この研究は、助成金なしによってサポートされていました。 SNUBH研究基金から14-2014-017。 著者は、この原稿の彼のプロボノ編集のためにJ.パトリック・バロン、名誉教授、東京大学医科大学と非常勤講師、盆唐ソウル大病院にお世話になっています。

注：すべての動物実験プロトコルは、盆唐ソウル大病院（BA1308-134 / 072から01）の施設内動物管理使用委員会によって承認されました。 ヒアルロン酸セラミドの1の合成（HACE） <ol><li>二重蒸留水（DDW）の60ミリリットルでヒアルロン酸の12.21ミリモル（HA）オリゴマー及びテトラn個の -butylammonium水酸化物（TBA）の9.77ミリモルを可溶化します。 30分間撹拌しました。 </li><li> DS-Y30リンカーを合成するために、テトラヒドロフラン25ミリリットル（THF）中のDS-Y30セラミドの8.59ミリモルおよびトリエチルアミン9.45ミリモルを溶解します。 THF中の4-クロロメチルベンゾイルクロリドの8.59ミリモルと混合。 60℃で6時間撹拌します。 </li><li> THFの混合物及びアセトニトリルでDS-Y30リンカーの合成されたHA-TBAの8.10ミリモル及び0.41ミリモル溶解（4：1、v / v）でした。 40℃で5時間撹拌します。 </li><li>フィルター剤で濾過することにより不純物を除去し、真空蒸着法により、有機溶媒を除去します。 （透析膜を使用して生成物を精製分子量カットオフ：3.5キロダルトン）および凍結乾燥。 </li></ol>ナノ粒子の作製<ol><li> HB 1mgのジメチルスルホキシド（DMSO）0.5mlのパクリタキセル1mgを溶解し、ボルテックス混合し、5分間によってDDW 0.5mlでブレンド。その後、さらに5分間ボルテックス混合することにより、その混合物中のHACEを可溶化します。 </li><li>窒素ガスの穏やかな流れの下で4時間、70℃で溶剤、熱を除去するために。 </li><li> DDW 1mlでHACE、HBからなる膜、及びパクリタキセルを再懸濁します。封入されていない薬物を除去するために、シリンジフィルター（孔径0.45μm）でフィルタします。 </li></ol> 3. インビトロ光毒性<ol><li>肺癌細胞株中のナノ粒子の取り込み<ol><li> RPMI-1640培地を含む10％の（v / v）のウシ胎児血清および1％（w / v）のペニシリン - ストレプトマイシンを準備します。 </li><li> 1×10 5細胞/ウェルの各々について（三連の密度で24ウェル細胞培養プレート中の種子A549細胞グループ）。加湿5％CO 2及び95％空気雰囲気中37℃で24時間インキュベートします。 </li><li>細胞付着後に、培地を除去し、リン酸緩衝生理食塩水（PBS）を1ml添加して細胞を洗浄します。 </li><li> 2μM/ mlのHBの最終濃度になるようにPBS中のナノ粒子を溶解させます。その後、暗所で4時間、各ウェルにPBS、空のNP、HB-のNP、またはHB-P-NPの1ミリリットルで細胞を培養します。 </li><li>ソリューションのすべてを削除し、冷PBSの1ミリリットルを追加することによって、細胞を洗浄。もう一度洗浄工程を繰り返します。新鮮な培地を追加します。 </li></ol></li><li>細胞生存率アッセイ<ol><li> （ウェルにPDTファイバからの距離から1cmで）PDT繊維の下で細胞培養プレートを置きます。レーザー安全メガネを着用し、種々の時間暗所でPDTレーザー（630 nmで、400ミリワット/ cm 2）を用いて細胞を照らす：0、5、10、20、30、および40秒（0、2、4 、8、12、16 J / cm 2）でした。その後、暗所で24時間細胞をインキュベートします。 </li><li>培地を吸引し、冷PBSを1mlを加えることによって細胞を洗浄します。もう一度洗浄工程を繰り返します。 </li><li>各ウェルに細胞毒性測定溶液10μlを加えます。 2時間暗所でインキュベートします。 </li><li>マイクロプレートリーダーを用いて450nmで吸光度を測定します。 </li></ol></li><li>顕微分析<ol><li> （ウェルにPDTファイバからの距離から1cmで）PDT繊維の下で細胞培養プレートを置きます。レーザー安全メガネを着用し、0、20、または40秒（0、8、または16 J / cm 2）を、暗所でのPDTレーザー（630 nmで、400ミリワット/ cm 2）を用いて細胞を照らします。その後、暗所で24時間細胞をインキュベートします。 </li><li>培地を吸引し、冷PBSを1mlを加えることによって細胞を洗浄します。もう一度洗浄工程を繰り返します。 </li><li>アネキシンV-FITCを50μlと4 &#39;、6-ジアミジノ-2-フェニルインドール（DAPI、/ mlの1.5μgの）の50μLを加えます。静かにプレートを振る、暗所で室温で15分間静置します。 注：アネキシンV-FIを使用してください蛍光顕微鏡キットからTC。 </li><li>冷PBSの1ミリリットルを追加することによって、細胞を洗浄。もう一度洗浄工程を繰り返します。新鮮なPBSで細胞を保管してください。 100Xの倍率で光学顕微鏡を用いて、アポトーシス細胞を識別します。 </li></ol></li><li> （FACS）分析、蛍光活性化細胞選別<ol><li> （ウェルにPDTファイバからの距離から1cmで）PDT繊維の下で細胞培養プレートを置きます。レーザー安全メガネを着用し、0、20、または40秒（0、8、または16 J / cm 2）をするためのPDTレーザー（630 nmで、400ミリワット/ cm 2）を用いて細胞を照らします。その後、暗所で24時間細胞をインキュベートします。 </li><li>培地を吸引し、冷PBSを1mlを加えることによって細胞を洗浄します。もう一度洗浄工程を繰り返します。 </li><li>転送100μlを、1×結合緩衝液1mlに細胞を再懸濁（0.1MのHepes / NaOHで（pHは7.4）、1.4 MのNaCl、および25mMのCaCl 2 9重量部を蒸留水10×結合緩衝液の1部を希釈） 5ミリリットルの試料溶液の培養管。 </li><li>アネキシンV-FITCおよびヨウ化プロピジウム（PI）を5μlの5μlを添加します。静かにチューブをボルテックスし、暗所で室温（RT）で15分間インキュベートします。 1×結合緩衝液の400μlのを追加します。 注：蛍光顕微鏡キットから、アネキシンV-FITCおよびPIを使用してください。 </li><li> FACS 14を使用して、アポトーシス細胞を識別します。 PIおよびアネキシンV-FITCの励起レーザー線は、それぞれ、488 nmおよび635 nmです。それぞれ、610±20 nmおよび660±20 nmでPIとアネキシンV-FITCの蛍光発光を測定します。 10,000事象ごとのフローサイトメーター上で獲得した細胞を収集します。 </li></ol></li></ol>担癌マウスのin vivo抗腫瘍効果4. <ol><li>肺癌誘発性マウスモデル<ol><li> 0.1ミリリットルRPMI-1640培地に1×10 6 A549細胞を準備し;氷の中に保管してください。 </li><li>キシラジンの腹腔内注射およびチレタミンとゾラゼパム（1の混合物をマウスに麻酔：2、1ミリリットル/kg）。優しくマウスの皮膚の小さな倍をつまんで適切な麻酔を確認してください。麻酔下ながら乾燥を防ぐために、目に獣医軟膏を使用してください。 注：移動が観察されない場合、動物は、実験を開始するのに十分な麻酔です。 </li><li> （ - 7週齢、20〜22グラム6）BALB / C雄ヌードマウスの左脇腹に皮下細胞を注入します。 </li><li>彼らはケージの周りに移動を開始するまで、マウスを観察してください。 注：それは胸骨横臥位を維持するのに十分な意識を取り戻したまで無人の動物を放置しないでください。それは完全に回復するまで他の動物の会社に手術を受けた動物を返さないでください。特定病原体フリー（SPF）の条件下でマウスを保管してください。 </li><li>毎日ノギスで腫瘍の大きさを測定します。腫瘍サイズが体積で約200ミリメートル3に達したとき（長さ×幅2）/ 2として腫瘍体積（mm 3で）を計算し 、実験を開始します。 </李&gt; </ol></li><li>抗がん効果の研究<ol><li>無作為に4群（各群n = 10）にマウスを分けます。 </li><li> （：2、1ミリリットル/キログラム1）キシラジンの腹腔内注射およびチレタミンとゾラゼパムの混合物をマウスに麻酔。優しくマウスの皮膚の小さな倍をつまんで適切な麻酔を確認してください。麻酔下ながら乾燥を防ぐために、目に獣医軟膏を使用してください。 注：移動が観察されない場合、動物は、実験を開始するのに十分な麻酔です。 </li><li> 2 mg / mlでのHBの最終濃度になるようにPBS中にナノ粒子を溶解します。 0日と7日に二回、尾静脈（HBとしての2mg / kg）を介して、PBS、無料のHB、HB-のNP、またはHB-P-のNPを注入します。 </li><li>彼らはケージの周りに移動を開始するまで、マウスを観察してください。 注：それは胸骨横臥位を維持するのに十分な意識を取り戻したまで無人の動物を放置しないでください。完全になるまで他の動物の会社に手術を受けた動物を返さないでください。回収されました。暗く、特定のSPF条件下でケージを保管してください。 </li><li> （：2、1ミリリットル/キログラム1）各注射後24時間は、キシラジンおよびチレタミンとゾラゼパムの混合物の腹腔内注射でマウスを麻酔します。優しくマウスの皮膚の小さな倍をつまんで適切な麻酔を確認してください。麻酔下ながら乾燥を防ぐために、目に獣医軟膏を使用してください。 注：移動が観察されない場合、動物は、実験を開始するのに十分な麻酔です。 </li><li> （腫瘍に対するPDTファイバからの距離から1cmで）PDT繊維の下に腫瘍部位を配置します。 、レーザー安全メガネを着用してスイッチをオフにして、1日目と8日に二回500秒（200 J / cm 2）をするためのPDTレーザー（630 nmで、400ミリワット/ cm 2）を用いて腫瘍を照らします。 </li><li>彼らはケージの周りに移動を開始するまで、マウスを観察してください。それは胸骨横臥位を維持するのに十分な意識を取り戻したまで無人の動物を放置しないでください。 undergonを持つ動物を返さないでください。他の動物の会社に電子手術は完全に回復するまで。 </li><li> SPF条件下でケージを保管してください。レーザー治療後24時間暗所でケージを維持します。 </li><li>視覚的に毎日の腫瘍体積と腫瘍部位での変化を監視します。ノギスで腫瘍の大きさを測定し、（長さ×幅2）/ 2（ミリメートル3）などの体積を計算します。 PDT後の腫瘍表面変化を確認するために、毎日の腫瘍部位の写真を撮ります。 </li><li> 16日目には、イソフルランを用いて、端末麻酔によって群当たり5匹のマウスを生け贄に捧げます。 </li><li>鉗子やハサミで、外側の皮膚をカットし、腫瘍15を露出させます。慎重にそれらを収穫。 、10％ホルマリンでそれらを修正し、パラフィン中にそれらを埋め込む、組織学的分析16のためにヘマトキシリンおよびエオシン（H＆E）でそれらを染色します。 </li><li>モニタリングの45日後に、イソフルランを用いて、端末麻酔によって残りのマウスを生け贄に捧げます。 </li></ol></li></ol>

