Name: induction and diagnosis of tumors in drosophila imaginal disc epithelia
Uploaded: 2017-07-25T14:00:00
Description: Watch this Scientific Journal Video about Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia at JoVE.com

Vi takker J. Vaughen for kritisk læsning af manuskriptet. Dette arbejde blev støttet af tilskud fra JSPS KAKENHI Grant Numbers 26891025, 15H01500 og The Takeda Science Foundation Research Grant til YT

1. Flykryds og kloninduktion <ol><li> Fjern alle fluer i hætteglasset 12 timer inden samling af jomfru fluer. </li><li> Bedøve fluerne i hætteglasset ved at injicere CO 2 -gas og placere fluer på en CO 2 flyveplade. </li><li> Overfør 10 - 20 jomfruhvaler og 10 hanner fra CO 2 flyvepuden i et frisk hætteglas og inkuber i 1 dag ved 25 ° C. </li><li> Overfør disse fluer i et frisk hætteglas og inkuber i 12 timer ved 25 ° C. BEMÆRK: Kassér det første hætteglas, da ufru...

<ol>
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GAL4-UAS-systemet er et af de mest kraftfulde genetiske værktøjer til målrettet genekspression i Drosophila 26 og letter i høj grad tumorcelleinduktion og -analyse in vivo 4 . Dette system muliggør dannelsen af ​​kloner, der bærer knockdown af tumor-suppressorgener eller overekspression af onkogener inden for vildtype epithelvæv, en situation, der ligner meget de indledende stadier af humant cancer, hvor transformerede pro-tumorceller er omgive...

Epitelvæv er højt organiserede systemer, der har den bemærkelsesværdige homeostatiske evne til at opretholde deres organisation gennem udvikling og celleomsætning. Dette robuste selvorganiserende system forstyrres imidlertid progressivt under tumorudvikling. Ved begyndelsen af ​​tumorudvikling fremkommer individuelle mutantceller, der opstår ved onkogenaktivering eller tumor-suppressor gen inaktivering inden for et epithelialag. Når denne transformerede &quot;pro-tumorcelle&quot; undgår et undertrykkende miljø, forstyrrer epitelorganisationen og begynder ukontrolleret proliferation, forekommer tumorigenese 1 . I løbet af de sidste par årtier har udestående teknologiske fremskridt inden for genetik og molekylærbiologi gjort bemærkelsesværdige fremskridt med kræftforskning. Specielt har nyere undersøgelser, der anvender de genetisk mosaiske analyseværktøjer i Drosophila melanogaster , såsom FLP-FRT (flippase recombinase / flippase recombinase target) mitotisk recombinAtion 2 og flip-out-GAL4-UAS (opstrøms aktiverende sekvens) systemer 3 har i høj grad bidraget til bedre forståelse af de genetiske mekanismer involveret i dannelsen og metastasen af ​​tumorer 4 , 5 , 6 . Undersøgelser af en gruppe af konserverede Drosophila- tumor-suppressor-gener, dødbringende larver ( lgl ), diske store ( dlg ) og scribble ( scrib ) fremhævede det kritiske forhold mellem tab af epithelial organisation og tumorudvikling, da disse gener spiller nøgleroller I regulering af apisk-basalcellepolaritet og celleproliferation i epitelvæv 7 . Mens Drosophila imaginale diske normalt er monolagede epithelia, forårsager homozygote mutationer i et hvilket som helst af disse tre gener celler at tabe struktur og polaritet, undlader at diffeLeje, overproliferere og til sidst danne flerlags amorfe masser, der smelter sammen med tilstødende væv 7 . Tilsvarende er forstyrrelse af disse gener i pattedyr involveret i udviklingen af ​​ondartede tumorer 8 , 9 . De neoplastiske fænotyper udstillet af de mutante væv har ført til klassificeringen af ​​disse tre gener som konserverede neoplastiske tumor-suppressor gener (nTSG&#39;er) 7 , 8 . Når homozygote nTSG-mutante celler imidlertid sporadisk genereres i udvikling af vildtype-imaginale diske ved anvendelse af FLP-FRT-medieret mitotisk rekombination, fjernes mutantceller fra vævet gennem c-Jun N-terminale kinase (JNK) -afhængig apoptose 10 , 11 , 12 , 13 , 14 , ekstrudering 15 </suP&gt; , 16 eller engulfment og fagocytose af naboer 17 . I denne genetisk mosaiske epithelia registreres apoptose for det meste i nTSG-mutante celler, der er placeret ved klongrænsen, hvilket tyder på, at tilstødende normale celler udløser apoptosen af ​​nTSG-mutante celler 10 , 11 , 12 , 18 . Nylige undersøgelser i mammale celler har bekræftet, at denne cellekonkurrenceafhængige eliminering af pro-tumorceller er et evolutionært bevaret epithelial selvforsvarsmekanisme mod cancer 19 , 20 , 21 , 22 , 23 . En nylig undersøgelse i Drosophila- imaginale diske viste imidlertid, at mosaik-nTSG-knockdown-kloner inducerer neoplastiske tumorer i specifikkeIc regioner af vinge imaginal diske 16 . Indledende tumordannelse blev altid fundet i det perifere &quot;hængsel&quot; -område og observeret aldrig i den centrale &quot;pose&quot; -region i vingeskiveepitelet, hvilket tyder på, at det tumorgenetiske potentiale af nTSG-knockdown-celler afhænger af det lokale miljø. Den centrale pose region fungerer som en &quot;tumor coldspot&quot;, hvor pro-tumor celler ikke viser dysplastisk overvækst, mens den perifere hængsel region opfører sig som en &quot;tumor hotspot&quot; 16 . I &quot;coldspot&quot; -poseregioner delaminerer nTSG-knockdown-celler fra basalsiden og gennemgår apoptose. I modsætning hertil, som &quot;hotspot&quot; hængselsceller har et netværk af robuste cytoskeletale strukturer på deres basale sider, delaminerer nTSG-knockdown-celler fra epitelens apikale side og initierer tumorigenisk overgrowth 16 . Derfor kræver analyse af tumorfænotyper i imaginal diske nøje konsistensDeration af den regionspecifikke modtagelighed for tumorigeniske stimuli. Her beskriver vi en protokol til at inducere dannelse af neoplastisk tumor i Drosophila- fløjens imaginale diske, der anvender GAL4-UAS- RNAi- systemet, hvorved nTSG-knockdown-celler genereres i normal wing disc epithelia. Selv om disse eksperimentelle systemer er nyttige til at studere de tidlige stadier af kræft, er en klar klassificeringsmetode til evaluering af stadierne af tumorprogression i imaginal disc epithelia ikke blevet tydeligt beskrevet tidligere. Derfor foreslår vi også en diagnosemetode til klassificering af pro-tumor-klonale fænotyper induceret i vingeskiveepithelia i tre kategorier: hyperplasi (akkumulering af et for stort antal normale fremtrædende celler med forøget proliferation), dysplasi (præalignant væv sammensat af abnormt forekommende Celler) og neoplasi (godartet eller ondartet tumor sammensat af celler med et unormalt udseende og unormalt proliferationsmønster).

