本研究は、復旦大学中山病院(2016ZSLC17)の臨床研究特別基金によって支援されました。著者らは、復旦大学中山病院の西林胡博士が英語執筆に対する非常に専門的な提案をしてくれたことに非常に感謝しています。

この単一センターの回顧研究は、復旦大学中山病院の地元の機関審査委員会によって承認されました。
1. インフォームド・コンセントの取得
<ol><li>TRA介入の前に、介入放射線科医(IRs)に、患者に対するTRAの利点および潜在的な合併症を説明してもらう。</li>
</ol>2. 患者の評価
<ol><li>インフォームド・コンセントを得た後、穿刺および缶詰の実現可能性についてRAを評価する。</li>
	<li>患者の病歴の総合的なレビューを行います。患者が重度の血管の強発性、重篤な末梢血管疾患、透析のための瘻孔、またはRA透析手術のための準備を有しているかどうかを確認する。これらは、TRA介入を受けている患者に対する相対的な禁忌である。</li>
	<li>RAの可視性を評価します。<ol>
<li>パルスオキシメトリーを使用してバーボー試験を行い、介入前に手側副動脈がどの程度目に見えるかを評価する。バーボーD波形はRAのカンヌレーションのための絶対的な禁忌と考えられています。</li>
		<li>バルボーC波形としてテストされた患者のために、前腕および手の副次循環の量についてのより信頼できる情報を提供するためにドップラー超音波検査を適用する。2mm未満の内径は禁忌と見なされます。 注:病歴評価およびRA評価後、禁忌患者は、イプシラテラRAを介して穿刺を控えるべきである。反側RAは、Barbeau試験評価を通じて特許であることが判明した場合、理想的な供給として機能する可能性があります。最初は、左側の RA が優先アクセス ルートとして選択されます。左のRAが不適当と判明した場合、右のRAが代替選択として機能する場合があります。</li>
	</ol>
</li>
</ol>3. 放射状動脈アクセス
<ol><li>患者を血管造影台の上の立場に置く。次に、左腕を患者の体に平行に置き、左ウエストに近づけ、カテーテルとワイヤーの配置を容易にし、オペレータの位置をTFAと同等にします。</li>
	<li>遠位RAパルスを触診でマークします。10%ポビドネヨウ素外科スクラブ液で皮膚表面をきれいにし、溶液を空気乾燥させます。手術用ドレープで左腕を覆います。<ol>
<li>潜在的な左RA穿刺障害の場合は、右腕または右のインジナル領域を殺菌してドレープすることによって代替アクセスルートを準備します。</li>
	</ol>
</li>
	<li>局所麻酔(すなわち、リドカイン1 mL2%)を適用する左RAの最も強力な脈動の軸に沿ってスタイロイドプロセスに近似。</li>
	<li>手首を伸ばし、変更されたセルディンガー技術を使用して20G針でRAを穿刺します。拍動性動脈血の戻りが観察されるとき、0.025インチ親水性ガイドワイヤーを導入する。<ol>
<li>ガイドワイヤーを引き込み、抵抗に遭遇した場合は針を再び取り付けます。ガイドワイヤーを挿入しないでください。デジタル減算血管造影(DSA)の助けを借りて、RAを強調し、親水性ガイドワイヤーを挿入するのに役立つコントラストの約1〜2 mLを注入します。</li>
	</ol>
</li>
	<li>アクセスが取得されたら、針を取り外し、ガイドワイヤーで4-フランスの親水性シースを導入します。シース挿入後、少量の動脈血を注射器で静かに汲み上げ、シース先端が血管内にあることを確認する。</li>
</ol>4. 抗凝固と血管拡張
<ol><li>血管拡張カクテル溶液(3,000 IUの未分離ヘパリン、0.1mgのニトログリセリン、リドカイン20mg)を10 mL用意します。</li>
	<li>8 mLの血管拡張カクテル溶液を、シースを通して0.5 mL/sの速度で投与する(図1)。 注:中等度または高い出血リスクを有する患者のためのヘパリンの用量を減らすか、または停止する。</li>
</ol>5. カテーテルの選択
<ol><li>4-フランス語、125センチの一般的なカテーテルと標準的な0.035インチx 180 cm親水性ワイヤーを使用して、鎖骨下動脈を横断し、下降大動脈を係合します。DSAフルオロソコピーを使用して、腕内のナビゲーション中に近位腋窩動脈を可視化し、動脈ループまたは血管損傷に対する潜在的な病変を避けます。 注:鎖骨下動脈には多くの動脈枝があります。血管造影誘導により、ガイドワイヤーカテーテルが逆行カテーテル法の間に副血管に入るのを防ぎます。いくつかの症例は、放射状動脈に動脈ループを有する。標準ワイヤがループを通過できない場合は、マイクロカテーテルと角度 0.016 インチまたは 0.018 インチマイクロワイヤの使用をお勧めします。</li>
	<li>4-フランス語、125 cmの一般的なカテーテルを標準の0.035インチワイヤーと組み合わせて使用して、ガイドワイヤーを下降大陸に向けて導くために横断アーチを交渉します。 注:大動脈と左鎖骨下動脈の間の角度が非常に鋭い場合、このターンを達成するためにコブラ2字型カテーテル(例えば、シモンズIまたはシモンズIIカテーテル)をお勧めします。</li>
	<li>下降大動脈のカテーテル後、同軸技術を介して共通のカテーテルを交換する。一般的なカテーテルが下降大間に挿入されたら、DSA透視検査の指導の下でセリアックトランクのカテーテルのためにカテーテル先端を腹腔内に操縦する。ほとんどの場合、セリアック幹、肝動脈、および優れた腸間膜動脈の血管造影をカテーテル化し、行うことが容易である。 注:セリアック動脈と下降大動脈の間の角度が非常に鋭い場合は、コブラカテーテルを使用して手順を完了します。</li>
	<li>肝塞栓術の場合、同軸技術を用いて超選択的カテーテル法および化学式化を行い、2.8-フランス語150cmマイクロカテーテルを、腫瘍に供給する肝動脈の標的枝に配置する(図2)。病気の負担と患者の好みに応じてTACEを実行します。</li>
	<li>十分な塞栓を確認するために高圧注入器を使用して、共通のカテーテルを介して血管造影を行います.カテーテル先端は通常、一般的な肝動脈に位置する。9~12 mLの造影剤を1sあたり3~4mLの割合で、蛍光顕微鏡時間は~15秒で注入します。次に、RAへの損傷を避けるためにガイドワイヤー上のカテーテルを取り外します。</li>
</ol>6. ラジアル動脈止血症
注: 非閉塞性止血症は、RAの愛息を維持するために特別な止血帯を使用して行われます(図3)。
<ol><li>残りの2mLの血管拡張カクテル溶液(セクション4)をRAシースを介して投与する。直後に、約5cmの鞘を取り出す。</li>
	<li>左手首の放射状アクセスサイトの上に止血帯を置き、付属の注射器を使用して空気で止血帯のエアバッグを十分に膨らまします。その後、シースを完全に取り外し、ゆっくりとエアバッグを収縮します。アクセス部位で漏れが見られる場合は、1 mLの空気をカフに戻します。通常、10~15 mLの空気をエアバッグに加え、止止めを維持します。</li>
	<li>出血や漏れがないことを確認します。同時に、遠位径動脈パルスが止血中に触知可能であることを確認する。パルスオキシメータ波形を使用して、左手親指の動脈波形を確認します。</li>
	<li>2時間ごとに2mLの空気嚢をゆっくりと6時間以下に膨らませる。止血帯が操作後6時間取り出されたら止血が成し遂がすることを再確認する。 注:デフレ時に穿刺部位からの出血や漏れが見られると、空気が30分間空気嚢に戻され、プロセスが繰り返されます。</li>
	<li>退院前に、BARBEAU検査を行い、RAの開腸を確認し、放射状動脈閉塞を有する患者を記録し、綿密にフォローアップする。</li>
</ol>7. フォローアップ
<ol><li>約1ヶ月後、TRA患者に左手首検査や脈拍検査を含む徹底的な身体検査を行う。潜在的に閉塞したRAを有する患者のために、前腕ドップラー超音波またはパルスオキシメトリーを使用して手の血液供給のその後の評価を行う。</li>
	<li>TACEの後にすべての患者を綿密にフォローアップします。新しい腫瘍結節がCTスキャンで明らかであり、最初の病変が再血管化したように見えた場合は、別のTACE治療を行う。</li>
</ol>

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D., 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=Systematic+review+and+cost-benefit+analysis+of+radial+artery+access+for+coronary+angiography+and+intervention.">Systematic review and cost-benefit analysis of radial artery access for coronary angiography and intervention.</a> Circulation: Cardiovascular Quality and Outcomes. 5 (4), 454-462 (2012).</li><li>Shoji, 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=Stroke+After+Percutaneous+Coronary+Intervention+in+the+Era+of+Transradial+Intervention.">Stroke After Percutaneous Coronary Intervention in the Era of Transradial Intervention.</a> Circulation: Cardiovascular Interventions. 11 (12), 006761 (2018).</li><li>Jurga, 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=Cerebral+microembolism+during+coronary+angiography:+a+randomized+comparison+between+femoral+and+radial+arterial+access.">Cerebral microembolism during coronary angiography: a randomized comparison between femoral and radial arterial access.</a> Stroke. 42 (5), 1475-1477 (2011).</li><li>Bishay, V. 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=Transradial+Approach+for+Hepatic+Radioembolization:+Initial+Results+and+Technique.">Transradial Approach for Hepatic Radioembolization: Initial Results and Technique.</a> AJR: American Journal of Roentgenology. 207 (5), 1112-1121 (2016).</li><li>Bernat, 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=ST-segment+elevation+myocardial+infarction+treated+by+radial+or+femoral+approach+in+a+multicenter+randomized+clinical+trial:+the+STEMI-RADIAL+trial.">ST-segment elevation myocardial infarction treated by radial or femoral approach in a multicenter randomized clinical trial: the STEMI-RADIAL trial.</a> Journal of the American College of Cardiology. 63 (10), 964-972 (2014).</li></ol>

