私たちは、原稿の批判的読解に対するJ.ヴォーゲンに感謝します。この研究は、日本学術振興会グラント番号26891025,15H01500およびYTへの武田科学財団研究助成金

1.フライクロスクローン誘導<ol><li>バイアルのハエを収集する前に、バイアル内のすべてのハエを12時間後に除去する。 </li><li>バイアル内のハエをCO 2ガスを注入して麻酔し、飛行船をCO 2フライパッドに置きます。 </li><li> CO 2フライパッドから10〜20匹の未処理の雌と10匹の雄を新鮮なバイアルに移し、25℃で1日間インキュベートする。 </li><li>これらのハエを新鮮なバイアルに移し、25℃で12時間インキュベートする。 注：最初の日に十分な卵を置いていないバージンの雌が最初のバイアルを捨てます。 </li><li>エンハンサー-GAL4系統については、成虫のハエを除去し、解剖まで25℃でバイアルをインキュベートする。 </li><li>フリップアウトGAL4によるモザイククローンの誘導のために、成虫のハエを除去し、バイアルを25℃で2〜4日間インキュベートする。熱ショック前のインキュベーション時間は、実験目的に依存する。卵の沈着の2日後の熱ショック（AED）は、未成熟の第2期にモザイククローンを生成する想像上皮を始める。 3または4日後の熱ショックAEDは、分化した早期から中期の第3齢の上皮上皮においてモザイククローンを生成する。 注：腫瘍原性表現型に対する熱ショックタイミングの変化の影響を調べることが重要である。 </li><li>フリップアウト-GAL4によるモザイククローンの誘導のために、バイアルを37℃の水浴中に10〜45分間浸漬し、25℃で2〜3日間インキュベートすることによってバイアルを熱ショックする。 注：各実験における熱ショック時間、タイミング、およびインキュベーション時間の情報は、図の凡例に示されています。 </li></ol> 2.幼虫の解剖<ol><li>蛍光立体顕微鏡下で適切な蛍光マーカー（ 例えば 、EGFP）で遺伝子型を木製スティックまたは鈍い鉗子で放浪している第3齢の幼虫を収集し、PBS（リン酸緩衝食塩水）を含む解剖皿に入れる。 </li><li> 2mLのプラスチックトランスファーピペットでピペッティングすることにより、PBS中の幼虫を洗浄する。 </li><li>1つの鉗子で幼虫の中心をピンチし、他の鉗子で半分に体を裂く。 </li><li> 1つの鉗子で前半の口のフックを挟み、他の鉗子で体に向かって口を押して、体を裏返しにします。 </li><li>唾液腺や脂肪体など不要な物質を鉗子で取り除きます。 </li></ol> 3.固定および抗体染色<ol><li>幼虫体の解剖学的前半部（imaginal wing discsを含む）を1.5mLプラスチックチューブに移し、穏やかに回転させながら暗所で室温で10分間Fix溶液（PBS中4％ホルムアルデヒド）1mLを固定する。 注意：ホルムアルデヒドは有毒で発癌性があります。皮膚に接触しないよう保護手袋と衣類を着用する。 注意：この部分では、特に明記されていない限り、すべてのステップは暗所で室温でナッターで行われます。 </li><li> Fixソリューションを削除して破棄します。組織を1mLのPBT（0.3％TritPBS中のX-100）上で3回、各15分間。 </li><li> PBTを除去し、ブロッキングのためにPBTG（0.2％ウシ血清アルブミンおよび5％正常ヤギ血清）1mLを添加し、室温で1時間または4℃で一晩インキュベートする。 </li><li> PBTGを除去し、PBTG（ 材料表を参照）で適切に希釈した一次抗体溶液を加え、4℃で一晩インキュベートする。 </li><li>一次抗体溶液を除去し、1mLのPBTで組織を3回、それぞれ15分間洗浄する。 </li><li> PBTを除去し、PBTG（1：400）で適切に希釈した二次抗体溶液を加える。室温で2時間、または4℃で一晩、ナットする。 </li><li>二次抗体溶液を除去し、1mLのPBTで組織を2回、それぞれ15分間洗浄する。 </li><li> F-アクチンを染色するために、PBTを除去し、PBS（1:40）で適切に希釈したPhalloidin溶液を加える。次に20分間寝かせる。ファロイジン溶液を除去し、1mLのPBTで組織を2回、それぞれ15分間洗浄する。 </li><li>核DNAを対比染色するために、PBTを除去し、DAPI（4 &#39;、6-ジアミジノ-2-フェニルインドール）溶液（PBS中0.5μg/ mLのDAPI）を加える。その後、10分間ナットする。 注意：DAPIは発癌性があります。皮膚に接触しないよう保護手袋と衣類を着用する。 </li><li> DAPI溶液を除去し、1mLのPBTで組織を2回、それぞれ15分間洗浄する。 </li><li>室温で10分間、1mLのPBSで1回すすいでください。 </li><li> PBSを除去し、プレマウント培地として500μLの100％グリセロールを添加する。 </li></ol> 4.顕微鏡スライドへの取り付け<ol><li> 2mLのプラスチックトランスファーピペットを用いて顕微鏡スライド上に染色した組織を置く。 </li><li>鉗子で別の顕微鏡スライド上のマウント媒体の滴に組織を転送します。 </li><li> 1つの鉗子で解剖組織の終わりを押さえ、脳と目の触角円板を他の鉗子で引き離します。 注：脳を翼の想像上のディスクの近くに置くように切開したままにしておきます。頭脳は、カバーガラスが羽の想像上のディスクを粉砕するのを防ぐプラットフォームとして機能する。 </li><li>翼の想像上のディスクを隔離するには、解剖された組織の端を1つの鉗子で押さえ、体の壁を静かに引っ掻き、他の鉗子で椎間板を引き裂く。 注：羽の想像上のディスクを見つけることが困難な場合は、後部から前側に気管を剥がします。ウィングの想像上の円板は気管にくっついている。 </li><li>穏やかにカバーガラスでイーガルディスクを覆い、マニキュアでシールします。 4℃で保存する。 </li></ol> 5.共焦点顕微鏡法<ol><li>発光波長の範囲、レーザ強度、ゲイン、オフセット、走査速度と画像サイズ24を含む共焦点顕微鏡設定された画像取得パラメータを用いた共焦点画像を取得します。 注：画像取得設定の詳細な手順については、各顕微鏡メーカーが提供する取扱説明書を参照してください。 </li><li>単一の共同をキャプチャするにはウイングイメージディスク全体の非焦点部分には、20倍の対物レンズを使用します。 </li><li> 40倍または60倍のレンズを使用して、0.5〜1.0μmのステップサイズでzスタックスキャンによって3次元画像を取得します。 注：細胞の表現型を高解像度で解析するには、画像サイズが512 x 512ピクセルより大きくなければなりません。 </li></ol> 6. ImageJを用いた画像解析<ol><li>共焦点zスタック画像を取得し、分析するために、フィジー、生体画像分析（https://fiji.sc/）25に集中オープンソースのImageJソフトウェアを使用します。 </li><li>垂直セクションを取得するには、ImageJでzスタックイメージを開き、メニューアイテム &quot;Image / Stacks / Reslice&quot;を選択します。 注：XZ軸またはYZ軸のいずれかは、[再分割]メニューで選択できます。垂直断面は、zスタック画像上に直線または矩形を描くことによって任意の方向に得ることもできる。 </li></ol> 7.新生物表現型の診断<ol><li>垂直を開く1組のz-スタック画像から得られた切片（XZまたはYZ軸）を分析し、形態学的表現型および抗体染色を分析する。 </li><li> （1）ノックダウンクローンを含む細胞塊が主上皮層から逸脱する場合、（2）接合細胞タンパク質の細胞内局在がこの細胞塊で変化した場合、腫瘍表現型の診断のために、 、および（3）この細胞塊の直径が4細胞より大きい場合。 </li><li> 図1に示すフローチャートに従って、腫瘍表現型を分類する。 </li></ol>

