The authors want to thank Ms. Cornelia Heis for her excellent technical assistance in immunohistochemistry and preparation of chicken embryos. This work was supported by the Austrian Science Fund (FWF Grant No. P19552) and the European Union (EU FP7 project Optatio No: 278570).

//：実験動物福祉のhatching.The NIH Officeは、この領域（HTTPで書かれたガイダンスを提供するまでオーストリアの法律、および米国公衆衛生サービスの実験動物福祉の事務所によると、鳥類の胚は生きた脊椎動物とは見なされませんwww.grants.nih.gov/grants/olaw/references/ilar91.htmおよびNIH公開番号：06から4515）。 1.細胞培養およびレンチウイルストランスフェクション<ol><li>培養MM細胞株OPM-2、RPMI-8226およびRPMI1640培地中の骨髄からヒト間葉系幹細胞、存在下で、10％ウシ胎児ウシ血清及び100 IU / mlペニシリン、100μg/ mlのストレプトマイシンおよび2mMグルタミン37℃、5％CO 2。 </li><li>トランスフェクション5×10 30μlのリポソームトランスフェクション試薬とトランスフェクション培地（10ml）を利用してウイルスパッケージングミックス（9μgの）DNAおよび3μgのをpLenti6 / V5 DESTのeGFPベクターと6 HEK 293FT細胞。 12時間後にトランスフェクション培地を除去し、10％ウシ胎仔血清および1％非必須アミノ酸（NEAA）で10ml DMEM培地を加えます。 </li><li> 5日後、水泳細胞（千XG、5分）の遠心分離後HEK293FT細胞の上清を収集します。他の場所28記載されているように、リアルタイムPCRによりウイルス力価を決定します。 </li><li>完全増殖培地中で24ウェルプレート中のeGFPレンチウイルス粒子（1×10 5粒子）で1×10 6 MM細胞をトランスフェクション。 3日後、培地を2 / mlのブラストサイジンを添加することにより、選択プロセスを開始します。市販のeGFPレンチウイルスの場合は、500 / mlのネオマイシンを使用しています。 </li><li>選択の2週間後、MM細胞をeGFP発現のクラスターが表示されます。遠心分離（千XG、5分）によって細胞を収集し、実験のためにOPM-2 EGFPおよびRPMI-8226 のeGFPサブラインとしてそれらを展開する（セクション2および3）。 </li></ol> 2. 3D-多発性骨髄腫スフェロイドモデル<ol><li>チルI型コラーゲン溶液および10×DMEM On個の氷。 </li><li>コラーゲンマトリックスに10×DMEM培地の1/10量を混ぜます。 7.4のpH値に酸性コラーゲン溶液を中和するために水酸化ナトリウム（0.2 N）を加えます。氷上ストアコラーゲン/媒体液。 </li><li>ヒト間葉系細胞（50,000細胞/スフェロイド、 すなわち、30μlの低下）と、トランスジェニックMM細胞株（スフェロイド250,000、OPM-2 のeGFPまたはRPMI-8226 のeGFP）を混ぜます。 </li><li> 15ミリリットルチューブ（1,000×gで、5分間）で遠心細胞混合物は、細胞ペレットにコールド用意コラーゲン混合物（1mLの）を追加し、（千μlの先端で）よく混ぜます。 </li><li>直ちに滅菌パラフィンフィルム上に24ウェルプレート中で（100μLチップで）コラーゲン/細胞混合物を30μlのピペットと細胞/コラーゲン混合物は、37℃（ 図1Aを参照）で30分間重合を可能にします。 </li><li> 1,10および100nMのボルテゾミブを含む1mLの培養培地でオーバーレイMMスフェロイド（ 図1B参照 ）。 </li><li> 37℃でのインキュベーションの72時間後、文書スフェロイドによって蛍光実体顕微鏡（ 図1C参照 ）。 </li><li> GFPを測定するための反応管に広い平坦なジョーを有する鉗子を使用して、各スフェロイドを転送（ 図1Dを参照）。 </li></ol> CAM 3. 3D多発性骨髄腫異種移植モデル<ol><li> 3日間37°Cおよび70％の湿度で、鳥類の卵のための特別なインキュベーター中で鶏の卵をインキュベートします。 </li><li>その後、オープン卵と移植胚は、細胞培養プレートの蓋をエタノール、正方形、10 cmのプラスチック製計量ボートで滅菌し、CAMが（ 図2A参照 ）を開発することができるように、さらに6日間「EX OVO」をインキュベートします。 </li><li>氷の上にI型コラーゲン溶液および10×DMEMを冷やし、コラーゲンマトリックスに10×DMEM培地の1/10量を混ぜて、7.4のpH値に酸性コラーゲン溶液を中和するために水酸化ナトリウム（0.2 N）を追加します。氷上ストアコラーゲン/媒体液。 </li><li>トランスジェニックMM細胞株（OPM-2 のeGFPまたはミックスRPMI-8226 のeGFP;ヒト間葉系細胞とスフェロイド）250,000（50,000細胞/スフェロイド）。 </li><li> 15ミリリットルチューブ（千XG、5分、各​​試験化合物1バイアル用）で遠心細胞は、細胞ペレットに（望ましい稼動濃度で）薬物と1mLの冷たい準備コラーゲン混合物を追加し、（千μLチップで）よく混ぜます。 </li><li> 37℃での細胞外マトリックスの重合を可能にするために30分間よくパテ6にコラーゲンが低下パラフィルム上（30μlの各）を配置します。 </li><li> （2センチメートル離れた胚）から9日齢のニワトリ胚のCAMの未処理面に鉗子を用いて、ステップ3.6からの転送「オンプラント」（各ニワトリ胚のための4オンプラント、 図2Bを参照してください） </li><li>蛍光実体顕微鏡によるインビボでの 37℃での卵のインキュベーター中で成長し、70％湿度、文書異種移植の5日後（ 図2Cを参照）。 5時間冷蔵庫で4℃での低体温によってニワトリ胚を安楽死させます。 </li><li>広い平坦な顎を持つ眼科はさみや鉗子による直下のCAM組織との異種移植片を取り除きます。 GFPの測定のためにそれらを使用します（セクション4、 図2Dを参照）または血管および/ ​​または侵入腫瘍細胞（第5節）の免疫組織化学的分析のため。 </li></ol> 4. ELISAによるEGFPタンパク質の定量<ol><li> 200 / mlのプロテ​​アーゼ阻害剤を含有する0.5ミリリットルのRIPAバッファーに各MMスフェロイドまたは切除された異種移植片を転送します。 </li><li>氷上の組織ホモジナイザーでスフェロイド/異種移植片を均質化。 </li><li>液体窒素および37℃の水浴三の凍結/解凍サイクルを実行します。 </li><li> 20分（12000グラム）とストア上清4℃で遠心分離ホモジネート。 </li><li>サンプルをELISAキットのアッセイ緩​​衝液（200μL）で1:20に希釈します。製造業者のプロトコルに従って、ビオチン化抗GFP抗体を用いた商用GFP ELISAキットによりGFP-レベルを測定します。 </li></ol> 5. Immunohi血管や侵入腫瘍細胞のstoc​​hemical分析<ol><li> 4℃で4％パラホルムアルデヒドO / NでCAM領域で切り出し異種移植片を修正しました。 </li><li>包埋カセットに固定された異種移植片を配置し、段階的な増加アルコールシリーズ（50％、70％、80％、95％エタノール、キシロール、パラフィン、各ステップ60分間）を用いて組織の埋め込みステーションに移します。 </li><li>卓上型ロータリーミクロトームを使用することにより、セクションの異種移植片（5ミクロン）。 O / N 56°Cでのガラススライド上のパラフィン切片焼きます。 </li><li>二重蒸留水に減少段階的アルコール系によって脱パラフィン切片（キシロール、95％、80％、70％、50％エタノール、再蒸留水;各ステップ10分）。 </li><li>抗原回復溶液（; pHは7.0、体積100μlのクエン酸塩緩衝液）との水浴（95℃、20分）で抗原回復を実行します。 </li><li> 30分間、100μlの3％H 2 O 2 /メタノールを用いて内因性ペルオキシダーゼ活性をブロックします。 </li><li>を含むPBSでブロックセクション45分（体積100μl）を、10％ウシ胎児血清。 </li><li>一次抗体の100μlで1時間染色を（1μgの/ ml）を、室温で、1％ウシ胎児血清を含むPBSで希釈しました。 </li><li> PBSで3回洗浄した後、室温で1％ウシ胎児血清を含むPBS中のビオチン化二次抗体（0.1μgの/ ml）で1時間インキュベートします。 </li><li> PBSで3回洗浄した後、製造業者の説明書に従って、アビジン/ビオチン複合体（ABC）とジアミノベンジジン（DAB）基質溶液によって呈色反応を行います。 </li><li> 5.11。ヘマトキシリン​​で二重蒸留水、対比にセクションを転送することによって反応を停止し、合成封入剤とのセクションをマウントします。 </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+CD34++cells+expressing+the+inv(16)+fusion+protein+exhibit+a+myelomonocytic+phenotype+with+greatly+enhanced+proliferative+ability.">Human CD34+ cells expressing the inv(16) fusion protein exhibit a myelomonocytic phenotype with greatly enhanced proliferative ability.</a> Blood. 108 (5), 1690-1697 (2006).</li><li>Hideshima, T., Mitsiades, C., Tonon, G., Richardson, P. G., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+multiple+myeloma+pathogenesis+in+the+bone+marrow+to+identify+new+therapeutic+targets.">Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets.</a> Nat.Rev.Cancer. 7 (8), 585-598 (2007).</li><li>Parikh, M. R., Belch, A. R., Pilarski, L. M., Kirshner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+tissue+culture+model+to+study+primary+human+bone+marrow+and+its+malignancies.">A three-dimensional tissue culture model to study primary human bone marrow and its malignancies.</a> J.Vis.Exp. (85), (2014).</li><li>Schuler, J., Ewerth, D., Waldschmidt, J., Wasch, R., Engelhardt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+of+multiple+myeloma:+a+critical+appraisal.">Preclinical models of multiple myeloma: a critical appraisal.</a> Expert.Opin.Biol.Ther. 13, S111-S123 (2013).</li><li>Kirshner, J., 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=A+unique+three-dimensional+model+for+evaluating+the+impact+of+therapy+on+multiple+myeloma.">A unique three-dimensional model for evaluating the impact of therapy on multiple myeloma.</a> Blood. 112 (7), 2935-2945 (2008).</li><li>Jilani, S. M., 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=Selective+binding+of+lectins+to+embryonic+chicken+vasculature.">Selective binding of lectins to embryonic chicken vasculature.</a> J.Histochem.Cytochem. 51 (5), 597-604 (2003).</li></ol>

