本論文では、国立トランスレーショナルサイエンス推進センターが、賞番号UL1TR003096(MDE)、国立心臓、肺、血液研究所肺疾患研修プログラム5T32HL134640(MLD)の下で支援を受けました。

ここに記載されているすべての方法は、バーミンガムのアラバマ大学の制度的動物ケアと使用委員会(IACUC)によって承認されています。
1. 実験プロトコル
<ol><li>承認された IACUC 方式を使用してマウスを犠牲にします。ここでは、5%イオブルランで麻酔したマウスの子宮頸部脱臼を用いた。研究に適切なマウスを使用します。ここでは、8週齢のFVBマウスを使用しています</li>
	<li>手術用ハサミを使用して、下腹部の真ん中に3.5〜5mmの水平切開を行います。次に、切開から作成された小さな穴に外科用ハサミを挿入し、マウスの首のすぐ下に中央の正中線を垂直に切断します。</li>
	<li>指で皮膚を引き戻し、腋窩リンパ節を検査します。</li>
	<li>手術用ハサミを使用して、3.5mmの側面切開を行い、腹腔を開き、胸部の底まで前方向に切断します。腹腔内の臓器を検査します:肝臓、脾臓、腎臓など。</li>
	<li>手術用ハサミの平らで、または鉗子を使用して、肝臓を動かして横隔膜を露出させる。横隔膜の腫瘍の成長や転移を検査します。その後、オペレータの右側にあるダイヤフラムをそっと切り取り、拡大します。横隔膜を右から左にそっと切り、胸腔と肺を露出させる。肺を切らないように注意してください。</li>
	<li>左肋骨ケージの横端(オペレータの右)を切り取って、肺の左葉を検査します。</li>
	<li>肺の右葉をそっと動かし、右肋骨ケージの横端を切り取り、リブケージを取り除きます。 注:リブケージの除去はオプションですが、除去は後の肺インフレのより明確なビューを可能にします。<ol>
<li>新鮮な肺組織または凍結した肺組織が必要な場合は、ヘモスタット鉗子を使用して左葉の気管支をクランプし、灌流前に外科用ハサミを使用して左肺を切除する。</li>
	</ol>
</li>
	<li>鉗子を使用して気管を覆う組織を持ち上げ、余分な組織を切り取る。その後、気管を裏打ちする薄い組織をそっと切り、気道を露出させる。</li>
	<li>外科用はさみで腎動脈を切り抜ける。</li>
	<li>肺を浸透させるために、22G針の3 mLシリンジを使用して、心臓の右心室に10 U/mLヘパリンを含む1x PBSを注入する。PBS/ヘパリンで約300μL/秒でゆっくりと肺を透過します。肺は頻繁に白くなります。このステップでは、通常、2.5 mL の PBS が使用されます。</li>
	<li>肺膨張のために、22G針を用いた3mLシリンジを使用し、今度は気管に平行に保持した。針を気管に入れ、肺が完全に膨張するまで流量が〜200μL/s以下の10%ホルマリンを注入します。肺が膨張すると、ホルマリンは気管から逆流します。針を数秒間所定の位置に保持し、引き出します。<ol>
<li>(オプション)針と肺の膨張を引き出す前に、縫合糸を使って気管を締め出してください。これを達成するには、小さな鉗子で糸のポイントを保持する縫合糸の4インチを使用してください。気管の裏側に糸を置き、引き抜いて針の周りにループを作ります。次に針の周りにオーバーハンドの結び目を作ります。結び目をしっかりと引っ張り、気管から針を取り除き、結び目を閉じます。</li>
	</ol>
</li>
	<li>鉗子を使って心臓を持ち上げ、肺の真後ろに外科用のはさみを挿入し、心臓を持ち上げて肺を切除しながら結合組織を切断する。</li>
	<li>肺からそれを取り除くために心臓をカットします。</li>
	<li>肺にマウスIDまたは研究IDでラベル付けされたカセットを置き、カセットを10%緩衝ホルマリンに入れ、24〜48時間固定します。肺は、必要に応じて1年以上固定剤に残すことができます。</li>
	<li>肺を含むカセットを70%エタノールに移し、占いの処理に進みます。</li>
</ol>

<ol>
	<li>Hsia, C. C., Hyde, D. M., Ochs, M., Weibel, E. R., Structure, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+official+research+policy+statement+of+the+American+Thoracic+Society/European+Respiratory+Society:+standards+for+quantitative+assessment+of+lung+structure.">An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure.</a> American Journal of Respiratory and Critical Care Medicine. 181 (4), 394-418 (2010).</li><li>Vasilescu, D. M., Knudsen, L., Ochs, M., Weibel, E. R., Hoffman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+murine+lung+preparation+for+detailed+structural+evaluation+via+micro-computed+tomography.">Optimized murine lung preparation for detailed structural evaluation via micro-computed tomography.</a> Journal of Applied Physiology. 112 (1), 159-166 (2012).</li><li>Blumler, P., Acosta, R. H., Thomas-Semm, A., Reuss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+fixation+for+the+preservation+of+air+spaces.">Lung fixation for the preservation of air spaces.</a> Experimental Lung Research. 30 (1), 73-82 (2004).</li><li>Oldmixon, E. H., Suzuki, S., Butler, J. P., Hoppin, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+dehydration+fixes+elastin+and+preserves+lung+air-space+dimensions.">Perfusion dehydration fixes elastin and preserves lung air-space dimensions.</a> Journal of Applied Physiology. 58 (1), 105-113 (1985).</li><li>Braber, S., Verheijden, K. A., Henricks, P. A., Kraneveld, A. D., Folkerts, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+fixation+methods+on+lung+morphology+in+a+murine+model+of+emphysema.">A comparison of fixation methods on lung morphology in a murine model of emphysema.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 299 (6), 843-851 (2010).</li><li>Renne, R., 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=Recommendation+of+optimal+method+for+formalin+fixation+of+rodent+lungs+in+routine+toxicology+studies.">Recommendation of optimal method for formalin fixation of rodent lungs in routine toxicology studies.</a> Toxicologic Pathology. 29 (5), 587-589 (2001).</li><li>Limjunyawong, N., Mock, J., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instillation+and+Fixation+Methods+Useful+in+Mouse+Lung+Cancer+Research.">Instillation and Fixation Methods Useful in Mouse Lung Cancer Research.</a> Journal of Visualized Experiments. (102), e52964 (2015).</li><li>Lum, H., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+10%+formalin+fixation+on+fixed+lung+volume+and+lung+tissue+shrinkage.+A+comparison+of+eleven+laboratory+species.">Effects of 10% formalin fixation on fixed lung volume and lung tissue shrinkage. A comparison of eleven laboratory species.</a> American Review of Respiratory Disease. 132 (5), 1078-1083 (1985).</li><li>Edmonds, M. 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=MicroRNA-31+initiates+lung+tumorigenesis+and+promotes+mutant+KRAS-driven+lung+cancer.">MicroRNA-31 initiates lung tumorigenesis and promotes mutant KRAS-driven lung cancer.</a> Journal of Clinical Investigation. 126 (1), 349-364 (2016).</li><li>Zhao, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wogonin+suppresses+melanoma+cell+B16-F10+invasion+and+migration+by+inhibiting+Ras-medicated+pathways.">Wogonin suppresses melanoma cell B16-F10 invasion and migration by inhibiting Ras-medicated pathways.</a> PLoS One. 9 (9), 106458 (2014).</li></ol>

著者らは開示するものは何もない。

マウス肺の灌流、膨張、および固定のために上記の手順は、肺腫瘍組織学および病理解析のためのマウス肺の迅速かつ効率的な調製に最適である。この手順は特別な装置を必要とせず、マウス1個につき6分以内に実行できます。この手順では、インフレや一定の流体圧力に固定体積は必要ありません。この手順は標準化されていないため、肺の立体学的または形態測定を行うことを望む人に?...