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	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hallmarks+of+cancer.">The hallmarks of cancer.</a> Cell. 100 (1), 57-70 (2000).</li><li>Xu, T., Rubin, 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=Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues.">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117 (4), 1223-1237 (1993).</li><li>Struhl, G., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organizing+activity+of+wingless+protein+in+Drosophila.">Organizing activity of wingless protein in Drosophila.</a> Cell. 72 (4), 527-540 (1993).</li><li>Potter, C. J., Turenchalk, G. S., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+in+cancer+research.+An+expanding+role.">Drosophila in cancer research. An expanding role.</a> Trends Genet. 16 (1), 33-39 (2000).</li><li>Miles, W. O., Dyson, N. J., Walker, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tumor+invasion+and+metastasis+in+Drosophila.">Modeling tumor invasion and metastasis in Drosophila.</a> Dis Model Mech. 4 (6), 753-761 (2011).</li><li>Tipping, M., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+context-dependent+tumorigenesis.">Drosophila as a model for context-dependent tumorigenesis.</a> J Cell Physiol. 229 (1), 27-33 (2013).</li><li>Bilder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+polarity+and+proliferation+control:+links+from+the+Drosophila+neoplastic+tumor+suppressors.">Epithelial polarity and proliferation control: links from the Drosophila neoplastic tumor suppressors.</a> Genes Dev. 18 (16), 1909-1925 (2004).</li><li>Humbert, P. O., 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=Control+of+tumourigenesis+by+the+Scribble/Dlg/Lgl+polarity+module.">Control of tumourigenesis by the Scribble/Dlg/Lgl polarity module.</a> Oncogene. 27 (55), 6888-6907 (2008).</li><li>Huang, L., Muthuswamy, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+protein+alterations+in+carcinoma:+a+focus+on+emerging+roles+for+polarity+regulators.">Polarity protein alterations in carcinoma: a focus on emerging roles for polarity regulators.</a> Curr Opin Genet Dev. 20 (1), 41-50 (2010).</li><li>Brumby, A. M., Richardson, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=scribble+mutants+cooperate+with+oncogenic+Ras+or+Notch+to+cause+neoplastic+overgrowth+in+Drosophila.">scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila.</a> EMBO J. 22 (21), 5769-5779 (2003).</li><li>Igaki, T., Pastor-Pareja, J. C., Aonuma, H., Miura, M., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+tumor+suppression+and+epithelial+maintenance+by+endocytic+activation+of+Eiger/TNF+signaling+in+Drosophila.">Intrinsic tumor suppression and epithelial maintenance by endocytic activation of Eiger/TNF signaling in Drosophila.</a> Dev Cell. 16 (3), 458-465 (2009).</li><li>Tamori, Y., 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=Involvement+of+Lgl+and+Mahjong/VprBP+in+cell+competition.">Involvement of Lgl and Mahjong/VprBP in cell competition.</a> PLoS Biol. 8 (7), e1000422 (2010).</li><li>Cordero, J. B., 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=Oncogenic+Ras+diverts+a+host+TNF+tumor+suppressor+activity+into+tumor+promoter.">Oncogenic Ras diverts a host TNF tumor suppressor activity into tumor promoter.</a> Dev Cell. 18 (6), 999-1011 (2010).</li><li>Yamamoto, M., Ohsawa, S., Kunimasa, K., Igaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ligand+Sas+and+its+receptor+PTP10D+drive+tumour-suppressive+cell+competition.">The ligand Sas and its receptor PTP10D drive tumour-suppressive cell competition.</a> Nature. 542 (7640), 246-250 (2017).</li><li>Vaughen, J., Igaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slit-Robo+Repulsive+Signaling+Extrudes+Tumorigenic+Cells+from+Epithelia.">Slit-Robo Repulsive Signaling Extrudes Tumorigenic Cells from Epithelia.</a> Dev Cell. 39 (6), 683-695 (2016).</li><li>Tamori, Y., Suzuki, E., Deng, W. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+Tumors+Originate+in+Tumor+Hotspots,+a+Tissue-Intrinsic+Microenvironment.">Epithelial Tumors Originate in Tumor Hotspots, a Tissue-Intrinsic Microenvironment.</a> PLoS Biol. 14 (9), e1002537 (2016).</li><li>Ohsawa, S., 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=Elimination+of+oncogenic+neighbors+by+JNK-mediated+engulfment+in+Drosophila.">Elimination of oncogenic neighbors by JNK-mediated engulfment in Drosophila.</a> Dev Cell. 20 (3), 315-328 (2011).</li><li>Men&#233;ndez, J., P&#233;rez-Garijo, A., Calleja, M., Morata, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tumor-suppressing+mechanism+in+Drosophila+involving+cell+competition+and+the+Hippo+pathway.">A tumor-suppressing mechanism in Drosophila involving cell competition and the Hippo pathway.</a> Proc Natl Acad Sci U S A. 107 (33), 14651-14656 (2010).</li><li>Hogan, C., 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=Characterization+of+the+interface+between+normal+and+transformed+epithelial+cells.">Characterization of the interface between normal and transformed epithelial cells.</a> Nat Cell Biol. 11 (4), 460-467 (2009).</li><li>Kajita, 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=Interaction+with+surrounding+normal+epithelial+cells+influences+signalling+pathways+and+behaviour+of+Src-transformed+cells.">Interaction with surrounding normal epithelial cells influences signalling pathways and behaviour of Src-transformed cells.</a> J Cell Sci. 123 (Pt 2), 171-180 (2010).</li><li>Norman, 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=Loss+of+Scribble+causes+cell+competition+in+mammalian+cells.">Loss of Scribble causes cell competition in mammalian cells.</a> J Cell Sci. 125 (1), 59-66 (2012).</li><li>Wagstaff, L., 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=Mechanical+cell+competition+kills+cells+via+induction+of+lethal+p53+levels.">Mechanical cell competition kills cells via induction of lethal p53 levels.</a> Nat Commun. 7, 1-14 (2016).</li><li>Kajita, M., Fujita, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EDAC:+Epithelial+defence+against+cancer-cell+competition+between+normal+and+transformed+epithelial+cells+in+mammals.">EDAC: Epithelial defence against cancer-cell competition between normal and transformed epithelial cells in mammals.</a> J Biochem. 158 (1), 15-23 (2015).</li><li>North, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing?+A+beginners'+guide+to+practical+pitfalls+in+image+acquisition.">Seeing is believing? A beginners' guide to practical pitfalls in image acquisition.</a> J Cell Biol. 172 (1), 9-18 (2006).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118 (2), 401-415 (1993).</li><li>Jory, A., 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+survey+of+6,300+genomic+fragments+for+cis-regulatory+activity+in+the+imaginal+discs+of+Drosophila+melanogaster.">A survey of 6,300 genomic fragments for cis-regulatory activity in the imaginal discs of Drosophila melanogaster.</a> Cell Rep. 2 (4), 1014-1024 (2012).</li><li>McGuire, S. E., Le, P. T., Osborn, A. J., Matsumoto, K., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+rescue+of+memory+dysfunction+in+Drosophila.">Spatiotemporal rescue of memory dysfunction in Drosophila.</a> Science. 302 (5651), 1765-1768 (2003).</li><li>Rodrigues, A. B., 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=Activated+STAT+regulates+growth+and+induces+competitive+interactions+independently+of+Myc,+Yorkie,+Wingless+and+ribosome+biogenesis.">Activated STAT regulates growth and induces competitive interactions independently of Myc, Yorkie, Wingless and ribosome biogenesis.</a> Development. 139 (21), 4051-4061 (2012).</li><li>Khan, S. 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=Epithelial+neoplasia+in+Drosophila+entails+switch+to+primitive+cell+states.">Epithelial neoplasia in Drosophila entails switch to primitive cell states.</a> Proc Natl Acad Sci U S A. 110 (24), E2163-E2172 (2013).</li><li>Pagliarini, R. A., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+screen+in+Drosophila+for+metastatic+behavior.">A genetic screen in Drosophila for metastatic behavior.</a> Science. 302 (5648), 1227-1231 (2003).</li><li>Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster:+a+model+and+a+tool+to+investigate+malignancy+and+identify+new+therapeutics.">Drosophila melanogaster: a model and a tool to investigate malignancy and identify new therapeutics.</a> Nat Rev Cancer. 13 (3), 172-183 (2013).</li><li>Patel, P. H., Edgar, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+design:+how+Drosophila+tumors+remodel+their+neighborhood.">Tissue design: how Drosophila tumors remodel their neighborhood.</a> Semin Cell Dev Biol. 28, 86-95 (2014).</li><li>Nakajima, Y. -. I., Meyer, E. J., Kroesen, A., McKinney, S. A., Gibson, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+junctions+maintain+tissue+architecture+by+directing+planar+spindle+orientation.">Epithelial junctions maintain tissue architecture by directing planar spindle orientation.</a> Nature. 500 (7462), 359-362 (2013).</li><li>Colombani, J., Andersen, D. S., L&#233;opold, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+peptide+Dilp8+coordinates+Drosophila+tissue+growth+with+developmental+timing.">Secreted peptide Dilp8 coordinates Drosophila tissue growth with developmental timing.</a> Science. 336 (6081), 582-585 (2012).</li><li>Garelli, A., Gontijo, A. M., Miguela, V., Caparros, E., Dominguez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaginal+discs+secrete+insulin-like+peptide+8+to+mediate+plasticity+of+growth+and+maturation.">Imaginal discs secrete insulin-like peptide 8 to mediate plasticity of growth and maturation.</a> Science. 336 (6081), 579-582 (2012).</li></ol>

著者は何も開示することはない。

GAL4-UASシステムは、 ショウジョウバエ 26の標的遺伝子発現のための最も強力な遺伝子ツールの1つであり、in vivoで腫瘍細胞の誘導と解析を大いに容易にします4 。このシステムは、野生型上皮組織内の腫瘍抑制遺伝子のノックダウンまたは癌遺伝子の過剰発現を有するクローンの生成を可能にする。これは、形質転換された前腫瘍細胞が正常?...

上皮組織は、発達および細胞代謝回転を通じて組織を維持する顕著なホメオスタシス能力を有する高度に組織化されたシステムである。しかしながら、この堅牢な自己組織化システムは、腫瘍発生の間に次第に破壊される。腫瘍発生の開始時に、腫瘍遺伝子活性化または腫瘍抑制遺伝子不活性化から生じる個々の変異細胞が上皮層内に出現する。この形質転換された「プロ腫瘍細胞」が抑制的環境を回避し、上皮組織を破壊し、制御されない増殖を開始すると、腫瘍形成が起こる1 。過去数十年の間に、遺伝学および分子生物学における優れた技術的進歩は、がん研究において顕著な進歩を遂げました。特に、FLP-FRT（フリッパーゼリコンビナーゼ/フリッパーゼリコンビナーゼ標的）有糸分裂組換え体のようなショウジョウバエメラノガスターにおける遺伝的モザイク分析ツールを用いた最近の研究エーション2とフリップアウト-GAL4-UAS（上流活性化配列）システム3、大幅に良好腫瘍4、5、6の形成および転移に関与する遺伝的メカニズムを理解することに貢献しています。 保存されたショウジョウバエ腫瘍サプレッサー遺伝子、 致死性の巨大幼虫 （ lgl ）、 大型ディスク （ dlg ）、 落書き （ scrib ）の研究は、これらの遺伝子が重要な役割を果たすため、上皮組織の消失と腫瘍発生の重要な関係を明らかにした上皮組織7で、頂端-基底細胞極性および細胞増殖の調節インチショウジョウバエの原始椎間板は通常単層上皮であるが、これらの3つの遺伝子のいずれかのホモ接合突然変異は、細胞が構造および極性を失い、過剰増殖し、​​最終的に隣接する組織と融合する多層アモルファス塊を形成する7 。同様に、哺乳動物におけるこれらの遺伝子の破壊は、悪性腫瘍8,9の開発に関与しています。変異体組織によって示される腫瘍性表現型は、保存、新生物、腫瘍抑制遺伝子（nTSGs）として、これら三つの遺伝子の分類7,8につながっています。ホモ接合nTSG変異細胞は散発的にFLP-FRT媒介有糸分裂組換えを使用して、野生型の成虫を開発する際に生成される場合しかし、変異細胞はc-Jun N末端キナーゼ（JNK）依存性アポトーシス10、11を介して組織から除去されます、12、13、14、15、押出し</sup&gt; 、 16 、または隣人による貪食および食作用17 。この遺伝的にモザイク上皮では、アポトーシスは主に隣接する正常細胞はnTSG変異細胞10、11、12、18のアポトーシスを引き起こすことを示唆し、クローンの境界に位置nTSG変異体細胞において検出されます。哺乳動物細胞における最近の研究では、プロの腫瘍細胞のこの細胞競争依存除去は癌19、20、21、22、23に対する進化的に保存された上皮の自己防御機構であることを確認しました。 しかし、最近のショウジョウバエの成虫の研究では、モザイクのnTSGノックダウンクローンが腫瘍性腫瘍を特異的に誘導することが示されています翼の想像上のディスクのIC領域16 。初期の腫瘍形成は末梢「ヒンジ」領域に常に見られ、ウイングディスク上皮の​​中央「ポーチ」領域では決して観察されず、nTSGノックダウン細胞の腫瘍形成能は局所環境に依存することが示唆された。中央ポーチ領域は、プロ腫瘍細胞が形成異常増殖を示さない一方で、周辺ヒンジ領域が「腫瘍ホットスポット」として振る舞う「腫瘍コールドスポット」として機能する16 。 「コールドスポット（cold spot）」ポーチ領域では、nTSG-ノックダウン細胞は基底面から剥離し、アポトーシスを起こす。対照的に、「ホットスポット」ヒンジ細胞は基底面に頑強な細胞骨格構造のネットワークを持っているため、nTSGノックダウン細胞は上皮の頂端側から剥離し、腫瘍原性の過増殖を開始する16 。したがって、原始椎間板における腫瘍表現型の解析には、注意深い検討が必要である腫瘍形成刺激に対する領域特異的感受性の低下。 ここでは、正常なウイングディスク上皮においてnTSGノックダウン細胞が生成されるGAL4-UAS- RNAiシステムを利用して、 ショウジョウバエのウイングのヘルニア椎間板に腫瘍性腫瘍形成を誘導するためのプロトコールについて説明する。これらの実験系は癌の初期段階を研究するのに有用であるが、椎間板上皮における腫瘍進行の段階を評価するための明確な分類方法はこれまで明らかにされていない。したがって、我々はまた、ウイングディスク上皮に誘導された前腫瘍クローン表現型を3つのカテゴリーに分類する診断法を提案する：過形成（増殖が増加した正常数の細胞の過剰な蓄積）、異形成（異常に出現する前悪性組織細胞）、および新生物（異常な外観および異常な増殖パターンを有する細胞からなる良性または悪性の腫瘍）。

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Wako</td>
			<td>162-19321</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Wako</td>
			<td>168-11805</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Wako</td>
			<td>064-00406</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>normal goat serum</td>
			<td>Sigma</td>
			<td>G6767</td>
			<td></td>
		</tr>
		<tr>
			<td>mounting medium, Vectashield</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse-anti-Dlg 4F3</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>4F3 anti-discs large, RRID:AB_528203</td>
