ハロルドバーマス、ブライアン c. ルイス、腫瘍形成の各、ダニー黄、シャロン パン、ミーガン ・ ウォン、Manasi m. Godbole を感謝いたします。Y.C.N.D. をサポートするには、DOD グラント W81XWH-16-1-0619 と NIH グラント 1R01CA204916。

<ol>
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S., Varmus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+for+hyaluronan-mediated+motility+isoform+B+promotes+liver+metastasis+in+a+mouse+model+of+multistep+tumorigenesis+and+a+tail+vein+assay+for+metastasis.">Receptor for hyaluronan-mediated motility isoform B promotes liver metastasis in a mouse model of multistep tumorigenesis and a tail vein assay for metastasis.</a> Proc Natl Acad Sci USA. 108 (40), 16753-16758 (2011).</li><li>Guernet, A., Grumolato, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+editing+of+the+genome+for+cancer+modeling.">CRISPR/Cas9 editing of the genome for cancer modeling.</a> Methods. , (2017).</li></ol>

著者が明らかに何もありません。

本研究ではマウスの強力なモデル、リップ-タグについて述べるRIP-tva鳥レトロ ウイルスの同定と転移の要因の解析を介して体細胞遺伝子配達を達成するために。ただし、リップ-タグ;RIP tvaマウス膵神経内分泌腫瘍を発症、このマウス モデルにおける転移因子も他の種類のがんの転移を促進するかもしれません。
我々 のアプローチの導入したがって散発的な...

ほとんどの癌は、体細胞変異1から発生します。従来の遺伝子組み換えマウス モデル (ジェム) は、腫瘍2に特定の遺伝子変異の貢献に重要な洞察力を提供しています。ただし、彼らはいくつかの制限があります。これらのモデルの主な欠点は、組織内の一部のセルだけが遺伝の変化を取得する人間の腫瘍の形成の散発的性質を複製しないこと。トランスジェニック、ノックアウト マウスの突然変異生殖開発に影響を与える可能性があります。また、これらマウス モデルの生成は高価で時間がかかるです。
転移は、がんの分野で重要な問題です。転移のモデリングはジェムで難しくなっています。自然転移は、マウスではまれです。浸透度は可変して転移3のジェムで長い待ち時間です。転移の翼列の初期の手順が排除されますので、実験的転移モデルはマウスの循環にセルの直接注入を採用しています。
Bitransgenic マウス モデル、リップ-タグ マウス モデルにおける転移因子を勉強の上記の制限の一部を克服するために開発しました。RIP tva4。戦略は、高い同期腫瘍進行マウス モデル、 RIP タグ5、および受容体サブグループ A トリ白血病ウイルス、tva 6、7の使用を組み合わせることに基づいています。このリップ-タグ;RIP tvaマウス モデルにより、単一の bitransgenic マウス緊張体細胞染色体導入する遺伝子。SV40 T 抗原 p53 および Rb の腫瘍抑制機能を抑制、マウスは過形成、血管新生、腺腫、浸潤癌などの段階で人間の腫瘍と同様の方法で膵神経内分泌腫瘍を開発します。このRIP タグモデルは、がん、膵神経内分泌腫瘍に限定されないの認刻極印の私達の理解に非常に有益されています。また、前臨床試験8で使用されています。
体細胞遺伝子伝リップ-タグに鳥レトロ ウイルス intracardiacally の注入のためのプロトコルを提案します。RIP tvaマウス。RCASBP 由来鳥レトロ ウイルスを感染ターゲットの積極的に増殖の細胞が必要です。したがって、我々 はリップ-タグを選んだRIP tva 7 週齢、膵島の約 50% に過形成を開発するときにマウス。高力価ウイルスの左心室心腔内注射は、10-20 4の感染効率を達成するために必要です。この配信方法は、ウイルス膵島に到達する前に循環内のウイルスの粒子の重要な希釈を低減します。
このアプローチを使用すると、以前 Bcl-xl がその抗アポトーシス機能4,9の独立したがんの転移を促進することを確認しました。Bcl xLリップ-タグに発癌性個体中すべての膵 β 細胞での遺伝子を介して発現していたときこの抗アポトーシス独立転移機能は観察されなかったRIP Bcl xLモデル10をマウスします。したがって、マウス モデルは、ユニークな機会を識別し、特徴付ける腫瘍形成の後の段階で表現される場合に遺伝子の機能を提供しています。小島の 2-4%リップ-タグで腫瘍に発展するのでRIP tvaウイルス感染とすべての前癌病変なし bitransgenic マウスは、RCASBP 由来の鳥のレトロ ウイルスに感染している、癌の自然経過の選択的優位性を与える要因のみを識別できます。特に、転移要因最も簡単に認識されますこのメソッドによって膵リンパ節や他の臓器への転移はリップ-タグで通常発生しませんのでRIP tvaマウス。

<table><tbody><tr><td>RCASBP-Y DV plasmid</td><td>Addgene</td><td>11478</td><td></td></tr><tr><td>RCAS-RNAi plasmid</td><td>Addgene</td><td>15182</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>fetal bovine serum</td><td>Atlanta Biologicals</td><td>25-005-CI</td><td></td></tr><tr><td>L-glutamine, 100x</td><td>Corning</td><td>25-005-CI</td><td></td></tr><tr><td>Penicillin-Streptomycin solution, 100x</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>PBS&lt;sup&gt;-/-&lt;/sup&gt;, 1x</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Superfect</td><td>Qiagen</td><td>301305</td><td></td></tr><tr><td>Polyallomer centrifuge tube</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>0.45 mm Nalgene&lt;br/&gt; Syringe Filters with PES Membrane</td><td>Thermo Scientific</td><td>194-2545</td><td></td></tr><tr><td>Insulin Syringes</td><td>BD</td><td>329461</td><td></td></tr><tr><td>synaptophysin</td><td>Vector Laboratories</td><td>VP-S284</td><td></td></tr><tr><td>VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG)</td><td>Vector Laboratories</td><td>PK-6101</td><td></td></tr><tr><td>AmpliTaq DNA Polymerase with Buffer II</td><td>Life Technologies</td><td>N8080153</td><td></td></tr><tr><td>MyTaq DNA Polymerase</td><td>Bioline</td><td>BIO-21106</td><td></td></tr></tbody></table>

identification and characterization of metastatic factors by gene transfer into the novel rip-tag; rip-tva murine model

転移がんは、固形腫瘍患者の死亡の 90% を占めています。がん転移のドライバーを理解して新たな治療標的を識別するために緊急の必要性があります。転移する癌から進行を駆動する分子のイベントを調査して、bitransgenic マウス モデル、リップ-タグを開発します。RIP tva。このマウス モデルでラットのインスリン プロモーター (RIP) は、膵 β 細胞における SV40 T 抗原 (タグ) およびサブグループ (tva) トリ白血病ウイルスの受容体の発現を駆動します。マウスは、過形成、血管新生、腺腫、浸潤癌などの段階で人間の腫瘍に類似している明確に定義された段階で 100% の浸透度を持った膵神経内分泌腫瘍を開発します。リップ-タグ;RIP tvaマウスは、転移性疾患を発症しない、転移を促進する遺伝の変化を簡単に識別することができます。体細胞遺伝子の tva 表現に、前癌病変は膵 β を増殖目的遺伝子変異をかくまっている鳥でレトロ ウイルスの心腔内注射によって実現されます。力価 &#62; 1 x 108感染単位 ml は生体内で感染症の適切なと見なされます。さらに、鳥のレトロ ウイルスリップ-タグで腫瘍由来細胞株に感染します。RIP tva高効率を持つマウス。細胞は、転移性の要因を明らかにも使用できます。ここで我々 はこのマウス モデルを利用し細胞の腫瘍の転移で候補者の遺伝子の機能を評価する方法を示します。

リップ-タグの生体内および生体外での感染率RIP tva RCASBP ベースのウイルスによる腫瘍細胞は ~ 20% 〜 80% とそれぞれ20。RIP-タグ;RIP tvaマウス モデル、各マウス 400 の膵島の約 4% に腫瘍20; 開発自然したがってウイルスによって遺伝子の潜在的な効果の組織学的表現型分析の各マウスにおける腫瘍細胞の十分?...

Watch this Scientific Journal Video about Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model at JoVE.com

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

リップ-タグを利用した体細胞遺伝子の転送システムを実証するためのプロトコルを提案します。RIP tvaマウス モデル転移遺伝子の機能を研究します。鳥のレトロ ウイルスは、成体マウスの膵 β 細胞の前癌性、非侵襲的病変への遺伝子導入を確保するため intracardiacally が配信されます。

同定と新規リップ-タグ; に遺伝子導入による転移因子の解析RIP tvaマウスモデル

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis ...

identification-characterization-metastatic-factors-gene-transfer-into

Research

JoVE Journal

Cancer Research

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

8.7K ビュー.  Weill Cornell Medicine. リップ-タグを利用した体細胞遺伝子の転送システムを実証するためのプロトコルを提案します。RIP tvaマウス モデル転移遺伝子の機能を研究します。鳥のレトロ ウイルスは、成体マウスの膵 β 細胞の前癌性、非侵襲的病変への遺伝子導入を確保するため intracardiacally が配信されます。

ビデオ: Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

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 ...

がん研究

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

虚血多灌流損傷の設定における転移性肝腫瘍のマウスモデル

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

尾静脈転移アッセイとリアルタイムインビボイメージングの併用による乳癌転移性コロニー形成と肺の負担の定量化

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

迅速かつ定量的なチック絨毛管内膜モデルを用いた転移性調節因子の発見

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

ヒト前立腺癌転移の同所マウスモデル

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

医学

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.

This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action.

メラノーマ転移の新規メディエーターの同定のための堅牢な発見プラットフォーム

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

乳癌の新規トランスジェニックマウスモデル：アデノ-Creを持つ乳管上皮の標的とすることにより、転移性乳癌の開始

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

定量的リアルタイムPCRによるマウス乳腺腫瘍モデルにおける肺転移の評価

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

ex vivoでのライブ肺転移のイメージングとその微小環境

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable model for tracing tumor progression and angiogenesis simultaneously in a visual and sensitive manner. Bioluminescence imaging displays its unique superiority in living imaging due to its advantages of high sensitivity, strong specificity, and accurate measurement. Presented here is a protocol to establish a tumor-bearing mouse model by injecting a Renilla luciferase-labeled murine breast cancer cell line 4T1 into the transgenic mouse with angiogenesis-induced Firefly luciferase expression. This mouse model provides a valuable tool to simultaneously monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging in a single mouse. This model may be widely applied in anti-tumor drug screening and oncology research.

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

腫瘍進行と血管新生の二重生物発光イメージング

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable ...