参加に同意すること、病院と患者のすべての医療および補助的なスタッフに感謝しますガブリエル白狼、エダンズ ・ グループ (www.edanzediting.com/ac) このレポートの下書きを編集してから、博士に感謝いたします。本研究は、ゲノム研究プロジェクト (容湖とミズーリ州) は、山梨県からの助成金から、安田医療財団 (容湖) 補助金によって支えられました。

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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=Osimertinib+or+Platinum-Pemetrexed+in+EGFR+T790M-Positive+Lung+Cancer.">Osimertinib or Platinum-Pemetrexed in EGFR T790M-Positive Lung Cancer.</a> N Engl J Med. 376 (7), 629-640 (2017).</li><li>Wong, S. Q., 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=Targeted-capture+massively-parallel+sequencing+enables+robust+detection+of+clinically+informative+mutations+from+formalin-fixed+tumours.">Targeted-capture massively-parallel sequencing enables robust detection of clinically informative mutations from formalin-fixed tumours.</a> Sci rep. 13 (3), 3493 (2013).</li><li>Wong, S. 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著者が明らかに何もありません。

本研究ではチックを使用して臨床病理標本から腫瘍 DNA を取得するための代替方法を提案します。TIC 準備非常にシンプルかつ FFPE のメソッドは、特別な楽器10の要件なしと比較してより少ない時間を必要があります。(図 1) 2 日以内は、DNA の抽出に TIC 準備からすべての手順を完了できます。このメソッド従って遺伝学的検査を行うため所要時間が短くなります。特に、これは分子の解析に必要な日数の短縮に重要な利点を提供します。この短い所要時間では、すぐに分子標的薬を必要とする進行がん患者に適切な治療を提供することが出来ます。このメソッドは、したがって腫瘍における遺伝子変化の解析およびがん治療の分子標的薬の管理のための利点を提供します。
TIC DNA を用いた遺伝学的解析の成功のいくつかの重要なポイントがあります。腫瘍組織は化学療法などの治療や他の治療のため壊死かもしれません。したがって、注意は、可能な限りの腫瘍内の壊死のセクションからのサンプリングを避けるために TIC 標本作成に必要です。さらに、初期の顕微鏡的評価は非常に重要な手順です。さらに、腫瘍組織の乾燥を防ぐことが成功した TIC のサンプル準備のため必要です。腫瘍組織が乾燥すると、これがスライド ガラスの少ない接続されたセルに発生します。また、腫瘍組織の変性につながる乾燥細胞型腫瘍と形態を観察することは困難こと。
TIC の DNA を使用するいくつかの潜在的な利点があります。まず、我々 は顕微鏡を用いた最初の評価中に腫瘍の細胞型を確認できます。間質細胞、リンパ球などの正常細胞がティッシュ サンプルの豊富なこれらの正常細胞の汚染は腫瘍細胞の体細胞変異を検出することを防ぐと思います。サンプルの迅速な微視的評価は、腫瘍細胞性の評価と適切な腫瘍 DNA サンプルされた DNA の抽出の前に TIC 標本から得られるかどうかの推定に役立つことがあります。第二に、我々 の結果を示した、TIC DNA が質・量ともに。FFPE の DNA は、ターゲット シーケンス、エキソーム配列、全ゲノム シーケンス16,17,18,,1920など次世代シーケンス解析のために使用されています。アーカイブの FFPE DNA も日常的に遺伝子解析21,22, FFPE DNA はホルマリン固定の中に断片化することができますが使用されます。これは pcr や DNA 抽出、シーケンスの範囲、対象地域での均一性の欠如の原因で問題が発生することができます、シーケンス エラー23,24のリスクを高めます。対照的に、TIC DNA が断片化されていなければ、核酸に影響を少なくしてアルコール固定のため可能性があります。FFPE DNA のようなない TIC DNA を断片化しているこれらのサンプル次世代シーケンス解析、リアルタイム PCR およびデジタル PCR のより適切ななります。確かに、我々 は以前に見せました TIC 腫瘍標本から DNA を次世代シーケンス解析、"TIC seq"と呼ばれる方法で使用できる、結果は正確に腫瘍の体細胞変異13を捕えることができました。第三に、この手法は材料の広い範囲のために適当です。現在のレポートでは、標本を使用しました。手術組織、リンパ節転移に加えて気管支または内視鏡的生検は TIC DNA を準備するため使用できます。
結論としては、チックのサンプルを使用して質の高い腫瘍 DNA を準備するための簡易迅速法を提案する.このメソッドは遺伝学的解析の分野で新たな可能性を拡大し、臨床の場面における精密医学を促進するため。

次世代シーケンシング技術提供している研究者の遺伝の変化、メンデルの病気、遺伝的素因、がん1,2,3 のゲノム情報を解析の飛躍的な発展.がんゲノム アトラス (TCGA) と国際がんゲノム コンソーシアム (ICGC) は、一般的な癌4のいくつかの種類の遺伝子変異の同定を追求してきた。何百もの重要なドライバー遺伝子は正常に識別されているといくつかこれらの分子の薬物開発1,5,6のターゲットにされています。
臨床場面における FFPE 標本は病理診断とがんを含むさまざまな病気の分子テストのため通常使用されます。ただし、ホルマリンで固定過程では、DNA 蛋白質または DNA のクロスリンクが発生した、DNA 断片化を誘導しました。したがって、FFPE DNA サンプルは常に遺伝学的解析に適した DNA7,8,9の低質と量のため。また、FFPE 標本を準備するまでに数日かかるし、技術的なスキルのセクションを正確に準備する必要があります。したがって、高品質そのままな DNA を求める簡便かつ迅速な手法を開発することをお勧めします。
細胞診、病理組織学的診断のための代替方法です。細胞サンプル準備は簡単、以下の FFPE 準備10と比較して高価で、かつ高速なアプローチです。TIC 手法は、センチネル リンパ節と周辺組織のいくつかの年11,12術中迅速診断乳がん患者に行われています。ただし、チックの標本から高品質の DNA を得ることができるかどうかを検討して、その後の遺伝学的解析のため使用するほとんどの報告がありません。細胞診標本が一般的パパニコロー (Pap) またはギムザ染色、染色し、我々 は以前報告量と TIC 標本 (特にギムザ染色サンプル) から抽出した DNA の品質は FFPE から採取した試料よりも優れて組織13。Pap 染色に比べると、少ない汚損プロシージャを必要とする利点を有するギムザ染色.Pap 染色の後、サンプルを固定されているし、ステンド グラス、彼らマウントしますする必要がある媒体 (例えばMalinol)、腫瘍細胞、正常細胞、炎症細胞を顕微鏡など、サンプルの内容を区別するためのマウント。Pap の標本は、取付手順なし準備だない試料を乾燥するために顕微鏡下で細胞を観察することはほとんど不可能。比較では、乾燥した状態で観察することができますギムザ染色、したがって、取り付け手順は迅速に携帯電話評価する必要ありません。レーザーマイクロダイ セクション、ギムザ染色より適切な乾燥標本が必要なため。
本報告では TIC ギムザ染色標本を準備するための簡易迅速法を導入し、チックが dna の FFPE 標本と比較してより良いソースであることを示す.

<table border="0" cellpadding="0" cellspacing="0" width="796">
	<tbody>
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			<td height="41" style="height:41px;width:283px;">FINE FROST white20 micro slide glass</td>
			<td style="width:191px;">Matsunami Glass ind, Ltd</td>
			<td style="width:143px;">SFF-011</td>
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			<td>Thermo Fisher Scientific</td>
			<td>LCM0522</td>
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			<td>Muto Pure Chemicals</td>
			<td>20571</td>
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			<td height="41" style="height:41px;">Cyto Quick B solution</td>
			<td>Muto Pure Chemicals</td>
			<td>20581</td>
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			<td height="41" style="height:41px;">May-Grunwald Solution</td>
			<td>Muto Pure Chemicals</td>
			<td>15053</td>
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			<td height="41" style="height:41px;">Giemsa solution</td>
			<td>Muto Pure Chemicals</td>
			<td>15002</td>
			<td></td>
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			<td height="41" style="height:41px;">QIAamp DNA FFPE tissue kit</td>
			<td>Qiagen</td>
			<td>56404</td>
			<td></td>
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			<td height="41" style="height:41px;">TaqMan Fast Advanced Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4444557</td>
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			<td height="41" style="height:41px;">TaqMan RNase P Detection Reagents Kit&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4316831</td>
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			<td height="41" style="height:41px;">TaqMan Assay from FFPE DNA QC Assay v2</td>
			<td>Thermo Fisher Scientific</td>
			<td>4324034</td>
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			<td height="41" style="height:41px;">MicroAmp Fast Optical 96-Well Reaction Plate&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4346907</td>
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			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4311971</td>
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			<td style="width:191px;">TITEC</td>
			<td style="width:143px;">0027765-000</td>
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			<td height="41" style="height:41px;width:283px;">ViiA 7 Real-Time PCR System</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">VIIA7-03</td>
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			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4468802</td>
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			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4475346</td>
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			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4480442</td>
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			<td>4471250</td>
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			<td>4484177</td>
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			<td>4488150</td>
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			<td height="41" style="height:41px;width:283px;">Ion PGM System</td>
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			<td>PGM11-001</td>
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			<td height="41" style="height:41px;width:283px;">Ion PGM Wash 2 Bottle kit</td>
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			<td>A25591</td>
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			<td height="41" style="height:41px;width:283px;">16-position Magnetic Stand</td>
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			<td height="41" style="height:41px;width:283px;">Nonstick, RNase-free Microfuge Tubes, 1.5 mL (Low binding tube)</td>
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			<td height="41" style="height:41px;width:283px;">Nuclease-free water</td>
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			<td height="41" style="height:41px;width:283px;">MicroAmp&#8482; Optical 96-well Reaction Plates</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
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			<td height="41" style="height:41px;width:283px;">MicroAmp&#8482; Clear Adhesive Film</td>
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			<td height="41" style="height:41px;width:283px;">Agencourt&#8482; AMPure&#8482; XP Kit</td>
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			<td height="41" style="height:41px;width:283px;">Sodium hydroxide (10M)</td>
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			<td>METTLER TOLEDO</td>
			<td>17014393</td>
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			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-10XLS+</td>
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			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-100XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014384</td>
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			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-200XLS+</td>
			<td>METTLER TOLEDO</td>
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			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-1000XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014382</td>
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			<td height="41" style="height:41px;">petit-change</td>
			<td>WAKEN</td>
			<td>MODEL8864</td>
			<td style="width:180px;">Mini centrifuge</td>
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			<td height="41" style="height:41px;">petit-incubator</td>
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			<td>WKN-2290</td>
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			<td height="41" style="height:41px;width:283px;">SensiCare Powder-free Nitrile Exam Gloves</td>
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			<td height="41" style="height:41px;width:283px;">Sterile gauze</td>
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			<td height="41" style="height:41px;width:283px;">Refrigerator MediCool</td>
			<td style="width:191px;">SANYO</td>
			<td style="width:143px;">MPR-312DCN-PJ</td>
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			<td height="41" style="height:41px;width:283px;">FEATHER TRIMMING BLAD</td>
			<td style="width:191px;">FEATHER</td>
			<td style="width:143px;">No.130</td>
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			<td height="41" style="height:41px;width:283px;">FEATHER TRIMMING BLAD</td>
			<td style="width:191px;">FEATHER</td>
			<td style="width:143px;">No.260</td>
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			<td height="41" style="height:41px;width:283px;">FEATHER&#160; S</td>
			<td style="width:191px;">FEATHER</td>
			<td style="width:143px;">FA-10</td>
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			<td height="41" style="height:41px;width:283px;">Vortex Genius 3</td>
			<td style="width:191px;">IKA</td>
			<td>41-0458&#160;</td>
			<td style="width:180px;">Vortex mixer</td>
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			<td height="41" style="height:41px;width:283px;">Pincette</td>
			<td style="width:191px;">NATSUME</td>
			<td style="width:143px;">A-5</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">1.5 mL microtube</td>
			<td style="width:191px;">BIOBIK</td>
			<td style="width:143px;">RC-0150</td>
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	</tbody>
</table>

simple and rapid method to obtain high-quality tumor dna from clinical-pathological specimens using touch imprint cytology

管理およびがん患者のための特定の分子標的薬の治療前にがんの突然変異の状態を確認する重要です。臨床設定では、ホルマリン固定パラフィン包埋 (FFPE) 組織は、遺伝学的検査のため活躍しています。ただし、FFPE の DNA は、ホルマリン固定過程における断片化や破損は、一般的に。したがって、FFPE DNA は時々 低品質と量の DNA のための遺伝学的検査の適切ではないです。顕微鏡下で観察することができますタッチ捺印細胞診がん細胞からゲノム DNA を取得する (TIC) の方法をご紹介します。細胞形態と癌細胞数は、チックの標本を使用して評価できます。さらに、2 日以内 TIC サンプルからゲノム DNA の抽出を完了ことができます。合計金額、このメソッドを使用して得られた TIC DNA の品質 FFPE DNA のそれよりも高かった。この迅速かつ簡単な方法では、研究者 (例えば、次世代シーケンス解析、デジタル PCR および量的なリアルタイム PCR) 遺伝学的検査の高品質 DNA を取得し、結果をレポートのターンアラウンド時間を短縮することができます。

図 1は、DNA の抽出に TIC 標本作製から全体のプロセスを示しています。特に、TIC サンプルからゲノム DNA を取得するのみ 2 日間を要します。スライド処理の前に腫瘍ストレージの効果を評価しました。組織検体はすぐにスライドに触れられたとき、および組織は、生理食塩水湿らせた滅菌・ ガーゼ 1 h (図 2) のために保たれた腫瘍細胞がスライド ガラス上に接続されていたことがわかった。しかし、組織は、室温で保たれた、時腫瘍細胞添付されませんでしたよくスライドに。準備されたスライドは、3 カ月、4 ° C で格納できます。したがって、乾燥のための標本を許可しないことが重要です。
我々 は本手法の有用性を検討したし、FFPE を用いて標本と比較します。14 の腫瘍の標本、TIC と FFPE サンプルの調製し、腫瘍内容と純度が TIC 標本から評価されることを確認しました。後、ギムザ染色を行い、顕微鏡で腫瘍細胞と腫瘍の形態の数で評価できます。したがって、定期的に腫瘍細胞を評価し、その後 DNA の品質チェックを実行することができた。
DNA 量と質は、量的なリアルタイム PCR13,14,15によって推定されました。我々 は DNA 量の絶対量とゲノム DNA の劣化度の指標である RQ 値を決定しました。FFPE 標本 (表 1) と比較して TIC 試料を用いた DNA の収量が達成されたことがわかった。また、TIC DNA の RQ 値が大幅に比べ FFPE DNA の (図 3p = 10-82-スチューデントの両側tテスト x 2.3)。チックと異なる腫瘍から抽出した TIC DNA は FFPE DNA (図 3) と比較して質の高いことがわかった FFPE DNA の RQ 値についても検討します。TIC DNA は FFPE DNA のよりも断片化が示唆されました。
次に TIC DNA を次世代シーケンス解析に使用できるかどうかを評価します。FFPE と TIC DNA は、大腸癌や転移性肝癌患者 (図 4 a) から得られるから調製しました。PCR 増幅とライブラリの準備をがんホット スポット パネルを使用して実施し、その後ターゲット シーケンスを行った。結果として、 APC Q1367 * 識別された FFPE のサイト 1 (表 2) から抽出された TIC DNA。さらに、両方の FFPE のAPC S1356 *、 KRAS G12D およびTP53 M237I を検出し、チックの DNA は、2、3、および 4 (表 2) サイトから抽出しました。同一体細胞突然変異が同じ腫瘍部位 (図 4 bと表 2) から調製したペアの FFPE と TIC DNA サンプルで識別されたことが示唆されました。特に、大腸癌 (サイト 2、サイト 1 ではない) とサイト 2 から腫瘍クローン転移肝臓 (図 4 bと表 2) ことを示唆している転移性肝癌の 2 つのサンプルの間同じ体細胞突然変異が検出されました。一緒にこれらの調査結果は TIC DNA が高い品質、次世代シーケンシングなど遺伝学的検査の広い範囲に適していますをお勧めします。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56943/56943fig1.jpg"> チックの試料から DNA を得るため 1. 概略図を図します。腫瘍組織は TIC サンプルを準備してスライド ガラスに触れられます。腫瘍形態, 顕微鏡下で内容を評価した後腫瘍 DNA を抽出、遺伝子検査に使用します。チックを使用することにより、腫瘍 DNA 得られる臨床病理標本 2 日以内。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56943/56943fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56943/56943fig2.jpg"> 図 2。TIC のサンプルの準備。切除腫瘍の標本がすぐに生理食塩水湿らせた滅菌・ ガーゼ 1 h (中央) のために通常のガラス スライド (左側のパネル) に触れるし、(右) 1 時間室温で保管します。サンプル #1 は肝細胞癌とサンプル 2 は乳がんだった。顕微鏡写真は顕微鏡にデジタル カメラを使用して撮影されました。スケール バー: 100 μ m.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56943/56943fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56943/56943fig3.jpg"> 図 3。定量的リアルタイム PCR 解析によって推定される DNA 品質チェックします。チックと FFPE DNA は大腸からの 14 の腫瘍組織から抽出した (n = 8)、胃 (n = 4)、および転移性肝癌 (n = 2)。TIC ギムザと彼 FFPE サンプル間の相対的な定量化スコアの比較。TIC DNA の RQ 値した FFPE DNA のそれよりも有意に高かった。2 つのグループ間の統計的解析を行ったとp-値を Excel を使用して対になっていない 2 尾 Student のt検定によって算出しました。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56943/56943fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56943/56943fig4.jpg"> 図 4。次世代シーケンス解析データ TIC と FFPE の DNA を使用しています。(A) マクロスコ ピックおよび顕微鏡画像。TIC ギムザとサンプル (サイト 3 および 4) 大腸癌 (サイト 1 と 2) と転移性肝臓癌から染色 FFPE 彼の代表的なイメージ。マクロ画像のスケール バー: 1 cm、顕微鏡画像のスケール バー: 100 μ m (B) 熱地図各腫瘍部位の体細胞突然変異の分布を示す (n = 8)。チックと FFPE DNA 標本間で同じ変異が検出されました。対立の分数の値は 100% (ピンク) 1% (ライトピンク) から卒業カラー スケールで示されます。灰色の列は、特定の突然変異を認めなかった.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56943/56943fig4large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td></td>
			<td colspan="4">TIC ギムザ (n = 14)</td>
			<td></td>
			<td colspan="4">FFPE 彼 (n = 14)</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td colspan="2">合計 DNA (ng)</td>
			<td></td>
			<td></td>
			<td></td>
			<td colspan="2">合計 DNA (ng)</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>サンプル</td>
			<td>腫瘍部位</td>
			<td>短い</td>
			<td>長い</td>
			<td></td>
			<td>RQ</td>
			<td></td>
			<td>短い</td>
			<td>長い</td>
			<td></td>
			<td>RQ</td>
		</tr>
<tr>
<td>"Site1"</td>
			<td>コロン</td>
			<td>1495</td>
			<td>1247</td>
			<td></td>
			<td>0.83</td>
			<td></td>
			<td>939</td>
			<td>349</td>
			<td></td>
			<td>0.37</td>
		</tr>
<tr>
<td>Site2</td>
			<td>コロン</td>
			<td>991</td>
			<td>1057</td>
			<td></td>
			<td>1.07</td>
			<td></td>
			<td>556</td>
			<td>204</td>
			<td></td>
			<td>0.37</td>
		</tr>
<tr>
<td>サイト 3</td>
			<td>肝臓</td>
			<td>467</td>
			<td>511</td>
			<td></td>
			<td>1.09</td>
			<td></td>
			<td>130</td>
			<td>39</td>
			<td></td>
			<td>0.3</td>
		</tr>
<tr>
<td>サイト 4</td>
			<td>肝臓</td>
			<td>2172</td>
			<td>2115</td>
			<td></td>
			<td>0.97</td>
			<td></td>
			<td>488</td>
			<td>127</td>
			<td></td>
			<td>0.26</td>
		</tr>
<tr>
<td>Site5</td>
			<td>コロン</td>
			<td>749</td>
			<td>598</td>
			<td></td>
			<td>0.8</td>
			<td></td>
			<td>529</td>
			<td>205</td>
			<td></td>
			<td>0.39</td>
		</tr>
<tr>
<td>Site6</td>
			<td>胃</td>
			<td>330</td>
			<td>286</td>
			<td></td>
			<td>0.86</td>
			<td></td>
			<td>211</td>
			<td>98</td>
			<td></td>
			<td>0.46</td>
		</tr>
<tr>
<td>Site7</td>
			<td>胃</td>
			<td>636</td>
			<td>499</td>
			<td></td>
			<td>0.78</td>
			<td></td>
			<td>154</td>
			<td>84</td>
			<td></td>
			<td>0.55</td>
		</tr>
<tr>
<td>Site8</td>
			<td>胃</td>
			<td>27</td>
			<td>27</td>
			<td></td>
			<td>1.01</td>
			<td></td>
			<td>135</td>
			<td>81</td>
			<td></td>
			<td>0.6</td>
		</tr>
<tr>
<td>Site9</td>
			<td>コロン</td>
			<td>1986</td>
			<td>1611</td>
			<td></td>
			<td>0.81</td>
			<td></td>
			<td>476</td>
			<td>163</td>
			<td></td>
			<td>0.34</td>
		</tr>
<tr>
<td>Site10</td>
			<td>コロン</td>
			<td>280</td>
			<td>218</td>
			<td></td>
			<td>0.78</td>
			<td></td>
			<td>209</td>
			<td>83</td>
			<td></td>
			<td>0.39</td>
		</tr>
<tr>
<td>Site11</td>
			<td>コロン</td>
			<td>1546</td>
			<td>575</td>
			<td></td>
			<td>0.37</td>
			<td></td>
			<td>366</td>
			<td>159</td>
			<td></td>
			<td>0.43</td>
		</tr>
<tr>
<td>Site12</td>
			<td>胃</td>
			<td>1501</td>
			<td>1200</td>
			<td></td>
			<td>0.8</td>
			<td></td>
			<td>274</td>
			<td>132</td>
			<td></td>
			<td>0.48</td>
		</tr>
<tr>
<td>Site13</td>
			<td>コロン</td>
			<td>1556</td>
			<td>1404</td>
			<td></td>
			<td>0.9</td>
			<td></td>
			<td>326</td>
			<td>179</td>
			<td></td>
			<td>0.55</td>
		</tr>
<tr>
<td>Site14</td>
			<td>コロン</td>
			<td>1565</td>
			<td>1210</td>
			<td></td>
			<td>0.77</td>
			<td></td>
			<td>680</td>
			<td>295</td>
			<td></td>
			<td>0.43</td>
		</tr>
<tr>
<td></td>
			<td>Mean±SD</td>
			<td>1093±682</td>
			<td>897±600</td>
			<td></td>
			<td>0.85±0.18</td>
			<td></td>
			<td>391±253</td>
			<td>157±86</td>
			<td></td>
			<td>0.42±0.10</td>
		</tr>
</tbody></table>テーブル 1。DNA 品質データ。ペア チック (n = 14) FFPE 標本 (n = 14) 胃ガン、肝臓、結腸から準備されました。TIC 染色ギムザと、ヘマトキシリンとエオシン (HE) 染色 FFPE サンプル。DNA サンプルが得られ、RNaseP 軌跡を増幅 2 つのプライマー組で量的なリアルタイム PCR による定量 (長い私アンプリコン (268 bp) と短い増幅 (87 bp))。RQ 価値は以下の通り計算された: 長い私アンプリコンの平均値が短い私アンプリコンの平均値を分割します。SD、標準偏差;RQ、相対定量
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>サンプル名</td>
			<td>場所</td>
			<td>準備</td>
			<td>遺伝子シンボル</td>
			<td>突然変異</td>
			<td colspan="2">位置</td>
			<td>参照</td>
			<td>バリアント</td>
			<td>コーディング</td>
			<td>カバレッジ</td>
			<td>対立遺伝子の割合</td>
		</tr>
<tr>
<td>大腸癌</td>
			<td>サイト 1</td>
			<td>FFPE</td>
			<td>APC</td>
			<td>Q1367 *</td>
			<td colspan="2">chr5:112175390</td>
			<td>C</td>
			<td>T</td>
			<td>c.4099C > T</td>
			<td>1999</td>
			<td>45%</td>
		</tr>
<tr>
<td>大腸癌</td>
			<td>サイト 1</td>
			<td>TIC</td>
			<td>APC</td>
			<td>Q1367 *</td>
			<td colspan="2">chr5:112175390</td>
			<td>C</td>
			<td>T</td>
			<td>c.4099C > T</td>
			<td>1011</td>
			<td>72%</td>
		</tr>
<tr>
<td>大腸癌</td>
			<td>サイト 2</td>
			<td>FFPE</td>
			<td>APC</td>
			<td>S1356 *</td>
			<td colspan="2">chr5:112175358</td>
			<td>C</td>
			<td>A</td>
			<td>c.4067C > A</td>
			<td>1968</td>
			<td>77%</td>
		</tr>
<tr>
<td>大腸癌</td>
			<td>サイト 2</td>
			<td>FFPE</td>
			<td>KRAS</td>
			<td>G12D</td>
			<td colspan="2">chr12:25398284</td>
			<td>C</td>
			<td>T</td>
			<td>c.35G > A</td>
			<td>1993</td>
			<td>52%</td>
		</tr>
<tr>
<td>大腸癌</td>
			<td>サイト 2</td>
			<td>FFPE</td>
			<td>TP53</td>
			<td>M237I</td>
			<td colspan="2">chr17:7577570</td>
			<td>C</td>
			<td>A</td>
			<td>c.711G > T</td>
			<td>1991</td>
			<td>73%</td>
		</tr>
<tr>
<td>大腸癌</td>
			<td>サイト 2</td>
			<td>TIC</td>
			<td>APC</td>
			<td>S1356 *</td>
			<td colspan="2">chr5:112175358</td>
			<td>C</td>
			<td>A</td>
			<td>c.4067C > A</td>
			<td>1967</td>
			<td>89%</td>
		</tr>
<tr>
<td>大腸癌</td>
			<td>サイト 2</td>
			<td>TIC</td>
			<td>KRAS</td>
			<td>G12D</td>
			<td colspan="2">chr12:25398284</td>
			<td>C</td>
			<td>T</td>
			<td>c.35G > A</td>
			<td>1994</td>
			<td>56%</td>
		</tr>
<tr>
<td>大腸癌</td>
			<td>サイト 2</td>
			<td>TIC</td>
			<td>TP53</td>
			<td>M237I</td>
			<td colspan="2">chr17:7577570</td>
			<td>C</td>
			<td>A</td>
			<td>c.711G > T</td>
			<td>1995</td>
			<td>86%</td>
		</tr>
<tr>
<td>転移性肝癌</td>
			<td>サイト 3</td>
			<td>FFPE</td>
			<td>APC</td>
			<td>S1356 *</td>
			<td colspan="2">chr5:112175358</td>
			<td>C</td>
			<td>A</td>
			<td>c.4067C > A</td>
			<td>1974</td>
			<td>92%</td>
		</tr>
<tr>
<td>転移性肝癌</td>
			<td>サイト 3</td>
			<td>FFPE</td>
			<td>KRAS</td>
			<td>G12D</td>
			<td colspan="2">chr12:25398284</td>
			<td>C</td>
			<td>T</td>
			<td>c.35G > A</td>
			<td>1995</td>
			<td>62%</td>
		</tr>
<tr>
<td>転移性肝癌</td>
			<td>サイト 3</td>
			<td>FFPE</td>
			<td>TP53</td>
			<td>M237I</td>
			<td colspan="2">chr17:7577570</td>
			<td>C</td>
			<td>A</td>
			<td>c.711G > T</td>
			<td>1296</td>
			<td>91%</td>
		</tr>
<tr>
<td>転移性肝癌</td>
			<td>サイト 3</td>
			<td>TIC</td>
			<td>APC</td>
			<td>S1356 *</td>
			<td colspan="2">chr5:112175358</td>
			<td>C</td>
			<td>A</td>
			<td>c.4067C > A</td>
			<td>1964</td>
			<td>97%</td>
		</tr>
<tr>
<td>転移性肝癌</td>
			<td>サイト 3</td>
			<td>TIC</td>
			<td>KRAS</td>
			<td>G12D</td>
			<td colspan="2">chr12:25398284</td>
			<td>C</td>
			<td>T</td>
			<td>c.35G > A</td>
			<td>1993</td>
			<td>64%</td>
		</tr>
<tr>
<td>転移性肝癌</td>
			<td>サイト 3</td>
			<td>TIC</td>
			<td>TP53</td>
			<td>M237I</td>
			<td colspan="2">chr17:7577570</td>
			<td>C</td>
			<td>A</td>
			<td>c.711G > T</td>
			<td>1998</td>
			<td>95%</td>
		</tr>
<tr>
<td>転移性肝癌</td>
			<td>サイト 4</td>
			<td>FFPE</td>
			<td>APC</td>
			<td>S1356 *</td>
			<td colspan="2">chr5:112175358</td>
			<td>C</td>
			<td>A</td>
			<td>c.4067C > A</td>
			<td>1965</td>
			<td>93%</td>
		</tr>
<tr>
<td>転移性肝癌</td>
			<td>サイト 4</td>
			<td>FFPE</td>
			<td>KRAS</td>
			<td>G12D</td>
			<td colspan="2">chr12:25398284</td>
			<td>C</td>
			<td>T</td>
			<td>c.35G > A</td>
			<td>1992</td>
			<td>60%</td>
		</tr>
<tr>
<td>転移性肝癌</td>
			<td>サイト 4</td>
			<td>FFPE</td>
			<td>TP53</td>
			<td>M237I</td>
			<td colspan="2">chr17:7577570</td>
			<td>C</td>
			<td>A</td>
			<td>c.711G > T</td>
			<td>1993</td>
			<td>92%</td>
		</tr>
<tr>
<td>転移性肝癌</td>
			<td>サイト 4</td>
			<td>TIC</td>
			<td>APC</td>
			<td>S1356 *</td>
			<td colspan="2">chr5:112175358</td>
			<td>C</td>
			<td>A</td>
			<td>c.4067C > A</td>
			<td>1960</td>
			<td>94%</td>
		</tr>
<tr>
<td>転移性肝癌</td>
			<td>サイト 4</td>
			<td>TIC</td>
			<td>KRAS</td>
			<td>G12D</td>
			<td colspan="2">chr12:25398284</td>
			<td>C</td>
			<td>T</td>
			<td>c.35G > A</td>
			<td>1992</td>
			<td>62%</td>
		</tr>
<tr>
<td>転移性肝癌</td>
			<td>サイト 4</td>
			<td>TIC</td>
			<td>TP53</td>
			<td>M237I</td>
			<td colspan="2">chr17:7577570</td>
			<td>C</td>
			<td>A</td>
			<td>c.711G > T</td>
			<td>1995</td>
			<td>95%</td>
		</tr>
</tbody></table>表 2。ペアの FFPE とターゲット シーケンス解析によって検出された TIC DNA の突然変異の比較。チックのペアと FFPE サンプルの調製 2 大腸癌 (サイト 1 と 2) と 2 転移性肝臓癌 (サイト 3 および 4)。これらの DNA のサンプルで対象のシーケンスを行い体細胞突然変異が同定されました。チックと FFPE DNA 標本間同一変異プロファイルであった。

Watch this Scientific Journal Video about Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology at JoVE.com

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

腫瘍組織から高品質 DNA を取得は、次世代シーケンシングによる遺伝子の変異を分析するための重要な第一歩です。この記事では腫瘍細胞を豊かにし、押印細胞診標本のタッチから無傷の DNA を取得する簡単かつ迅速な方法を提案する.

タッチ捺印細胞診を用いた臨床病理標本から質の高い腫瘍 DNA を取得する簡易法

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

simple-rapid-method-to-obtain-high-quality-tumor-dna-from-clinical

Nanyang Technological University

Research

JoVE Journal

Cancer Research

10.8K ビュー.  Yamanashi Central Hospital. 腫瘍組織から高品質 DNA を取得は、次世代シーケンシングによる遺伝子の変異を分析するための重要な第一歩です。この記事では腫瘍細胞を豊かにし、押印細胞診標本のタッチから無傷の DNA を取得する簡単かつ迅速な方法を提案する.

ビデオ: Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

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

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

キャプチャと血液から生存循環腫瘍細胞のリリース

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

循環腫瘍細胞を取り込むため医療用ワイヤー線を用いた腫瘍細胞の特性解析: 蛍光抗体法や DNA 魚に基づく 3 D アプローチ

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

放射性核種蛍光レポーターの遺伝子齧歯動物腫瘍モデルで腫瘍の進行を追跡するイメージング

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

アンチ PD L1 抗体は生検で非小細胞肺癌患者からの細胞のサンプル集中 22 3 を使用して

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

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

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

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

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

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

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者の生体流体における腫瘍関連循環DNAの検出とモニタリング

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

患者前立腺癌骨転移標本及びその異種移植片に由来する3次元(3D)オルガノイドの確立と解析

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the spheroid at the cellular level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. This protocol presents a new method of mounting and clearing spheroids that address these issues while allowing for confocal analysis of the inner structure of spheroids. In contrast to existing approaches, this protocol provides for rapid mounting and clearing of a large number of spheroids using standard equipment and laboratory supplies. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimizes size distortion common to other similar techniques. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional (3D) confocal images. The present article elaborates how the method yields high-quality two- and 3D images that provide information about inter-cell variability and inner spheroid structure.

Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing.

腫瘍スフェロイドの半ハイスループット分析のための迅速な光学的クリアリング

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the ...

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of mucin-secreting tumor cells in the peritoneal cavity. PMP can arise from various types of cancers, including appendiceal, ovarian, and colorectal, though appendiceal neoplasms are by far the most common etiology. PMP is challenging to study due to its (1) rarity, (2) limited murine models, and (3) mucinous, acellular histology. The method presented here allows real-time visualization and interrogation of these tumor types using patient-derived ex vivo organotypic slices in a preparation where the tumor microenvironment (TME) remains intact. In this protocol, we first describe the preparation of tumor slices using a vibratome and subsequent long-term culture. Second, we describe confocal imaging of tumor slices and how to monitor functional readouts of viability, calcium imaging, and local proliferation. In short, slices are loaded with imaging dyes and are placed in an imaging chamber that can be mounted onto a confocal microscope. Time-lapse videos and confocal images are used to assess the initial viability and cellular functionality. This procedure also explores translational cellular movement, and paracrine signaling interactions in the TME. Lastly, we describe a dissociation protocol for tumor slices to be used for flow cytometry analysis. Quantitative flow cytometry analysis can be used for bench-to-bedside therapeutic testing to determine changes occurring within the immune landscape and epithelial cell content.

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

We describe a protocol for the production, culture, and visualization of human cancers, which have metastasized to the peritoneal surfaces. Resected tumor specimens are cut using a vibratome and cultured on permeable inserts for increased oxygenation and viability, followed by imaging and downstream analyses using confocal microscopy and flow cytometry.