			<td>dilute in PBTG, 1:40</td>
		</tr>
		<tr>
			<td>mouse-anti-MMP1</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>3A6B4, RRID:AB_579780</td>
			<td>3 mixed 1:1:1 and dilute in PBTG, 1:40</td>
		</tr>
		<tr>
			<td>mouse-anti-MMP1</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>3B8D12, RRID:AB_579781</td>
			<td>3 mixed 1:1:1 and dilute in PBTG, 1:40</td>
		</tr>
		<tr>
			<td>mouse-anti-MMP1</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>5H7B11, RRID:AB_579779</td>
			<td>3 mixed 1:1:1 and dilute in PBTG, 1:40</td>
		</tr>
		<tr>
			<td>mouse-anti-atubulin</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>AA4.3, RRID:AB_579793</td>
			<td>dilute in PBTG, 1:100</td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 Phalloidin</td>
			<td>Molecular probes</td>
			<td>A22283</td>
			<td>dilute in PBS, 1:40</td>
		</tr>
		<tr>
			<td>goat anti-mouse IgG antibody, Alexa Fluor 546</td>
			<td>Molecular probes</td>
			<td>A11030</td>
			<td>dilute in PBTG, 1:400</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Fly strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sd-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#8609</td>
			<td>recombined with UAS-EGFP</td>
		</tr>
		<tr>
			<td>upd-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#26796</td>
			<td>recombined with UAS-EGFP</td>
		</tr>
		<tr>
			<td>UAS-lgl-RNAi</td>
			<td>Vienna Drosophila RNAi center</td>
			<td>#51247</td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-scrib-RNAi</td>
			<td>Vienna Drosophila RNAi center</td>
			<td>#105412</td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-RasV12</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#64196</td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-Yki3SA</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#28817</td>
			<td></td>
		</tr>
		<tr>
			<td>hsFLP</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#6</td>
			<td></td>
		</tr>
		<tr>
			<td>Act&#62;CD2&#62;GAL4 (flip-out GAL4)</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#4780</td>
			<td>recombined with UAS-EGFP</td>
		</tr>
		<tr>
			<td>UAS-EGFP</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#5428</td>
			<td>X chromosome</td>
		</tr>
		<tr>
			<td>UAS-EGFP</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#6658</td>
			<td>third chromosome</td>
		</tr>
		<tr>
			<td>UAS-Dicer2</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#24650</td>
			<td>second chromosome</td>
		</tr>
		<tr>
			<td>UAS-Dicer2</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#24651</td>
			<td>third chromosome</td>
		</tr>
		<tr>
			<td>vkg-GFP</td>
			<td>Morin et al. 2001</td>
			<td></td>
			<td>GFP protein trap</td>
		</tr>
	</tbody>
</table>

induction and diagnosis of tumors in drosophila imaginal disc epithelia

癌の初期段階では、形質転換された突然変異細胞は細胞学的異常を示し、制御されない過増殖を開始し、組織組織化を漸進的に破壊する。 Drosophila melanogasterは、腫瘍発生の遺伝的メカニズムおよび細胞メカニズムを研究するために、癌生物学における一般的な実験モデルシステムとして登場しました。特に、 ショウジョウバエ （ Drosophila） imaginal disc（幼生の上皮発生）のための遺伝的ツールは、正常な上皮組織内で形質転換された前腫瘍細胞の生成を可能にする。これはヒト癌の初期段階と同様の状況である。しかし、 ショウジョウバエの翅の椎間板の腫瘍形成に関する最近の研究では、腫瘍の開始は組織固有の細胞構造および局所微小環境に依存することが示され、imaginalの腫瘍表現型を評価する際の腫瘍原性刺激に対する領域特異的感受性を考慮することが重要であるディスク。腫瘍進行の表現型分析を容易にするためにGAL4-UASシステムを用いた遺伝子実験のプロトコールについて説明します。この実験では、翅の椎間板に腫瘍性腫瘍を誘導します。以前には報告されていなかった腫瘍の進行の様々な段階（肥厚、形成異常または腫瘍形成）を識別するための明確な分類方法として、想像上皮に誘発されたクローン病変の表現型を分類するための診断方法をさらに紹介する。これらの方法は、 ショウジョウバエの種々の臓器における腫瘍表現型のクローン分析に広く適用可能であろう。

ショウジョウバエのウイング上の椎間板におけるRNAi媒介性のnTSGノックダウンによって実験的に誘導された腫瘍形成を実証するために、3種の異なるGAL4ドライバを用いてlglまたはscribに対するUAS-RNAiを発現さ せた ：（1） sd-GAL4ウィングポーチ、およびヒンジ領域における軽度の発現（ 図2およ...

Watch this Scientific Journal Video about Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia at JoVE.com

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

ショウジョウバエの上椎間板上皮におけるモザイククローン解析は、腫瘍形成の遺伝的メカニズムおよび細胞メカニズムを研究するための強力なモデルシステムです。ここでは、GAL4-UASシステムを用いて、 ショウジョウバエの翅の椎間板に腫瘍を誘導するプロトコールについて説明し、腫瘍の表現型を分類するための診断法を紹介する。

腫瘍の誘導および診断<em&gt;ショウジョウバエ</em&gt; Imaginal Disc Epithelia

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue ...