著者らは、全く競合する金融利害関係はありません

難治性MMのための新たな治療薬の開発は、薬物に対するヒトMM細胞の感受性を評価するために少ない時間がかかり、高価な生体内のシステムを必要とします。これまで、わずか数の生体内のシステムは、新しい抗骨髄腫治療の前臨床評価のために利用可能です。それらのすべては、化合物ライブラリー29の大規模スクリーニングのためにそれらの限界があります。 ヒトMM細胞のための最高の現行モデルは、高度免疫不全マウス7,13,30と七面 ​​鳥の胚29です。 SCIDマウスおよび鳥類胚異種移植モデルの両方は、MMの生物学を研究し、新規の治療化合物を試験するために使用することができます。しかし、マウス系は、近交系の遺伝子型を含む、技術的に要求の厳しい手順、長い観察期間と高いコストを、いくつかの制限があります。 本研究では、新規な3Dスフェロイドおよびトリ異種移植モデルは、ヒトのMESの存在を伴うヒトMM細胞の生物学を研究するために提示されています支持部品としてenchymal幹細胞及び細胞外マトリックス。 MM細胞が強く、それぞれの微小環境に依存して生存すると31-33を増殖する間質由来増殖因子、サイトカインおよびECM成分の存在、すなわち。また、薬物は非効率的であるか、または骨髄腫細胞は、それらの局所微小環境31,34により保護されていたときに、変化した活性を示すことがあります。 マウスモデルと比較して、in vitro及びin vivoモデル系で記述さ3Dは、高価で迅速、かつ取り扱いが容易ではありません。また、ニワトリ胚の3D MMスフェロイドの移植は骨髄腫誘導血管形成の分析を可能にします。我々のシステムの限界は、MM細胞増殖の短時間であり、したがって、鳥の骨への転移の分析は不可能です。我々のモデルのさらなる制限は、我々は肝臓の酵素によって全身適用および薬物代謝回転/変更が反映されない場合がありますCAMにおける薬物の局所適用では動作していることです。でさらには、移植するまで約50％の生存率が原因ニワトリ胚の成長条件卵元に観察されました。 IHC分析のための内皮マーカーに関しては、内皮細胞を染色するために、ヒトおよびマウスのセクションで使用されているほとんどのマーカーは、ニワトリの血管に固有のものではありません。したがって、我々は、ニワトリ胚に壁画細胞マーカーデスミン/ ASMAまたはレクチンの注射のために35を染色することをお勧めします。いない使用可能なすべてのヒト骨髄腫細胞株は、ニワトリ宿主組織に侵襲的に成長し、表面的な成長が表示されます。これは、ミクロトームのセクションを切断した後に破壊された組織になります。また、特別なケア（選択抗生物質）は、トランスフェクトされた細胞は、永久遺伝子再配列またはDNAメチル化プロセスに起因する時間内のGFP導入遺伝子の発現を、失わないように注意しなければなりません。 結論として、間質支持体と、ヒトMM細胞の我々のニワトリ胚移植モデルは、STUに再現可能かつ予測可能な生体内モデルを提供しますDYヒトMM細胞の増殖と血管新生。これは、MMモデルは開発時間および新規薬物のコスト削減に貢献し、より速い生体内の抗MM剤のスクリーニングを容易にすることができる処理します。このような骨置換材料としての更なる改善のステップ、と、複雑なECMマトリックスおよびシステムは、患者のサンプルに対しても治療法を試験するために改善されるかもしれないサイトカイン。これは、パーソナライズされたがん医療、個々の耐火MM患者における薬剤感受性の試験のための前提条件です。

Multiple myeloma (MM) is characterized by proliferation of malignant plasma cells in the bone marrow, bone lesions and immunodeficiency 1. Although new treatment options such as proteasome inhibitors (bortezomib) and immune modulatory drugs (pomalidomide and lenalidomide) are available, MM still remains an incurable malignancy with a grim prognosis 2. The bad prognosis might be explained by the extraordinary heterogeneity of MM cell clones that contributes to variable responses to therapy, in particular under long time treatment and selection pressure of MM clones 3.
Preclinical testing of new drugs and their combinations in vitro and in vivo is a critical and time-consuming step for future drug development. Thus, useful in-vivo models of MM are required to gain a better understanding of the biology of the disease and to enable the discovery of new drugs. Actually, the best xenotransplantation models for hematological malignancies and therapeutics are immune-deficient mice, such as the severe-combined immunodeficient (SCID) mice 4-7, the non-obese diabetic/SCID (NOD/SCID) mice 8,9 or the &#946;-microglobulin-knockout NOD/SCID mice 10,11.
Although murine models of human MM in some aspects can resemble the phenotype of human disease, immune-deficient mice are inbred, therefore simulate only one individual response to a drug and costs are very high. Due to immunosuppression animals require special maintenance conditions and the engraftment of human MM in mice requires 6 weeks to 2 months 9,12, unless cells are grafted directly to the bone marrow using a technically demanding procedure with lower rates of animal survival 7,13. Therefore, new methods using stem-cell based organoid models 14, tissue engineering 15 or sophisticated 3D cell culture models 16 have been established. They will compete in the near future with classical animal experiments for preclinical drug testing, but cannot replace systemic toxicity tests in living organisms.
The chicken embryo has been demonstrated before to be a suitable organism for xenotransplantation of human cells and tissues due to lack of adaptive immune response until hatching 17-19. Moreover, each chicken embryo reflects an individual reaction to applied drugs or tumor cells due to genetic diversity within the chicken population. The chorioallantoic membrane (CAM) is a well-established system to study tumor-dependent angiogenesis 20-22. When solid tumors are grafted to the CAM, they display many characteristics of cancers in vivo, including proliferation, invasion, angiogenesis and metastasis 23-27. 
Based on the previous experience of our group with CAM xenograft models20,26,27, a human MM model was established that combines the advantage of a human 3D culture system with the model of ex ovo developing chicken embryos. This MM model system allows real time monitoring of MM growth progression, quantification of cell mass and preclinical drug testing.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>RPMI-8226 cells</td>
			<td>DSMZ</td>
			<td>ACC 9</td>
			<td>STR profiled</td>
		</tr>
		<tr>
			<td>OPM-2 cells</td>
			<td>DSMZ</td>
			<td>ACC 50</td>
			<td>STR profiled</td>
		</tr>
		<tr>
			<td>Human mesenchymal stem cells&#160;</td>
			<td>PromoCell</td>
			<td>PC-C-12974</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293FT cells&#160;</td>
			<td>Invitrogen</td>
			<td>R700-07</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 Medium</td>
			<td>Sigma Aldrich</td>
			<td>R0883</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160; HyClone</td>
			<td>ThermoScientific</td>
			<td>SH30070.03</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glut- Pen- Strep solution</td>
			<td>Sigma</td>
			<td>G6784</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Medium</td>
			<td>Gibco</td>
			<td>31966</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Sigma Life Sciences</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection Medium/Opti-MEM&#160;</td>
			<td>Gibco</td>
			<td>51985</td>
			<td></td>
		</tr>
		<tr>
			<td>eGFP lentiviral particles</td>
			<td>GeneCopoeia</td>
			<td>LPP-EGFP-LV105</td>
			<td>Ready to use viral particles</td>
		</tr>
		<tr>
			<td>pLenti6/V5Dest6 eGFP vector</td>
			<td>Invitrogen</td>
			<td>PN 35-1271</td>
			<td>from authors</td>
		</tr>
		<tr>
			<td>ViralpowerTM packaging mix&#160;</td>
			<td>Invitrogen</td>
			<td>P/N 35-1275</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection reagent/ Lipofectamin 2000</td>
			<td>Invitrogen</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Invitrogen</td>