肺腫瘍発生や癌転移から肺への様々なマウスモデルは、複雑なGEMMから発がん性および異種移植片モデルに至るまで、癌細胞が心臓内、胸腔内、尾静脈、または肺内に腫瘍を確立する他の方法を介して注入される同種および異種移植片モデルに至るまで存在する。これらのモデルはすべて、肺組織学と病理の組織学的評価に共通する必要性を共有しています。したがって、余分な血液を除去するために肺を浸透させ、肺を膨らませて肺のアーキテクチャを明確に可視化しながら、マウスの壊死を行う堅牢かつ迅速な方法が必要である。速度は、単一の時点で数十匹のマウスから肺を収集する必要があるため、この手順の重要な要素です。この手順は、マウスあたり6分以内に実行できます。
この手順は腫瘍組織学を評価するのに十分ですが、肺のステテロロジーまたは形態測定を行いたい人には推奨されません。このような測定では、肺の絶対表面積、絶対体積、歯槽サイズおよび番号1の計算と同様に、肺の膨張を標準化する必要があります。この方法は、一部のイメージングアプローチにも最適ではありません。例えば、ex vivoモルフォメトリック解析のためにμCTを介して肺をイメージングするには、肺が空気2で満たされたままである必要があります。空気空間および寸法の保存が第一の関心事である場合、過灌流脱水技術3、4によって肺を固定することが推奨される。このモデルの最大の懸念の1つは、肺胞壁の破裂の可能性であり、肺気腫の研究における使用を減らす。しかし、肺気腫の研究のための肺の固定のための推奨手順は、一定の流体圧力下で(ここで説明するプロトコルに類似した)10%ホルマリンの気管内点眼点眼によって、またはその場で固定5で肺を固定することが推奨されるように、まだ非常に似ています。
ここで説明した手順の利点は、一定の流体圧力を必要とせず、肺が完全に膨張するまで膨張し、処置に必要な時間を短縮することです。ここで説明する手順は、毒物学病理学会の軍備間膜によって推奨される方法によく似ており、毒物学研究のための肺固定の最良の方法を推奨する小委員会が結成された。この小委員会内の科学者の大半は、肺が固定剤6に残された時間に様々な勧告があったが、注射器で気管内点眼によって肺を固定することを推奨した。従って、肺の膨張および固定の様々な方法が存在する一方で、本明細書に記載される方法は、下流腫瘍組織学的評価のために肺を迅速に膨張および固定する最適な方法であると提案される。

<table>
	<tbody>
		<tr>
			<td>10% buffered formalin</td>
			<td>Fisher</td>
			<td>23-245685</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G Needle</td>
			<td>BD</td>
			<td>305155</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>BD</td>
			<td>309656</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Decon</td>
			<td>2405</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Harvard Apparatus</td>
			<td>72-8595</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fisher</td>
			<td>H19</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Harvard Apparatus</td>
			<td>72-8428</td>
			<td></td>
		</tr>
	</tbody>
</table>

perfusion and inflation of the mouse lung for tumor histology

肺の病態を評価する能力は、肺がん研究やがん転移の分野で重要です。調達した組織の質を犠牲にすることなく、研究から迅速かつ効率的に壊死を行うことも同様に重要です。このプロトコルの目的は、下流の組織学的分析のためにマウス肺を迅速に穿ファズ、膨張、および固定する方法を提示することです。この方法は、肺の膨張を標準化しません。したがって、特別な手順や機器を必要とせず、心臓を通して灌流した後の気管を通して直接固定液を植え付けるだけです。これにより、腫瘍のサイズ、組織学、およびスコアリングの十分な推定が可能になります。これはまた肺組織の固定の前に凍結組織のコレクションを可能にする。この方法は、後で肺の形態学的定量を可能にしないために制限されています。しかし、遺伝子組み換えマウスモデル(GEMM)、異種モデル、異種移植片腫瘍および転移研究による肺腫瘍分析には十分です。

上記のプロトコルはマウス肺の速い潅流、膨張および固定を可能にする。以下の図は、各ステップの重要性を表しています。 図1 は、灌流ステップがスキップされたか、肺が正しく穿フューする失敗したPBSおよび肺と浸透したH&E染色された肺を示している。示されているように、不十分な浸透性肺の過剰な血液は理想的な構造学よりも少なく、肺アーキテクチャを完全?...

Watch this Scientific Journal Video about Perfusion and Inflation of the Mouse Lung for Tumor Histology at JoVE.com

Perfusion and Inflation of the Mouse Lung for Tumor Histology

この方法の目的は、肺腫瘍病理の検査および肺への転移の評価のためのマウス肺の灌流、膨張、および固定のための簡単で効率的な方法を提示することである。

腫瘍組織学におけるマウス肺の多灌流とインフレーション

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform ...

perfusion-and-inflation-of-the-mouse-lung-for-tumor-histology

Research

JoVE Journal

Cancer Research

17.9K ビュー.  University of Alabama at Birmingham. この方法の目的は、肺腫瘍病理の検査および肺への転移の評価のためのマウス肺の灌流、膨張、および固定のための簡単で効率的な方法を提示することである。

ビデオ: Perfusion and Inflation of the Mouse Lung for Tumor Histology

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

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

ザ<em&gt;ショウジョウバエ</em&gt;成虫盤腫瘍モデル：遺伝的モザイクを用いた遺伝子発現と腫瘍侵襲性の可視化と定量化

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

大腸-26癌腫瘍を有するがん悪液質の研究のためのモデルとしてマウスを

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

単一のセル下流解析のターゲット細胞前濃縮と全体のゲノム増幅

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

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 ...

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

肺癌のマウスモデルにおける糖代謝の動的変化を測定する18F-FDG PET/CT イメージングと定量組織学を活用

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

無細胞性と細胞の肺転移モデル

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

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 of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

超音波画像によるマウスにおける肺腫瘍の進行の検出

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

腫瘍細胞の横尾静脈注入後の肺転移の病理学的解析

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

腫瘍組織学におけるマウス肺の多灌流とインフレーション

要約

さらに動画を探す

この動画の章

肺腫瘍細胞募集試験

ザ

大腸-26癌腫瘍を有するがん悪液質の研究のためのモデルとしてマウスを

単一のセル下流解析のターゲット細胞前濃縮と全体のゲノム増幅

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

肺癌のマウスモデルにおける糖代謝の動的変化を測定する¹⁸F-FDG PET/CT イメージングと定量組織学を活用

無細胞性と細胞の肺転移モデル

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

超音波画像によるマウスにおける肺腫瘍の進行の検出

腫瘍細胞の横尾静脈注入後の肺転移の病理学的解析

腫瘍組織学におけるマウス肺の多灌流とインフレーション

要約

さらに動画を探す

この動画の章

肺腫瘍細胞募集試験

ザ

大腸-26癌腫瘍を有するがん悪液質の研究のためのモデルとしてマウスを

単一のセル下流解析のターゲット細胞前濃縮と全体のゲノム増幅

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

肺癌のマウスモデルにおける糖代謝の動的変化を測定する18F-FDG PET/CT イメージングと定量組織学を活用

無細胞性と細胞の肺転移モデル

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

超音波画像によるマウスにおける肺腫瘍の進行の検出

腫瘍細胞の横尾静脈注入後の肺転移の病理学的解析

肺癌のマウスモデルにおける糖代謝の動的変化を測定する¹⁸F-FDG PET/CT イメージングと定量組織学を活用

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