induction-diagnosis-tumors-drosophila-imaginal-disc

Research

JoVE Journal

Cancer Research

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

14.8K ビュー.  SOKENDAI.  ショウジョウバエの上椎間板上皮におけるモザイククローン解析は、腫瘍形成の遺伝的メカニズムおよび細胞メカニズムを研究するための強力なモデルシステムです。ここでは、GAL4-UASシステムを用いて、 ショウジョウバエの翅の椎間板に腫瘍を誘導するプロトコールについて説明し、腫瘍の表現型を分類するための診断法を紹介する。 

ビデオ: Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

固体と液体の腫瘍におけるアクショナブル変異の検出のための次世代シーケンシング

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

がん研究

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

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

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

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

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

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

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

破骨細胞形成乳房癌細胞と共培養末梢血単球からの人間の前臨床モデルの開発

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

患者由来の軟部肉腫の主な文化の確立

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

濃縮と確立された皮下マウス腫瘍における腫瘍免疫と免疫のない微小のキャラクタリゼーション

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

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

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

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

虚血多灌流損傷の設定における転移性肝腫瘍のマウスモデル

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

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

婦人科腫瘍および乳がん腫瘍から癌幹細胞球体を取得する

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

乳癌の前臨床モデルとしての乳房腫瘍の移植性

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties make it an excellent in vivo experimental model to study angiogenesis, tumor growth, drug delivery systems, or photodynamic diagnosis (PDD) and photodynamic therapy (PDT). At the same time, this model addresses the requirement for the replacement of experimental animals with a suitable alternative. Ex ovo cultivated embryo allows easy substance application, access, monitoring, and documentation. The most frequently used is chick CAM; however, this article describes the advantages of the Japanese quail CAM as a low-cost and high-throughput model. Another advantage is the shorter embryonic development, which allows higher experimental turnover. The suitability of quail CAM for PDD and PDT of cancer and microbial infections is explored here. As an example, the use of the photosensitizer hypericin in combination with lipoproteins or nanoparticles as a delivery system is described. The damage score from images in white light and changes in fluorescence intensity of the CAM tissue under violet light (405 nm) was determined, together with analysis of histological sections. The quail CAM clearly showed the effect of PDT on the vasculature and tissue. Moreover, changes like capillary hemorrhage, thrombosis, lysis of small vessels, and bleeding of larger vessels could be observed. Japanese quail CAM is a promising in vivo model for photodynamic diagnosis and therapy research, with applications in studies of tumor angiogenesis, as well as antivascular and antimicrobial therapy.

The chorioallantoic membrane (CAM) of the avian embryo is a very useful and applicable tool for various areas of research. A special ex ovo model of Japanese quail CAM is suitable for photodynamic treatment investigation.

ウズラ絨毛尿膜 - 光線力学診断と治療のためのツール

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties ...

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes significantly. Genetically engineered murine models that mimic ATC&#39;s progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; BrafCA/wt; Trp53&#916;ex2-10/&#916;ex2-10&#160;transgenic ATC model was developed. The ATC murine model was induced by an intraperitoneal injection of tamoxifen with overexpression of BrafV600E and deletion of Trp53, and the tumors were generated within about 1 month. High-resolution ultrasound was applied to investigate the tumor initiation and progression, and the dynamic growth curve was obtained by measuring the tumor sizes. Compared to magnetic resonance imaging (MRI) and computed tomography scanning, ultrasound has advantages in observing the ATC murine model, such as being noninvasive, portable, in real-time, and without radiation exposure. High-resolution ultrasound is suitable for dynamic and multiple measurements. However, ultrasonographic examination of the thyroid in mice requires relevant anatomical knowledge and experience. This article provides a detailed procedure for utilizing high-resolution ultrasound to scan tumors in the transgenic ATC model. Meanwhile, ultrasonic parameter adjustment, ultrasound scanning skills, anesthesia and recovery of the animals, and other elements that need attention during the process are listed.

The present protocol describes high-frequency ultrasonography for visualizing the entire mouse thyroid gland and monitoring the growth of anaplastic thyroid carcinoma.

遺伝子改変マウスモデルにおける同所性ATC腫瘍の解析のための高解像度超音波検査

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes ...