私達は誠意をこめて、MATLABのプログラムとニュージャージー病理組織学的機能と顕微鏡施設のがん研究所と彼女の助けを博士Wenjinチェンに感謝します。我々はまた、彼らのコメントと提案についてのレビューに感謝します。本研究では、レイモンドとビバリー·サックラー研究財団によってサポートされています。

<ol>
	<li>Taal, B. G., Visser, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+neuroendocrine+tumours.">Epidemiology of neuroendocrine tumours.</a> Neuroendocrinology. 80, 3-7 (2004).</li><li>Lee, G. Y., Kenny, P. A., Lee, E. H., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+models+of+normal+and+malignant+breast+epithelial+cells.">Three-dimensional culture models of normal and malignant breast epithelial cells.</a> Nat. Methods. 4, 359-365 (2007).</li><li>Friedrich, J., Seidel, C., Ebner, R., Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid-based+drug+screen:+considerations+and+practical+approach.">Spheroid-based drug screen: considerations and practical approach.</a> Nat. Protocol. 4, 309-324 (2009).</li><li>Chan, T. F., Vese, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+contours+without+edges.">Active contours without edges.</a> IEEE Trans Image Process. 10, 266-277 (2001).</li><li>Olert, J., Wiedorn, K. H., Goldmann, T., K&#252;hl, H., Mehraein, Y., Scherthan, H., Niketeghad, F., Vollmer, E., M&#252;ller, A. M., M&#252;ller-Navia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HOPE++Fixation:+A+Novel+Fixing+Method+and+Paraffin-embedding+Technique+for+Human+Soft+Tissues.">HOPE Fixation: A Novel Fixing Method and Paraffin-embedding Technique for Human Soft Tissues.</a> Pathology-Research and Practice. 197, 823-826 (2001).</li><li>Baradari, V., Huether, A., H&#246;pfner, M., Schuppan, D., Scher&#252;bl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiproliferative+and+proapoptotic+effects+of+histone+deacetylase+inhibitors+on+gastrointestinal+neuroendocrine+tumor+cells.">Antiproliferative and proapoptotic effects of histone deacetylase inhibitors on gastrointestinal neuroendocrine tumor cells.</a> Endocr. Relat. Cancer. 13, 1237-1250 (2006).</li><li>Foty, 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+Simple+Hanging+Drop+Cell+Culture+Protocol+for+Generation+of+3D+Spheroids.">A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids.</a> J. Vis. Exp. (51), e2720 (2011).</li><li>Shaw, K. R., Wrobel, C. N., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+three-dimensional+basement+membrane+cultures+to+model+oncogene-induced+changes+in+mammary+epithelial+morphogenesis.">Use of three-dimensional basement membrane cultures to model oncogene-induced changes in mammary epithelial morphogenesis.</a> J. Mammary Gland Biol. Neoplasia. 9, 297-310 (2004).</li><li>Ivascu, A., Kubbies, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+generation+of+single-tumor+spheroids+for+high-throughput+cell+function+and+toxicity+analysis.">Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis.</a> J. Biomol. Screen. 11, 922-932 (2006).</li><li>Napolitano, A. P., Chai, P., Dean, D. M., Morgan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+Self-Assembly+of+Complex+Cellular+Aggregates+on+Micromolded+Nonadhesive+Hydrogels.">Dynamics of the Self-Assembly of Complex Cellular Aggregates on Micromolded Nonadhesive Hydrogels.</a> Tissue Engineering. 13, 2087-2094 (2007).</li><li>do Amaral, J. B., Rezende-Teixeira, P., Freitas, V. M., Machado-Santelli, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCF-7+cells+as+a+three-dimensional+model+for+the+study+of+human+breast+cancer.">MCF-7 cells as a three-dimensional model for the study of human breast cancer.</a> Tissue Eng. Part C Methods. 17, 1097-1107 (2011).</li><li>Hirschhaeuser, F., Menne, H., Dittfeld, C., West, J., Mueller-Klieser, W., Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+tumor+spheroids:+An+underestimated+tool+is+catching+up+again.">Multicellular tumor spheroids: An underestimated tool is catching up again.</a> Journal of Biotechnology. 148, 3-15 (2010).</li></ol>

利害の衝突が宣言されません。

我々はアガロースでコーティングされた24ウェルプレートを用いたヒト神経内分泌腫瘍細胞からの3次元多細胞スフェロイドを形成するための、シンプルで信頼性が高く、再現可能な方法を実証した。この手法の成功は、セルの固定の番号から6日目で均質な大きさと均一な形状を持つ単一のスフェロイドを取得する機能があります。重要なステップは、めっき後の最初の16から24時間NETの細胞を用いたプレートのゆっくり攪拌します。また、このメソッドは、一般的なヒト乳癌細胞株MCF-7、ヒト結腸腺癌細胞株HT-29、ヒト非小細胞肺癌細胞株H1299とヒト胚腎臓細胞株HEK-293に適用することができます。彼らはすべての治療薬の画面（データは示さず）のための統一とコンパクトな3Dスフェロイドを形成することができます。いくつかの方法は、3D回転楕円体を生成することが報告されていますが、7月10日方法は、特殊なプレートや試薬を必要としないここで報告し、その結果、シンプルで費用対効果です。私たちの未発表データdemonstrat単層2次元のセルと比較した場合、アガロース上に成長させたNET 3D回転楕円体は、幾何学的および分子生体 NET異種移植腫瘍における模倣した電子。これは、他のヒト癌細胞株で作られた3D回転楕円体について報告されているものと似ています。3D細胞培養モデルの11,12制限は、それが完全に任意の薬剤または治療的処置の in vivoでの有効性のテストで置き換えることができないということですので、 3D回転楕円体は、この面でのin vivoで腫瘍内と異なっている、血管腫瘍のようなものです。それにもかかわらず、このメソッドはいくつかの側面における in vivo、in vitroでからNET治療薬のギャップと評価を埋めるのに役立ちます。 多細胞スフェロイドにおける免疫蛍光染色は、浮動小数点3D回転楕円体と共焦点顕微鏡の限界の大きなサイズとジオメトリーのために実行することは非常に困難である。ここでは、パラフィン包埋のためにHistoGelを処理する方法を示しています3D回転楕円体のシリアル切片スライドの免疫組織化学的染色を可能にして、埋め込む。また、私たちのカスタム開発されたMATLABプログラムは、正確かつ再現性、スフェロイドの大きさを推定する効率的な薬効を監視するのに役立ちますし、NET患者のための薬の開発におけるハイスループットスクリーニングに組み込まれる可能性があります。

<table border="1"><tbody><tr><td> 名 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> DMEM/F12メディア</td><td>ギブコ</td><td> 10565-018 </td><td></td></tr><tr><td> TrypLE </td><td>ギブコ</td><td> 12604 </td><td></td></tr><tr><td> 24ウェルプレート</td><td>ファルコン</td><td> 353047 </td><td></td></tr><tr><td> 70mMのセルストレーナー</td><td>フィッシャー·サイエンティフィック</td><td> 22363548 </td><td></td></tr><tr><td>滅菌シールテープ</td><td>ヌンク</td><td> 236366 </td><td></td></tr><tr><td>トリコスタチンA </td><td>シグマ</td><td> T8552 </td><td></td></tr><tr><td>オービタルシェーカー</td><td>フィッシャー·サイエンティフィック</td><td> 11-402-12 </td><td></td></tr></tbody></table>

human neuroendocrine tumor cell lines as a three-dimensional model for the study of human neuroendocrine tumor therapy

神経内分泌腫瘍では、（ネット）は年間100、000、個人ごとに2つの発生率と、まれな腫瘍であり、彼らはすべてのヒト悪性腫瘍の0.5％を占めています。1は、限局性疾患を呈する少数の患者の手術以外にも、あります全身療法のほとんど、またはまったく延命効果。したがって、優れたネットの生物学を理解し、特に切除不能または転移性神経内分泌腫瘍の患者さんのための新しい治療標的を定義するための優れた必要がある。 3D細胞培養は、 インビボで腫瘍組織の組織と微小環境でのモデリングで、その妥当性に起因する薬物のスクリーニングのための一般的な方法となっています。2,3次元多細胞スフェロイドが直接2Dから進んでよりタイムリーかつ安価な方法で貴重な情報を提供することができます動物（マウス）モデルへの細胞培養実験を行った。 NETパットのための新しい治療薬の発見を容易にするためにients、我々はヒトNET細胞株を用いたin vitroの3D多回転楕円体モデルで開発してきました。 NET細胞は非接着性アガロースでコーティングされた24ウェルプレートに播種し、播種後16から24時間のために非常にゆっくりと攪拌しながら、生理的条件下（5％CO 2、37℃）下でインキュベートされています。細胞は、第3または4日目に始まる多細胞スフェロイドを形成しています。スフェロイドは、薬物治療が開始される時点で、6日目 、することで、より球状になります。 NETスフェロイドに対する薬物治療の有効性は、位相差光学顕微鏡によるスフェロイドの形態、形状、サイズに基づいて監視されています。スフェロイドのサイズが自動的にアクティブな輪郭アルゴリズムに基づいてカスタム開発されたMATLABプログラムを用いて推定されています。さらに、我々は標準組織学的および免疫組織化学的手法を使用することができ、これらの3D回転楕円体上のHistoGel埋め込みを処理する簡単な方法を示しています。 <pクラス= &quot;jove_content&quot;&gt;これは、治療薬の効果を調べるためにNET細胞株を用いた3次元回転楕円体を生成する上で最初の報告である。また、これらの3次元回転楕円体で組織を行い、NETスフェロイドの成長と増殖に対する単剤の効果の例を表示しています。我々の結果は、スフェロイドNETは、NET生物学と医薬品開発のさらなる研究のために貴重であることをサポートしています。

Watch this Scientific Journal Video about Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy at JoVE.com

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

我々は、神経内分泌癌細胞株を用いた3次元多細胞スフェロイドを成長させるシンプルなアガロースオーバーレイプラットフォームを提示します。このメソッドは、神経内分泌腫瘍細胞に治療薬の効果を調べるために非常に便利な方法を提供します。それはまた、私たちはがん治療のための人間の神経内分泌腫瘍のスフェロイドを確立するのに役立つ可能性があります。

人間の神経内分泌腫瘍療法の研究のための三次元モデルとしての人間の神経内分泌腫瘍細胞株

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

human-neuroendocrine-tumor-cell-lines-as-three-dimensional-model-for

Research

JoVE Journal

Medicine

28.5K ビュー.  Raymond and Beverly Sackler Foundation. 我々は、神経内分泌癌細胞株を用いた3次元多細胞スフェロイドを成長させるシンプルなアガロースオーバーレイプラットフォームを提示します。このメソッドは、神経内分泌腫瘍細胞に治療薬の効果を調べるために非常に便利な方法を提供します。それはまた、私たちはがん治療のための人間の神経内分泌腫瘍のスフェロイドを確立するのに役立つ可能?...

ビデオ: Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

新規高分解能<em生体内で&gt;</em腫瘍増殖と治療に頭蓋内構造物の動的応答を研究する&gt;イメージング技術

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

医学

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

初代ヒト骨髄およびその悪性腫瘍を研究する三次元組織培養モデル

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脳Microslicesの興奮毒性刺激<em&gt;インビトロ</em&gt;脳卒中のモデル

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

3次元（3D）モデルを用いた乳癌細胞浸潤の定量化

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

腫瘍間質相互作用のための三次元共培養モデル

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

皮下腫瘍モデルにおける腫瘍浸潤白血球サブセットの評価

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

腫瘍の増殖、浸潤及び血管新生を研究するために、ニワトリでのヒト多発性骨髄腫異種移植モデルの確立

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三次元（3D）腫瘍スフェロイド浸潤アッセイ

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable risks for normal tissue injury and the hazards of physical tumor miss. A first-of-its-kind SBRT platform achieves high-precision ablative radiation treatment through a combination of versatile real-time imaging solutions and sophisticated tumor tracking capabilities. It uses dual-diagnostic kV x-ray units for stereoscopic open-loop feedback of cancer target intrafraction movement occurring as a consequence of respiratory motions and heartbeat. Image-guided feedback drives a gimbaled radiation accelerator (maximum 15 x 15 cm field size) capable of real-time &#177;4 cm pan-and-tilt action. Robot-driven &#177;60&#176; pivots of an integrated &#177;185&#176; rotational gantry allow for coplanar and non-coplanar accelerator beam set-up angles, ultimately permitting unique treatment degrees of freedom. State-of-the-art software aids real-time six dimensional positioning, ensuring irradiation of cancer targets with sub-millimeter accuracy (0.4 mm at isocenter). Use of these features enables treating physicians to steer radiation dose to cancer tumor targets while simultaneously reducing radiation dose to normal tissues. By adding respiration correlated computed tomography (CT) and 2-[18F] fluoro-2-deoxy-&#7429;-glucose (18F-FDG) positron emission tomography (PET) images into the planning system for enhanced tumor target contouring, the likelihood of physical tumor miss becomes substantially less1. In this article, we describe new radiation plans for the treatment of moving lung tumors.

Stereotactic ablative body radiation therapy (SBRT) involves the precise delivery of high-dose radiation to cancer tumor targets. A novel SBRT platform offers a first-of-its-kind gimbaled radiation accelerator mounted within a pivoting O-ring gantry capable of dynamic image-guided tumor tracking. Here, we describe dynamic tumor tracking for lung targets.

定位アブレーションボディ放射線治療のための動的肺腫瘍の追跡

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

腫瘍細胞移動の研究および抗浸潤性薬物の患者特異的効果の研究のためのヒト神経膠芽腫の器官型スライス培養モデル

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...