			<td>R210-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomycin</td>
			<td>Biochrom</td>
			<td>A2912</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen-Type1&#160; Rat Tail</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM powder</td>
			<td>Life Technologies</td>
			<td>Art.Nr. 10338582</td>
			<td></td>
		</tr>
		<tr>
			<td>plitidepsin</td>
			<td>Pharmamar</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bortezomib</td>
			<td>LKT Lab., Inc.</td>
			<td>B5871</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">SPF-white hen eggs</td>
			<td rowspan="3">Charles River</td>
			<td rowspan="3"></td>
			<td rowspan="3">Fertilized&#160; white Leghorn&#160; chicken eggs</td>
		</tr>
		<tr>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Plastic weighing boats</td>
			<td rowspan="2">neoLab</td>
			<td rowspan="2">Art.Nr. 1-1125</td>
			<td rowspan="2">for ex-ovo culture</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Petridish square (Lids)</td>
			<td>Simport</td>
			<td>D210-16</td>
			<td>for ex-ovo culture</td>
		</tr>
		<tr>
			<td>RIPA Buffer (10x)</td>
			<td>Cell Signaling</td>
			<td>#9806</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Tablets</td>
			<td rowspan="2">Roche</td>
			<td rowspan="2">11 836 170 001</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Complete Mini EDTA-free</td>
		</tr>
		<tr>
			<td>GFP ELISA</td>
			<td>Cell Biolabs, Inc.</td>
			<td>AKR-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Histocette II</td>
			<td>Simport</td>
			<td>M493-6</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA&#160; 37%</td>
			<td>Roth</td>
			<td>7398.1</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Lonza</td>
			<td>BE17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolut</td>
			<td>Normapur</td>
			<td>20,821,321</td>
			<td></td>
		</tr>
		<tr>
			<td>Roti-Histol</td>
			<td>Roth</td>
			<td>Art.Nr.6640.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Sigma</td>
			<td>A6330</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">SuperFrost Microscope Slides</td>
			<td>R. Langenbrinck&#160;</td>
			<td>Art.-Nr.</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Labor- u. Medizintechnik</td>
			<td>03-0060</td>
		</tr>
		<tr>
			<td rowspan="2">DakoCytomation Wash Buffer 10x</td>
			<td rowspan="2">DakoCytomation</td>
			<td>Code-Nr.</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>S 3006</td>
		</tr>
		<tr>
			<td rowspan="2">Target Retrieval Solution (10x)&#160; pH 6,1</td>
			<td rowspan="2">DAKO</td>
			<td>Code-Nr.</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>S 1699</td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu ASMA clone 1A4</td>
			<td>DAKO</td>
			<td>M0851</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu CD138 clone MI15</td>
			<td>DAKO</td>
			<td>M7228</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu Vimentin clone V9</td>
			<td>DAKO</td>
			<td>M0725</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu Desmin clone D33</td>
			<td>DAKO</td>
			<td>&#160;M0760</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu Ki67&#160; clone MIB-1&#160;&#160;</td>
			<td>DAKO</td>
			<td>&#160;M7240</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated goat- anti-mouse IgG</td>
			<td>Vector Laboratories Inc.</td>
			<td>BA-9200</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain Elite ABC Kit</td>
			<td>Vector Laboratories Inc.</td>
			<td># PK-6100</td>
			<td></td>
		</tr>
		<tr>
			<td>FAST DAB Tablet Set.</td>
			<td>Sigma Biochemicals</td>
			<td># D4293</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#8217;s haemalaun solution</td>
			<td>Merck</td>
			<td>1,092,490,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Roti Histokitt</td>
			<td>Roth</td>
			<td>Art.Nr.6638.2</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bench top rotary microtome</td>
			<td>Thermo Electron, Shandon Finesse ME+</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue embedding station</td>
			<td>Leica, TP1020</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Egg-Incubator</td>
			<td>Grumbach&#160;</td>
			<td>BSS160</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo fluorescence microscope equipped with an connected with a digital camera (Olympus E410) and flexible cold light&#160;</td>
			<td>Olympus, SZX10</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Turrax&#160;</td>
			<td>IKA T10</td>
			<td></td>
			<td>Homogenizer</td>
		</tr>
	</tbody>
</table>

establishment of a human multiple myeloma xenograft model in the chicken to study tumor growth, invasion and angiogenesis

多発性骨髄腫（MM）、悪性形質細胞疾患は、不治のままであり、新規の薬剤は、患者の予後を改善するために必要とされます。これにより、骨の微小環境と自動/パラクリン成長因子の欠如により、ヒトMM細胞を育成することは困難です。したがって、ヒトMM細胞に対する新規治療薬の作用を研究するために、in vitroおよびin vivo培養系において適切な確立が急務となっています。ここでは、in vitroおよびin vivoで 、複雑な3D環境内でヒト多発性骨髄腫細胞を増殖するためのモデルを提示します。 MM細胞株OPM-2及びRPMI-8226は、GFP導入遺伝子を発現するようにトランスフェクトされた三次元スフェロイド、ヒト間葉系細胞とコラーゲンI型基質の存在下で培養しました。加えて、スフェロイドは、ニワトリ胚の漿尿膜（CAM）に移植した腫瘍の成長は、ステレオ蛍光顕微鏡法によってモニターしました。どちらのモデルも、新たな治療DRUの研究を可能にします複雑な3D環境と導入遺伝子に特異的なGFP-ELISAにおける移植片の均質化後の腫瘍細胞塊の定量化でGS。また、下にある宿主組織へのホストおよび腫瘍細胞の浸潤の血管新生応答は、実体顕微鏡により毎日モニターし、ヒト腫瘍細胞（KI-67、CD138、ビメンチン）、または宿主壁細胞被覆血管に対する免疫組織化学染色によって分析することができます（デスミン/ ASMA）。 結論として、onplantシステムは、複雑な3D環境でのMM細胞の増殖と血管新生を研究することができますし、MM細胞の生存および増殖を標的とする新規治療化合物のスクリーニングを可能にします。

3D多発性骨髄腫スフェロイドアッセイにおける標的化合物のインビトロでの分析で これにより、in vitroで初代ヒトMM細胞を培養する制限に、我々は、細胞外成長マトリックスおよび骨髄（ 図1A、B）から支持初代ヒト間葉系細胞を利用したヒトMM細胞株のためのインビトロ培養モデルにおける新しい3Dを確立しました。 EGFPトランスジェニックMM細胞株は、スフェロイド内の3D成長後に視覚化し、MM腫瘍塊の定量化を可能にします。両方のMM細胞株OPM-2 EGFPおよびRPMI-8226 のeGFPステレオ蛍光顕微鏡でGFPの発現によって可視化したボルテゾミブ腫瘍の漸増濃度（1〜100nMの）の存在下で3日間培養した（ 図1C） 。腫瘍細胞塊は、スフェロイドの均質化後に定量化し、GFP-ELISA（ 図1でのeGFPの内容を測定しました。D）。 ニワトリ胚における多発性骨髄腫異種移植片における標的化合物の生体内分析で 三日目のニワトリ胚は6日間とMM細胞（ 図2A）のグラフトに使用9日目の元OVO培養しました。 EGFPトランスジェニック骨髄腫細胞（OPM-2 EGFP）、ヒト骨髄間葉系細胞は、細胞外マトリックス成 ​​分としてコラーゲンI型に包埋した一緒。標的物質ボルテゾミブは、1ナノモル（ 図2B）で局所的に適用しました。各動物4「オンプラント」のニワトリ胚の漿尿膜（CAM）に移植しました。 5日後、MM異種移植片は、eGFPの発現によって可視化することができた腫瘍を形成しました。対照と比較して、ボルテゾミブは、異種移植片において、MM細胞の増殖を阻害しました。移植片は、よりグリーンMM腫瘍細胞塊（ 図2C）を示しました。目からの単一のMM異種移植片REE異なる動物（N = 12）は、均質化を切除し、その後、GFP ELISAによって測定しました。対照と直接比較はボルテゾミブで処理した異種移植片は、有意に減少し、骨髄腫細胞塊（ 図2D）を有していました。 ニワトリ胚における血管新生応答および骨髄腫異種移植片の浸潤の生体内分析で オンプラントの周りの血管新生応答は、実体顕微鏡により観察することができます。異種移植片の血管新生が大幅に薬物処置異種移植片（1ナノモルPlitidepsin、 図3）に低下しました。血管新生応答は、Ribatti ら 21によってゼラチンスポンジアッセイについて記載したようにonplant出芽血管をカウントすることによって定量しました。 侵略の分析のために、異種移植片は、隣接するCAM領域で切り出しました。異種移植片は、固定され、パラフィンに包埋し、切片を作製した（ 図4 </強いです&gt;）。切片を、鶏の血管を覆う壁細胞を検出するためにASMA /デスミンに対する抗体で、および増殖およびニワトリ宿主組織におけるヒト腫瘍細胞に侵入する（ 図4）を検出するために、ヒトのKi-67、ビメンチンおよびCD138に対する抗体で染色しました。 <img alt="図1" src="/files/ftp_upload/52665/52665fig1.JPG" /> 図1. 3D多発性骨髄腫スフェロイド。細胞外マトリックス成 ​​分として（A）OPM-2 のeGFPの生成および一次ヒト骨髄間葉系細胞とコラーゲンI型を含むRPMI-8226 のeGFPスフェロイド。（B）スフェロイドを培地とボルテゾミブのそれぞれの濃度とインキュベートしました（1- 100 nM）を、（C）MMスフェロイドを3日間増殖させ、ステレオ蛍光顕微鏡で撮影しました。バーが500μmを示す。（D）シングルSPHeroidsは、溶解緩衝液中でホモジナイズし、その後、GFP ELISAで測定しました。単一のスフェロイドのGFP濃度は、（N = 5、平均±SEM）を算出しました。星は、P &lt;0.05の値を示しています。 （株）=対照; BZB =ボルテゾミブ。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52665/52665fig1large.jpg" target="_blank">この図の拡大版を表示するには、こちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/52665/52665fig2.JPG" /> 2.多発性骨髄腫異種移植モデル図。 （A）発生の日に9 元卵ニワトリ胚を実験グラフトのために使用した。（B）、OPM-2 EGFPおよびRPMI-8226 eGFPの細胞外マトリックス成 ​​分としてコラーゲンタイプI、一次ヒト骨髄間葉系細胞と混合し、そしてボルテゾミブの1 nMのです。凝固スフェロイド（N = 4）をニワトリ胚の漿尿膜上に移植した後にS。（C）5日後CAMにおけるMM移植片のeGFPの発現を可視化することができます。異種移植片は、ステレオ蛍光顕微鏡で毎日撮影することができます。バーは500μmで示す。（D）シングルMM異種移植片は、下にあるCAM組織と切除溶解緩衝液中で均質化し、GFP ELISAで測定しました。単一の腫瘍のGFP濃度は、（N = 12、平均±SEM）を算出しました。星は、P &lt;0.05の値を示しています。 BZB =ボルテゾミブ。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52665/52665fig2large.jpg" target="_blank">この図の拡大版を表示するには、こちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/52665/52665fig3.JPG" /> 骨髄腫血管新生の図3.分析。MM異種移植片（OPM-2 のeGFP）は 5日、ニワトリ胚のCAMに移植した後に記録しました。移植片を対照と比較して、treatmplitidepsin（1nMol、N = 10）との耳鼻咽喉科は、CAM組織による小さい血管が新生した表面的に増殖する腫瘍をもたらしました。血管タイプのグラフィカルモデルに示すようにオンプラント（赤）に成長する血管を計数しました。バーは、1ミリメートルを示す。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52665/52665fig3large.jpg" target="_blank">この図の拡大版を表示するには、こちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/52665/52665fig4.JPG" /> 骨髄腫細胞浸潤の図4.分析。下にあるCAMホスト組織とMM異種移植片（OPM-2 EGFP）の免疫組織化学的分析（対照に対してボルテゾミブ処理されました）。増殖したヒトMM細胞は、キ= 67、CD138およびビ​​メンチンについて陽性に染色し、細胞クラスターが組織をホストとして侵入します。鶏の血管が壁画細胞マーカーASMA（大きな血管と動脈）とデスミン（毛細血管）で染色します。 Magnificati200Xに、アスタリスクは、CAMの血管を示している。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52665/52665fig4large.jpg" target="_blank">この図の拡大版を表示するには、こちらをクリックしてください。</a>

Watch this Scientific Journal Video about Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis at JoVE.com

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

ヒト多発性骨髄腫（MM）細胞は、間葉系細胞と生存および増殖のための細胞外マトリックス成分の支援微小環境が必要です。我々は、腫瘍の増殖、浸潤および血管形成に対する抗がん剤の効果を研究するために移植されたヒト骨髄腫および間葉細胞とin vivoでニワトリ胚モデルを確立しました。

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 ...

establishment-human-multiple-myeloma-xenograft-model-chicken-to-study

Nanyang Technological University

Research

JoVE Journal

Medicine

13.0K ビュー.  Innsbruck Medical University. ヒト多発性骨髄腫（MM）細胞は、間葉系細胞と生存および増殖のための細胞外マトリックス成分の支援微小環境が必要です。我々は、腫瘍の増殖、浸潤および血管形成に対する抗がん剤の効果を研究するために移植されたヒト骨髄腫および間葉細胞とin vivoでニワトリ胚モデルを確立しました。 

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

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

アンインビトロ</em転移性増殖に腫瘍の休眠とスイッチを研究する>システム

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

医学

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

口腔扁平上皮癌の同所性マウスモデルにおける腫瘍細胞の浸潤の多光子イメージング

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

血管新生研究のための角膜ポケットアッセイのマウスモデル

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

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

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 malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

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 ...

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 ...

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 ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

変更された<em&gt;インビトロ</em媒介する癌細胞浸潤におけるホルモン、サイトカインおよび/または成長因子の潜在的な役割を決定するために&gt;浸潤アッセイ

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

腫瘍増殖を研究するためにマウスの乳房脂肪パッド中に乳癌細胞の同所注入。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into 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 ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.