WZとZPYによるコンセプトとデザインを学ぶ。ND、ZHZおよびMJYによるデータの取得;ZPYによって資金を得た。著者らは関連する財務上の開示を持っていません。

TRA介入療法は近年、特に診断および介入的な心臓病の手順12において、世界的に著しく成長している。また、末梢血管疾患介入に対する注目度が高まっている。手続き上の成功率を損なうことなく、TRAから心臓介入は、TFA13,14と比較して出血および血管合併症の割合を効果的に減少させることができる。TFAと比較して、TRAは処置後?...

肝細胞癌(HCC)は非常に一般的な悪性腫瘍であり、世界で6番目に高い発生率を有する。また、世界1の癌死亡率の第2位の原因です。治癒療法を受けることができる患者の5%~20%に過ぎないため、眼下血栓塞栓術(TACE)は、未分除菌性HCC2患者に対して最も一般的な緩和治療である。TACEは、中間段階3でHCC患者に対して最も一般的かつ効果的な治療アプローチとして認識されている。トランスフェモラルアクセス(TFA)ケマンボラ化は、TACE4の最も一般的なアプローチです。しかし、アクセス部位での出血や主要な血管合併症5を含むTFA介入に関連するリスクがある。これらの合併症は、長期の入院とコストの増加につながります。さらに、TFAは少なくとも6時間の固定化を必要とし、患者に対する不快感および不満を増加させる。
経放射型アクセス (TRA) は、経皮的冠動脈介入 (PCI) で 20 年以上5,6に使用されてきた代替アプローチです。TRA PCIには、プロシージャの快適性の向上、アクセス部位関連の出血の減少、主要な血管合併症の減少、および死亡率7、8の減少など、いくつかの利点があります。放射状動脈(RA)は、その表面的な位置7のためにアクセスし、穿刺が容易である。止血は介入後に行うのは簡単であり、厳格な不モリ化は9 .心臓カテーテル法におけるTRA介入の証拠を奨励しているにもかかわらず、現在までに末梢疾患介入においてTRAを使用した研究はごくわずかである。悪性肝腫瘍に対するTRA介入はさらにまれである。ここで、TRA肝塞栓の臨床実現可能性および安全性が分析される。提供されるステップバイステップのTRAプロトコルに関する1つの機関の経験も説明されています。

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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Reagents</td>
			<td style="width:127px;"></td>
			<td style="width:123px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Embosphere</td>
			<td style="width:127px;">Merit</td>
			<td style="width:123px;">20173776165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Gelfoam</td>
			<td style="width:127px;">Alicon</td>
			<td style="width:123px;">20143771056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Heparin</td>
			<td style="width:127px;">Hepatunn</td>
			<td style="width:123px;">H51021209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Injection syringe</td>
			<td style="width:127px;">KDL</td>
			<td style="width:123px;">20163150518</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:141px;">Iodinated oil</td>
			<td style="width:127px;">Yantai Luyin Pharmaceutical Co.Ltd</td>
			<td style="width:123px;">H37022398</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:141px;">Lidocaine</td>
			<td style="width:127px;">Shandong Hualu Pharmaceutical Co.Ltd</td>
			<td style="width:123px;">H37022147</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:141px;">Lobaplatin</td>
			<td style="width:127px;">Hainan Changan International Pharmaceutical Co.Ltd</td>
			<td style="width:123px;">H20050308</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:141px;">Nitroglycerin</td>
			<td style="width:127px;">Brijing Yimin Pharmaceutical Co.Ltd</td>
			<td style="width:123px;">H11020289</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:141px;">Normal saline</td>
			<td style="width:127px;">Anhui Shuanghe Pharmaceutical Co.Ltd</td>
			<td style="width:123px;">H34023609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Pharmorubicin</td>
			<td style="width:127px;">Pfizer</td>
			<td style="width:123px;">H20000496</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Ultravist 370</td>
			<td style="width:127px;">Bayer</td>
			<td style="width:123px;">H20171333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:141px;">Hydrophilic Guide Wire</td>
			<td style="width:127px;">Merit</td>
			<td style="width:123px;">LWSTDA38180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Injection syringe</td>
			<td style="width:127px;">KDL</td>
			<td style="width:123px;">20163150518</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:141px;">Maestro Microcatheter</td>
			<td style="width:127px;">Merit</td>
			<td style="width:123px;">28MC24150SN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MPA1 (I) catheter</td>
			<td style="width:127px;">Cordis</td>
			<td style="width:123px;">451-406P0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Sheath Introducer</td>
			<td style="width:127px;">Merit</td>
			<td style="width:123px;">PSI-4F-11-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Steerable Guidewire</td>
			<td style="width:127px;">Merit</td>
			<td style="width:123px;">TNR2411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">TR Band</td>
			<td style="width:127px;">Terumo</td>
			<td style="width:123px;">XX*RF06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">DSA</td>
			<td style="width:127px;">Toshiba</td>
			<td style="width:123px;">INFX-9000V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Ultrasonic machine</td>
			<td style="width:127px;">SonoScape</td>
			<td style="width:123px;">20172231180</td>
			<td></td>
		</tr>
	</tbody>
</table>

transradial access chemoembolization for hepatocellular carcinoma patients

動脈間化学化(TACE)は、中間段階で肝細胞癌(HCC)の治療に最も一般的なモダリティである。TACEは、典型的には、トランスフェモラルアクセス(TFA)を介して行われる。しかしながら、経放射状アクセス(TRA)は、合併症および死亡率の低下による冠動脈介入において好ましい。TRAの利点をTACEに適用できるかどうかは調査が必要です。
単一のセンターでTRA TACEを受けている患者は、遡及的に研究のために登録された。手続き上の詳細、技術的な成功、放射状動脈閉塞(RAO)率、およびアクセス部位関連出血合併症を評価した。2017年10月から2018年10月にかけて、112人の患者が160回のTRA TACE処置を受けた。全体的な技術的成功率は95.0%(152/160)でした。TRAからTFAへのクロスオーバー率は1.9%でした。いずれの場合も、アクセス部位関連の出血合併症は見つからなかった。無症候性RA閉塞は3人の患者(2.7%)で起こった。TFAと比較して、TRAはアクセス部位関連の出血の合併症を減らしながら安全および患者の満足度を高めることができる。さらに、TRAの介入は、高齢、肥満、または出血合併症のリスクが高い患者に利益をもたらす可能性があります。

2017年10月から2018年10月にかけて、112人の患者が160回のTRA TACE処置を受け、全体的な技術的成功率は95.0%(152/160)でした。8つのケースが技術的な失敗に見られました。このうち5例は左RA穿刺障害によって引き起こされ、その後右RAアクセスでTACEが成功した。他の3つのケースは、缶めの失敗によって引き起こされ、その後、右FAアクセスへのクロスオーバーによる介入に成功しました。FAアクセスへ...

Watch this Scientific Journal Video about Transradial Access Chemoembolization for Hepatocellular Carcinoma Patients at JoVE.com

Transradial Access Chemoembolization for Hepatocellular Carcinoma Patients

動脈間化学塞栓術(TACE)は、肝細胞癌の中間段階における患者に対する標準的な治療法であり、典型的には大腿動脈へのアクセスを通じて行われる。経大的なアクセスと比較して、経放射状アクセス(TRA)は出血の合併症の率を減少させ、忍耐強い許容を改善できる。放射状動脈アクセスを介して動脈間化学式化を行う方法をここに提示する。

肝細胞癌患者に対する経放射型アクセスケミオボリ

Transarterial chemoembolization (TACE) is the most common modality for treatment of hepatocellular carcinoma (HCC) at the intermediate stage. TACE is ...

transradial-access-chemoembolization-for-hepatocellular-carcinoma

Research

JoVE Journal

Cancer Research

5.3K ビュー.  Fudan University. 動脈間化学塞栓術(TACE)は、肝細胞癌の中間段階における患者に対する標準的な治療法であり、典型的には大腿動脈へのアクセスを通じて行われる。経大的なアクセスと比較して、経放射状アクセス(TRA)は出血の合併症の率を減少させ、忍耐強い許容を改善できる。放射状動脈アクセスを介して動脈間化学式化を行う方法をここに提示する。

ビデオ: Transradial Access Chemoembolization for Hepatocellular Carcinoma Patients

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models.
In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects.

The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.

ヒト肝細胞癌細胞株HepG2細胞と三次元肝モデルに再増殖におけるウイルスベクターの研究

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector ...

がん研究

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.

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, ...

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4&#8242;,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

ホルマリン固定癌組織標本の腫瘍免疫研究の自動多重蛍光パネル

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

肺扁平上皮癌進行をモデル化する多次元共培養システム

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

ゼブラフィッシュにおける非アルコール性脂肪性肝疾患関連肝細胞癌の早期進行過程における肝微小環境の解析

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

肝細胞癌における腫瘍間質相互作用を調べる三次元スフェロイドモデル

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

肝細胞癌の同所性モデルマウスにおける非侵襲的PET/MRイメージング

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

Establishment and Characterization of Patient-Derived Xenograft Models of Anaplastic Thyroid Carcinoma and Head and Neck Squamous Cell Carcinoma

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its heterogeneity. Pharmacodynamic results based on PDX models are highly correlated with clinical practice. Anaplastic thyroid carcinoma (ATC) is the most malignant subtype of thyroid cancer, with strong invasiveness, poor prognosis, and limited treatment. Although the incidence rate of ATC accounts for only 2%-5% of thyroid cancer, its mortality rate is as high as 15%-50%. Head and neck squamous cell carcinoma (HNSCC) is one of the most common head and neck malignancies, with over 600,000 new cases worldwide each year. Herein, detailed protocols are presented to establish PDX models of ATC and HNSCC. In this work, the key factors influencing the success rate of model construction were analyzed, and the histopathological features were compared between the PDX model and the primary tumor. Furthermore, the clinical relevance of the model was validated by evaluating the in vivo therapeutic efficacy of representative clinically used drugs in the successfully constructed PDX models.

The present protocol establishes and characterizes a patient-derived xenograft (PDX) model of anaplastic thyroid carcinoma (ATC) and head and neck squamous cell carcinoma (HNSCC), as PDX models are rapidly becoming the standard in the field of translational oncology.

未分化甲状腺癌および頭頸部扁平上皮癌の患者由来異種移植片モデルの確立と特性評価

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its ...

標的同定と医薬品開発のためのHCCオルガノイド形成の最適化

Development and Optimization of A Human Hepatocellular Carcinoma Patient-Derived Organoid Model for Potential Target Identification and Drug Discovery

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents necessitate further research into its pathogenesis and treatment. Organoids, a novel model that closely resembles native tumor tissue and can be cultured in vitro, have garnered significant interest in recent years, with numerous reports on the development of organoid models for liver cancer. In this study, we have successfully optimized the procedure and established a culture protocol that enables the formation of larger-sized HCC organoids with stable passaging and culture conditions. We have comprehensively outlined each step of the procedure, covering the entire process of HCC tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation, and provided detailed precautions in this paper. These organoids exhibit genetic similarity to the original HCC tissues and can be utilized for diverse applications, including the identification of potential therapeutic targets for tumors and subsequent drug development.

著者スポットライト:患者由来オルガノイドを用いた肝臓がんの病因の調査 

We provide a comprehensive overview and refinement of existing protocols for hepatocellular carcinoma (HCC) organoid formation, encompassing all stages of organoid cultivation. This system serves as a valuable model for the identification of potential therapeutic targets and the assessment of drug candidate effectiveness.

ヒト肝細胞癌患者由来オルガノイドモデルの開発と最適化による標的同定と創薬

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents ...