><li>Soo, K. C., 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=Prognostic+implications+of+perineural+spread+in+squamous+carcinomas+of+the+head+and+neck.">Prognostic implications of perineural spread in squamous carcinomas of the head and neck.</a> Laryngoscope. 96 (10), 1145-1148 (1986).</li><li>Parsons, J. T., Mendenhall, W. M., Stringer, S. P., Cassisi, N. J., Million, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+factors+influencing+the+outcome+of+postoperative+irradiation+for+squamous+cell+carcinoma+of+the+oral+cavity.">An analysis of factors influencing the outcome of postoperative irradiation for squamous cell carcinoma of the oral cavity.</a> Int J Radiat Oncol Biol Phys. 39 (1), 137-148 (1997).</li><li>Liao, C. 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=Does+adjuvant+radiation+therapy+improve+outcomes+in+pT1-3N0+oral+cavity+cancer+with+tumor-free+margins+and+perineural+invasion.">Does adjuvant radiation therapy improve outcomes in pT1-3N0 oral cavity cancer with tumor-free margins and perineural invasion.</a> Int J Radiat Oncol Biol Phys. 71 (2), 371-376 (2008).</li><li>Fan, K. H., 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=Treatment+results+of+postoperative+radiotherapy+on+squamous+cell+carcinoma+of+the+oral+cavity:+coexistence+of+multiple+minor+risk+factors+results+in+higher+recurrence+rates.">Treatment results of postoperative radiotherapy on squamous cell carcinoma of the oral cavity: coexistence of multiple minor risk factors results in higher recurrence rates.</a> Int J Radiat Oncol Biol Phys. 77 (4), 1024-1029 (2010).</li><li>Dai, H., 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=Enhanced+survival+in+perineural+invasion+of+pancreatic+cancer:+an+in+vitro+approach.">Enhanced survival in perineural invasion of pancreatic cancer: an in vitro approach.</a> Hum Pathol. 38 (2), 299-307 (2007).</li><li>Ceyhan, G. O., 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=Neural+invasion+in+pancreatic+cancer:+a+mutual+tropism+between+neurons+and+cancer+cells.">Neural invasion in pancreatic cancer: a mutual tropism between neurons and cancer cells.</a> Biochem Biophys Res Commun. 374 (3), 442-447 (2008).</li><li>Gil, Z., 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=Paracrine+regulation+of+pancreatic+cancer+cell+invasion+by+peripheral+nerves.">Paracrine regulation of pancreatic cancer cell invasion by peripheral nerves.</a> J Natl Cancer Inst. 102 (2), 107-118 (2010).</li><li>He, 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=GFRalpha1+released+by+nerves+enhances+cancer+cell+perineural+invasion+through+GDNF-RET+signaling.">GFRalpha1 released by nerves enhances cancer cell perineural invasion through GDNF-RET signaling.</a> Proc Natl Acad Sci U S A. 111 (19), 2008-2017 (2014).</li><li>He, 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+chemokine+(CCL2-CCR2)+signaling+axis+mediates+perineural+invasion.">The chemokine (CCL2-CCR2) signaling axis mediates perineural invasion.</a> Mol Cancer Res. 13 (2), 380-390 (2015).</li><li>Bakst, R. L., 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=Radiation+impairs+perineural+invasion+by+modulating+the+nerve+microenvironment.">Radiation impairs perineural invasion by modulating the nerve microenvironment.</a> PLoS One. 7 (6), 39925 (2012).</li><li>Ayala, G. E., 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=In+vitro+dorsal+root+ganglia+and+human+prostate+cell+line+interaction:+redefining+perineural+invasion+in+prostate+cancer.">In vitro dorsal root ganglia and human prostate cell line interaction: redefining perineural invasion in prostate cancer.</a> Prostate. 49 (3), 213-223 (2001).</li><li>Na'ara, S., Gil, Z., Amit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Modeling+of+Cancerous+Neural+Invasion:+The+Dorsal+Root+Ganglion.">In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion.</a> J Vis Exp. (110), (2016).</li></ol>

The authors have no competing financial interests.

プロトコル内の重要なステップ このプロトコル内の最も重要なステップは、後根神経節の正確な解剖と抽出されています。 2半棘に脊柱と正中線縦部門の適切離断は、DRGを大量に得るために重要です。個々のDRGの解剖時には、神経節は、直接処理するべきではありませんが、むしろ周囲の筋膜は、微細な鉗子で把持する必要があります。これを行うに失敗す...

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the US, with 10,000 deaths per year nationally and 300,000 deaths per year worldwide1. The overall prognosis for HNSCC has remained unchanged at 50% for the past several decades. Perineural invasion (PNI) is one of the most prominent pathological features that portend a poor prognosis in patients with HNSCC. Unfortunately, PNI is a frequent occurrence in HNSCC and can be found in up to 40% of HNSCC patients2,3. 
PNI is the process by which malignant cells track along nerves to adjacent tissues, allowing for higher rates of local and distant spread. Accordingly, PNI-positive HNSCC tumors have higher rates of locoregional recurrences and distant metastases, resulting in lower overall survival compared to HNSCC patients without PNI4-8.
Although the treatment of patients with PNI is typically maximized by employing surgery, radiation, and chemotherapy, the overall survival rates of these patients are still decreased by up to 40% compared to patients without PNI9-11. Thus, it is clear that the current treatment modalities for HNSCC are ineffective in improving the adverse prognosis associated with PNI. The approach of developing targeted therapy against PNI in HNSCC has been hindered by the poor understanding of the factors that regulate this process. This is, in part, a consequence of the lack of a consistent in vitro model for the study of PNI in HNSCC. 
In recent years, several groups have been utilizing an in vitro model for studying PNI in predominantly pancreatic and prostate cancers12-19. This model uses the neurites generated from dorsal root ganglia isolated from mice or rats as a surrogate for large-nerve invasion. The dorsal root ganglia are fixed in a factor-depleted semisolid matrix, which is a solubilized basement membrane protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells. This matrix allows for the outgrowth of the neurites and the tracking of single cancer cells along these neurites. Described here is the adaption of this model for the examination of PNI in HNSCC.

<table border="0" cellpadding="0" cellspacing="0" width="879">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">DMEM/F-12 50/50 Mix with L-glutamine &#38; 15mM HEPES</td>
			<td style="width:207px;">Corning Cellgro</td>
			<td style="width:139px;">10-090-CV</td>
			<td style="width:157px;">Manassas, VA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td>Atlanta biologicals</td>
			<td>S11150</td>
			<td>Flowery Branch, GA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA (1x)</td>
			<td>Life Technologies Corporation</td>
			<td>25200056</td>
			<td>Grand Island, NY</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered Saline 1x</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td>Manassas, VA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel hESC-Qualif Mouse</td>
			<td>Corning Incorporated</td>
			<td>354277</td>
			<td>Bedford, MA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gamma Irradiated 35mm glass bottom culture dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>Ashland, MA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SteREO Discovery.V8 Operating Microscope</td>
			<td>Carl Zeiss Microimaging</td>
			<td>495015-0021-000&#160;</td>
			<td>Thornwood, NY</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Schott ACE I light source</td>
			<td>Schott</td>
			<td>A20500</td>
			<td>Germany</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTracker&#160;</td>
			<td>Life Technologies Corporation</td>
			<td>C2925</td>
			<td>Carlsbad, CA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD PrecisionGlide Needle 18G and 21G</td>
			<td>BD</td>
			<td>305195</td>
			<td>Franklin Lakes, NJ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Microdissecting Tweezer</td>
			<td>Harvard Apparatus</td>
			<td>60-3851</td>
			<td>Holliston, MA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Fine Operating Standard Scissors</td>
			<td>Harvard Apparatus</td>
			<td>52-2789</td>
			<td>Holliston, MA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Spring Scissors</td>
			<td>Harvard Apparatus</td>
			<td>60-3923</td>
			<td>Holliston, MA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dressing Forceps</td>
			<td>Harvard Apparatus</td>
			<td>72-8949</td>
			<td>Holliston, MA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Athymic nude mice (002019)</td>
			<td>Jackson Laboratory</td>
			<td>002019</td>
			<td>Bar Harbor, ME</td>
		</tr>
	</tbody>
</table>

a model for perineural invasion in head and neck squamous cell carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

DRGの解剖とマトリックス液滴内で配置した後、アッセイの外観は、DRGが完全な円ではなく、それをマトリックス液滴内の中心に位置していることを、図1（注）のようになります。これは、部分的に、図2に示す360度における神経突起の伸長を可能にします。 DRGの特定の部分は、典型的には、遠心性と求心性神経の枝が入力され、それぞれ、DR...

Watch this Scientific Journal Video about A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma at JoVE.com

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

頭頸部扁平上皮癌における神経周囲の浸潤のためのモデル

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

a-model-for-perineural-invasion-head-neck-squamous-cell

Research

JoVE Journal

Cancer Research

10.5K ビュー.  University of Pittsburgh Medical Center. Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

ビデオ: A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

定量的および前臨床治療の縦評価のための転移性腎細胞癌の同系マウスモデル

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

がん研究

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

ホルマリン固定パラフィン包埋ヒト卵黄扁平上皮癌試料におけるT細胞亜集団の四色蛍光免疫組織化学

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

上に移動: シンプルかつ柔軟なIn Vitro三次元侵略試金のプロトコル

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&amp;E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

We describe an in vivo murine model of perineural invasion by injecting syngeneic pancreatic cancer cells into the sciatic nerve. The model allows for quantification of the extent of nerve invasion, and supports investigation of the cellular and molecular mechanisms of perineural invasion.

神経周囲浸潤のIn Vivoマウスの坐骨神経モデル

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

治療の頭頸部扁平上皮癌に対する回転楕円体に基づく 3 D 細胞培養モデルのテスト

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

舌の細胞外マトリックスと舌扁平上皮癌生体外の再構成の作製

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

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

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

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 ...

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 ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

マウス腎臓と鶏絨毛性膜に確立された転移性透明細胞腎細胞癌モデルの比較

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.