Name: isolation and propagation of circulating tumor cells from a mouse cancer model
Uploaded: 2015-10-09T15:00:00.000+00:00
Duration: 5 min 22 s
Description: Watch this Scientific Journal Video about Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model at JoVE.com

Work in Dr. Ogunwobi&#8217;s laboratory is supported by a Research Centers in Minority Institutions Program grant from the National Institute on Minority Health and Health Disparities (MD007599) of the National Institutes of Health. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NIMHD or the NIH.

倫理の声明：すべての動物実験は、ニューヨーク市立大学ハンターカレッジの施設内動物管理使用委員会（IACUC）によって承認されました。 1.前の実験手順<ol><li>動物施設に到着すると、最初にフィルタトップと木材チップの寝具をケージに3匹のBALB / cマウスを収容します。マウスは食品、ピュリナ齧歯類固形飼料および水を自由にアクセスできることを確認します。ケージは週に一度クリーニングする必要がありながら、寝具や食品を週に2回変更する必要があります。 </li><li>毎分1リットルの一定流量で酸素と一緒にイソフルラン気化器を使用して、マウスでの全身麻酔を誘導します。システムは、自立であり、精密な気化器が含まれています。 </li><li>精密気化器とそれに接続されたガス/麻酔システムを持っているプラ​​スチック製の箱の位置動物。動物は2に気化器を下げ、その時点で、意識不明になるまで、最初は2.5に気化器を設定します。 </li><li>その時、スイッチバルブライン、ボックスにそれをシールし、ノーズコーンにそれを開きます。 </li><li>ノーズコーン（メンテナンス麻酔のために）適切に配置し、接続して、準備領域に動物を移動します。 </li><li>マウス当たりの移植のために5×10 6 BNL 1ME A.7R.1マウスHCC細胞を準備します。 BNL 1ME A.7R.1培地中で増殖されるべきである（10％熱不活化FBS、1％ペニシリン/ストレプトマイシンおよび2mM L-グルタミン）空気及び5％の加湿インキュベーター中、37℃で細胞培養皿中でインキュベートCO 2。 </li><li>シリンジ20 G針を有する、BALB / cマウスの肝臓の葉に5×10 6 BNL 1ME A.7R.1マウス肝細胞癌細胞を含むPBSを100μlのインプラントは、原発腫瘍を生成します。移植後、社内着床前と同じ家の要件にケージ内のBALB / cマウス。 </li><li>実験のエンドポイントで、肝細胞癌の臨床的証拠は、身体条件の有意な減少として観察されますスコア（通常は4週間後に検出されます）。この時点では、転移性の堆積物が肺に形成されているが、一次腫瘍は肝臓で開発している可能性があります。その後、循環腫瘍細胞の単離および増殖のプロセスを開始します。 </li></ol>腫瘍細胞の単離を循環させるための全血の2コレクション注：適切に手術領域を消毒し、それが乱雑フリーであることを確認してください。すべての手術器具をオートクレーブするか、製造元の推奨に従って消毒剤に浸し<ol><li>実験のエンドポイントでの人道的な安楽死（腫瘍発生の臨床的証拠）によりマウスを生け贄に捧げます。マウスの安楽死は、CO 2窒息を関与させるべきです。麻酔が正しく配布されて確認するために、適切にマウスを守ってください。呼吸停止を起こし、10分 - 二酸化炭素は5かけてチャンバー内にゆっくりと放出されるべきです。心臓内放血とcervicによってフォローアル脱臼。5 </li><li>全血の収集のための1mlシリンジに取り付けた25 G針を使用してください。 0.01ミリリットルヘパリンと注射器をHeparanize。約45°の角度でxyphisternumに劣る針とすぐにパンク肌。