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The authors have nothing to disclose.

波長、パワー、照射時間：この研究の中で最も重要なステップは、適切なレーザー条件を選択されています。特定の光増感剤に適した光の適切な波長は、PDTのために必要です。私たちは、出力電力が多くのパイロット研究に基づいて400ミリワット/ cm 2に設定したもう一つの重要な要因であったヒポクレリンBについて適切であった630 nmのレーザーを使用していました。 400 mWの/ cm 2<...

Photodynamic therapy (PDT) is composed of three major factors: photosensitizers, light, and oxygen. PDT is reported as a promising treatment for various cancers3. When the photosensitizers are administered into the cancer patient, they selectively accumulate in the tumor tissues. When the proper wavelength of light is applied, the highly reactive singlet oxygen and other free radicals lead to tumor cell damage4. 
Lung cancer was introduced as one of the first applications for PDT in the early 1980s5. PDT provides several advantages in treating lung cancer. Since PDT is a non-invasive and non-surgical treatment, it is an attractive alternative choice for the patients in whom surgical resection is inappropriate.
There have been many challenges to enhance the cancer-targeting efficacy of the photosensitizers. Increasing photosensitizer accumulation in cancer sites and decreasing accumulation in normal tissues are the identical goals for the cancer-targeting studies. A variety of targeted drug delivery systems, such as polymers, liposomes, and nanoparticles are adopted as photosensitizer carriers6-8. In our previous studies, nanoparticles effectively increased the cancer-targeting abilities of the photosensitizers9,10. Nanoparticles are ideal cancer-targeting carriers since they possess both passive and active targeting abilities. The leaky tumor vessels provide opportunity for nano-sized carriers to accumulate easily in tumors, which is well-known as the enhanced permeability and retention (EPR) effect11,12. The interaction between the nanoparticles and the specific receptors on cancer cells enables active cancer targeting. In this study, we prepared hyaluronic acid-based nanoparticles to interact with CD44, the major hyaluronic acid receptor that is overexpressed on lung cancer cells13. 
To maximize the anticancer efficacy, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may permit an increased therapeutic effect. Furthermore, decreased doses of both the photosensitizer and the anticancer drug can diminish adverse effects. We selected hypocrellin B (HB) as a novel photosensitizer in the present study. HB is isolated from Chinese medicinal fungus Hypocrella bambuase. Shang et al. reported that HB-based PDT possesses a higher anticancer efficacy when compared to hematoporphyrin derivative-based PDT1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for various cancers, including lung cancer2. 
Herein, we compared the anticancer efficacies of photosensitizer (hypocrellin B, HB) solution, photosensitizer-encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>oligo hyaluronic acid</td>
			<td>Bioland Co., Ltd.</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>DS-Y30 (ceramide 3B; mainly N-oleoyl-phytosphingosine)</td>
			<td>Doosan Biotech Co., Ltd.</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>adipic acid dihydrazide</td>
			<td>Sigma Aldrich</td>
			<td>A0638</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(3-dimethylaminopropyl)-N&#8242;-ethylcarbodiimide</td>
			<td>Sigma Aldrich</td>
			<td>39391</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(chloromethyl)benzoyl chloride</td>
			<td>Sigma Aldrich</td>
			<td>270784</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Tokyo Chemical Industry Co., Ltd.</td>
			<td>T0546</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe filter</td>
			<td>Sartorius Stedim Biotech GmbH</td>
			<td>17762</td>
			<td>15 mm, RC, PP, 0.45 &#181;m</td>
		</tr>
		<tr>
			<td>triethylamine</td>
			<td>Sigma Aldrich</td>
			<td>T0886</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-GeBAflex tubes</td>
			<td>Gene Bio-Application Ltd.</td>
			<td>D070-12-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paclitaxel</td>
			<td>Taihua Corporations</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco Life Technologies, Inc.</td>
			<td>11875</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin&#8211;streptomycin</td>
			<td>Gibco Life Technologies, Inc.</td>
			<td>15070</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco Life Technologies, Inc.</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>Celite (Filter agent)</td>
			<td>Sigma Aldrich</td>
			<td>6858</td>
			<td>See step 1.4</td>
		</tr>
	</tbody>
</table>

anticancer efficacy of photodynamic therapy with lung cancer-targeted nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

私たちは、上記の技術とHB-のNPとHB-P-NPの両方を用意しました。 HB-のNPとHB-P-NPの平均直径はそれぞれ220.9±3.2 nmおよび211.9±1.6nmでした。 光照射（0〜16 J / cm 2）を、続いてPBS、空のNP、HB-のNP、及びHB-P-のNPとのインキュベーションの4時間後のA549細胞の細胞生存率は、光がない。 図1に示されています、 HB-P...

Watch this Scientific Journal Video about Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles at JoVE.com

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

肺癌標的化ナノ粒子と光線力学療法の抗がん効能

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

anticancer-efficacy-photodynamic-therapy-with-lung-cancer-targeted

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

8.9K ビュー.  Seoul National University Bundang Hospital. Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

ビデオ: Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

肺腫瘍細胞募集試験

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

がん研究

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

膵臓がんの蛍光同所性マウスモデル

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

真空安定化撮像窓と肺における長期高分解能生体顕微鏡

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

ひと下垂体腺腫の組織のプロテオームの分析のための質量分析法と相まって二次元ゲル電気泳動

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

肺組織に腫瘍のセルの視覚化を循環させるため肺植民地化アッセイの確立

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines respond to therapeutic compounds in 2D (dimensional) monolayer cell cultures, 3D culture systems were developed to understand the efficacy of drugs in more physiologically relevant models. In recent years, a paradigm shift was observed in pre-clinical research to validate the potency of new molecules in 3D culture systems, more precisely mimicking the tumor microenvironment. These systems characterize the disease state in a more physiologically relevant manner and help to gain better mechanistic insight and understanding of the pharmacological potency of a given molecule. Moreover, with the current trend in improving in vivo cancer models, zebrafish has emerged as an important vertebrate model to assess in vivo tumor formation and study the effect of therapeutic agents. Here, we investigated the therapeutic efficacy of hydroxycoumarin OT48 alone or in combination with BH3 mimetics in lung cancer cell line A549 by using three different 3D culture systems including colony formation assays (CFA), spheroid formation assay (SFA) and in vivo zebrafish xenografts.

Here, we present the preclinical screening of anticancer coumarins using 3D culture and zebrafish.

回転楕円体、コロニー形成アッセイとゼブラフィッシュ異種移植片による抗癌性クマリン OT48 の生物活性の臨床評価

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

抗がん剤抗体抗悪性腫瘍薬による効果の増強: 組み合わせインデックス式を用いた抗体医薬の相乗作用の検出

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

それらの相互作用を研究する歯髄組織と癌細胞との共培養から間葉系幹細胞の分離

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

腫瘍神経膠芽腫でフィールド療法を治療の臨床応用

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

A method for establishing afatinib-resistance cell lines from lung adenocarcinoma PC-9 cells was developed, and resistant cells were characterized. The resistant cells can be used to investigate epidermal growth factor receptor tyrosine kinase inhibitor-resistance mechanisms, applicable for patients with non-small cell lung cancer.

アファチニブ耐性肺腺癌PC-9細胞株の確立と特徴付けは、アファチニブの用量の増加とともに開発された

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in ...