心を穿刺するために、ゆっくりと針を進め、その後、針の角度を下げます。着実な場所に針と注射器を保持し、500μlの引き出し - 心臓から全血を1000μLを。 </li><li>注射器と針を外し、前標識された1.5ミリリットルの発熱物質を含まないチューブに血液を移します。 5分間1167×gで1.5ミリリットルの発熱物質を含まないチューブに採取した全血を遠心分離します。 注：遠心分離を3層に全血試料を分離：赤血球、軟膜の中間薄層、および上部血漿層からなる下部層またはヘマトクリット。 </li><li>ピペットで慎重にプラズマを取り外し、フュルトのための独立した1.5​​ミリリットルの発熱物質を含まないチューブにそれを集めますそのような無細胞核酸の検出7としてER実験。 </li><li>循環腫瘍細胞（CTCの）を単離するために、赤血球（RBC）溶解の準備のために別々の1.5 mlの発熱物質を含まないチューブに軟膜を収集します。これは、バフィーコート層中の残留赤血球を除去する必要があります。 </li></ol>赤血球（RBC）溶解バッファー3.レシピ<ol><li> RBC溶解緩衝液1 L溶液を作ります。 </li><li>塩化アンモニウムの500ミリリットルとトリスの500ミリリットルを行います。その後、塩化アンモニウムの155 mMのトリスと10mMの最終濃度でRBC溶解緩衝液の1リットルの溶液を作成するために混合します。 </li><li> 7.5のpHに解決策をバッファ。 2 +8℃でのRBC溶解バッファを保存し、使用前に、すぐに室温にもたらします。 </li></ol>軟膜の4 RBC溶解<ol><li>収集したバフィーコートを含む滅菌した発熱物質を含まない1.5mlチューブにRBC溶解緩衝液1mlを追加します。 </li><li>により、チューブの内容物を混合複数回転倒。ベンチに室温で5分間チューブの混合された内容物をインキュベートします。遠心1167×gで5分間混合した内容物。 </li><li>遠心分離後、白っぽいペレットが得られます。ペレットを中断することなく、ゆっくりと慎重に、10％の漂白剤に赤みを帯びた上清を捨てます。 3.5 - ペレットが白っぽくていない場合は、再doが3.1を繰り返します。 10にRBC溶解緩衝液中でインキュベーション時間を増やす - 15分することも有用であろう。 </li><li> 10％の漂白剤での生物学的廃棄物の除染は、24時間後に完了すると、安全に生物学的廃棄物を処分します。 </li></ol>細胞培養物中の5伝播注：腫瘍細胞は、通常、急速に細胞をシャーレに播種した細胞のオリジナルミックスに豊富に存在する白血球を拡大しています。媒体の繰り返し変更、CTCのその後の継代の後、全ての白血球が除去され、CTCの比較的純粋な集団が残ります。 <ol><li>後10％の漂白剤に上清を廃棄し、少なくとも一回1 mlのリン酸緩衝生理食塩水（PBS）中に白っぽいペレットを洗浄し、細胞培養における増殖のための完全培地中で再懸濁します。 </li><li>洗浄が完了した後、ペレットに完全BNL 1ME A.7R.1培養培地1ml（FBS 10％熱不活性化、1％ペニシリン/ストレプトマイシンおよび2mM L-グルタミン）を添加し、培養液中にペレットを再懸濁します。 </li><li>細胞培養皿への細胞の再懸濁ミックスをシード。空気および5％CO 2の加湿インキュベーター中で37℃で細胞培養皿をインキュベートします。 </li><li> 2〜3日ごとに細胞培養培地を変更します。 5~7日後のCTCは、細胞培養皿に付着し、増殖し始めています。これらの細胞は25継代を超えて生存したままで繰り返し凍結融解サイクル5の後になります。 </li></ol>肝細胞癌の6検証は、腫瘍細胞株を循環します<ol><li> APを実行しますolymerase連鎖反応（PCR）の新規確立CTCの細胞株は、元の移植BNL 1ME A.7R.1 HCC線のようなマウス細胞であることを確認するために、マウスβグロビン遺伝子の1つの特定のDNAセグメントを増幅するためのベースの方法。方法は、もともとSteube らによって記載され、検証された。5,8 </li><li>特異的な抗体を用いて、タンパク質3のような3（CREB3L3）に結合する肝細胞特異的なマーカーcAMP応答要素の免疫染色を行います。これは、確立されたCTCは、肝細胞移植元のBNL 1ME A.7R.1細胞株のような実際にあるその小説を確認します。5 </li></ol>

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	<li>Ashworth, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+case+of+cancer+in+which+cells+similar+to+those+in+the+tumors+were+seen+in+the+blood+after+death.">A case of cancer in which cells similar to those in the tumors were seen in the blood after death.</a> Australian Medical Journal. 1869, 146-147 (14).</li><li>van de Stolpe, A., den Toonder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+Tumor+Cells:+What+Is+in+It+for+the+Patient?+A+Vision+towards+the+Future.">Circulating Tumor Cells: What Is in It for the Patient? 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The authors declare that they have no competing financial interests.

In this study, how to isolate and propagate CTCs from the whole blood of a syngeneic mouse with HCC were described. The objective of this work is to enhance the ongoing studies of the mechanisms of cancer metastasis. 
A factor that contributes to the poor prognosis of many cancers is the lack of timely detection and consequent widespread dissemination of the malignancy3. CTCs originate from primary cancers and spread via the blood stream to distant organs. As such, CTCs are importan...

The cancer research community has known of the existence of circulating tumor cells (CTCs) since first being observed by Thomas Ashworth in 18691. Since then, CTCs have been shown to be important in tumor metastasis and disease progression2-5. Today, solid tumors are a major cause of morbidity and mortality worldwide. CTCs are rare cells that originate from primary tumors and travel through the blood stream to different organs of which only a small fraction ultimately develop into metastasis2-5. Notably, there is positive correlation between tumor size and CTC number3,4. 
An understanding of CTC biology can contribute to the search for targeted therapy. Furthermore, CTCs may have diagnostic applications. To achieve these potential clinical applications, one needs to overcome some current challenges to studying CTCs. One challenge is related to the fact that CTCs may be present as single cells or as clusters and they may even be able to change their phenotype in response to the blood microenvironment2. Moreover, detection can be very challenging, in part, due to the low count of CTCs (a few to hundreds per milliliter) among one billionhematologic cells per milliliterin the blood6. Nevertheless, in recent years, research into the potential clinical applications of CTCs from solid organ cancers has intensified.
Despite these efforts, the challenges of studying and understanding the role of CTCs persist due to the rarity of CTCs and the inadequacy of the technological tools currently available. Despite these challenges, the tremendous potential for clinical applications continues to be an incentive to pursue research into the role of CTCs in cancer metastasis.
We were recently successful in isolating and propagating in cell culture CTCs from an orthotopic syngeneic mouse model of hepatocellular carcinoma (HCC) metastasis5. The purpose of the current paper is to describe in detail all aspects of the successful methodology. The significance of this methodology lies in the fact that this approach may be modified in order to successfully isolate and propagate in culture human CTCs, thus enhancing the possibility of in vitro studies of CTC biology. 
There are multiple potential clinical applications for the use of CTCs. CTCs may be useful for prognosis, response monitoring, screening, dynamic monitoring of tumor molecular alterations, and personalized therapy4. Therefore, a better understanding of the biology of CTCs has high potential for clinical impact.

<table><tbody><tr><td>Heparin Sodium Salt 1 g</td><td>VWR</td><td>89508-852</td><td></td></tr><tr><td>BTX Tube Micro 1.5 ml Clear NS</td><td>VWR</td><td>89511-254</td><td>1.5 ml&amp;nbsp; pyrogen-free eppendorf Tubes</td></tr><tr><td>Needle Sterile Disp BD 25 G x 1 in</td><td>VWR</td><td>BD305125</td><td>25 G Needle</td></tr><tr><td>Slp Tip SRNG 1 ml 200 each per pack</td><td>VWR</td><td>BD309659</td><td>1 ml syringe tip</td></tr><tr><td>Syringe 1 ml leur lok Pk 100</td><td>VWR</td><td>BD309628</td><td>1 ml syringe</td></tr><tr><td>VWR Forceps Tissue 6</td><td>VWR</td><td>82027-446</td><td>Forceps</td></tr><tr><td>Cyromold intrm 15 x 15 x 5 mm PK 100</td><td>VWR</td><td>25608-924</td><td>Cyromold</td></tr><tr><td>Cryo-oct compund 4 oz</td><td>VWR</td><td>25608-930</td><td>Oct compound</td></tr><tr><td>VWR Slide sprfrst 25 x 75 mm PK72</td><td>VWR</td><td>48311-703</td><td>Slides</td></tr><tr><td>VWR Cover Glass #2 22 x 50 mm</td><td>VWR</td><td>48-382-128</td><td>Cover Glass</td></tr><tr><td>VWR Slide Box True North Fm Pu</td><td>VWR</td><td>89140-278</td><td>Slide Box</td></tr><tr><td>Super HT PAP Pen</td><td>VWR</td><td>89427-058</td><td>PAP pen</td></tr><tr><td>Water RNASe and DNAse free 2 L</td><td>VWR</td><td>101454-204</td><td>Nuclease Free Water</td></tr><tr><td>Buffer Tris Ultra Pure Grade 500 g</td><td>VWR</td><td>97061-796</td><td>Tris Buffer</td></tr><tr><td>Ammonium Chloride ACS Grade 2, 5 Kg</td><td>VWR</td><td>97062-048</td><td>Ammonium Chloride Buffer</td></tr><tr><td>Falcon Tissue Culture Dish 60 x 15 mm Style polystyrene</td><td>VWR</td><td>353002</td><td>Tissue Culture Dish</td></tr><tr><td>Clorox Germicidal Bleach, Regular</td><td>VWR</td><td>89501-620</td><td>Clorox Bleach</td></tr><tr><td>PBS, 1x (Phosphate-Buffered Saline) without calcium &amp;amp; magnesium (500 ml)</td><td>Thermo Fischer Scientific</td><td>21-040-CV</td><td>PBS, 1x</td></tr><tr><td>DMEM, 1x&amp;nbsp; with 4.5 g/L glucose &amp;amp; L-glutamine without sodium pyruvate</td><td>Thermo Fischer Scientific</td><td>10-017-CV</td><td>DMEM 1x media for BNL 1ME A.7R.1 cells</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fischer Scientific</td><td>35-010-CV</td><td>FBS</td></tr><tr><td>Penicillin Streptomycin Solution, 100x</td><td>Thermo Fischer Scientific</td><td>30-002-Cl</td><td>Penicillin Streptomycin</td></tr><tr><td>Sorvall Biofuge pico</td><td>Thermo Fischer Scientific</td><td>75002411</td><td>13,000 rpm Centrifuge</td></tr></tbody></table>

isolation and propagation of circulating tumor cells from a mouse cancer model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

ここで説明する方法は、CTCを単離することができることを示しています。マウスは、人道的に、実験終了時点で安楽死させました。プロセスは、二酸化炭素窒息、心臓内瀉血、および頸椎脱臼を含みました。手順の重要なステップの概略図を図2に示した。心臓内放血により採取した全血試料は、上記のプロトコルを用いて処理しました。その後、遠心分離は、RBCの?...

Watch this Scientific Journal Video about Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model at JoVE.com

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

マウス癌モデルからの循環腫瘍細胞の単離と伝播

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

isolation-propagation-circulating-tumor-cells-from-mouse-cancer

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12.0K ビュー.  Hunter College of The City University of New York. Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

ビデオ: Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

結腸直腸癌モデルマウスにおける循環腫瘍細胞の単離

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

がん研究

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

下流の分子分析と転移性電位評価のための循環腫瘍細胞のマイクロマニピュレーション

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

マウス横紋筋肉腫から単離された原発性腫瘍細胞からの腫瘍球誘導と治療

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

遺伝学

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice.
Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo.
In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

Culturing CTCs allows a deeper functional characterization of cancer, through assaying specific marker expression, and assessing drug resistance and the ability to colonize the liver among other possibilities. Overall, CTC culture could be a promising clinical tool for personalized medicine to improve patient outcome.

循環腫瘍細胞株:基礎的・翻訳的研究のための革新的なツール

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an ...

マウスにおける腫瘍のガイド下接種とマルチモーダルイメージング

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

著者スポットライト:高解像度生体内イメージングとMRIを統合して、定位放射線治療計画を強化する

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

トランスレーショナルがん研究における生体内顕微鏡検査と磁気共鳴画像法の併用のための背側Skinfold Window Chamber腫瘍マウスモデル

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

原発腫瘍と肺転移を研究するためのMEF細胞の単離

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

医学

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

肝細胞癌転移の「患者様」同所同系マウスモデル

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

腫瘍特異的内皮細胞の単離および培養拡大

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

マウス癌モデルからの循環腫瘍細胞の単離と伝播

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結腸直腸癌モデルマウスにおける循環腫瘍細胞の単離

下流の分子分析と転移性電位評価のための循環腫瘍細胞のマイクロマニピュレーション

マウス横紋筋肉腫から単離された原発性腫瘍細胞からの腫瘍球誘導と治療

循環腫瘍細胞株:基礎的・翻訳的研究のための革新的なツール

トランスレーショナルがん研究における生体内顕微鏡検査と磁気共鳴画像法の併用のための背側Skinfold Window Chamber腫瘍マウスモデル

トランスレーショナルがん研究における生体内顕微鏡検査と磁気共鳴画像法の併用のための背側Skinfold Window Chamber腫瘍マウスモデル

トランスレーショナルがん研究における生体内顕微鏡検査と磁気共鳴画像法の併用のための背側Skinfold Window Chamber腫瘍マウスモデル

原発腫瘍と肺転移を研究するためのMEF細胞の単離

肝細胞癌転移の「患者様」同所同系マウスモデル

腫瘍特異的内皮細胞の単離および培養拡大