<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

I de tidlige stadier af kræft viser transformerede mutante celler cytologiske abnormiteter, begynder ukontrolleret overvækst og gradvist forstyrrer vævsorganisation. Drosophila melanogaster er opstået som et populært eksperimentelt modelsystem i kræftbiologi for at studere tumoregenesens genetiske og cellulære mekanismer. Især aktiverer genetiske værktøjer til Drosophila imaginale diske (udviklende epithelier i larver) skabelsen af ​​transformerede pro-tumorceller inden for et normalt epithelvæv, en situation svarende til de indledende stadier af menneskelig cancer. En nylig undersøgelse af tumorigenese i Drosophila- vinge-imaginale diske viste imidlertid, at tumorinitiering afhænger af den vævsintrinsiske cytoarkitektur og det lokale mikromiljø, hvilket tyder på, at det er vigtigt at overveje den regionspecifikke modtagelighed for tumorigeniske stimuli ved evaluering af tumorfænotyper i imaginal diske. At lette fænotypisk analyse af tumorprogressiPå imaginal diske, her beskriver vi en protokol for genetiske eksperimenter ved hjælp af GAL4-UAS-systemet til at fremkalde neoplastiske tumorer i vinge-imaginale diske. Vi introducerer yderligere en diagnosemetode til klassificering af fænotyper af klonale læsioner induceret i imaginal epithelia, da en klar klassificeringsmetode til at diskriminere forskellige stadier af tumorprogression (såsom hyperplasi, dysplasi eller neoplasi) ikke tidligere var beskrevet. Disse metoder kan være bredt anvendelige til klonanalysen af ​​tumorfænotyper i forskellige organer i Drosophila .

For at demonstrere neoplastisk tumordannelse induceret eksperimentelt med RNAi- medieret nTSG-knockdown i Drosophila- vinge-imaginale diske, blev tre forskellige GAL4-drivere brugt til at udtrykke UAS-RNAi for lgl eller scrib : (1) sd-GAL4, som driver stærkt UAS-udtryk i Vingepose og mildt udtryk i hængselregionerne ( figur 2 og figur 3A ); (2) upd-GAL4, som kører...

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

Mosaik klonanalyse i Drosophila imaginal disc epithelia er et kraftfuldt model system til at studere tumoregenesens genetiske og cellulære mekanismer. Her beskriver vi en protokol til at fremkalde tumorer i Drosophila- vinge-imaginale diske ved hjælp af GAL4-UAS-systemet og indføre en diagnosemetode til klassificering af tumorfænotyperne.

Induktion og diagnose af tumorer i<em&gt; Drosophila</em&gt; Imaginal Disc Epithelia

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

Research

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Cancer Research

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

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

Next Generation Sequencing til påvisning af søgsmål mutationer i fast og flydende tumorer

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

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

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

Det<em&gt; Drosophila</em&gt; Imaginære Disc Tumor Model: Visualisering og Kvantificering af genekspression og tumorindtrængen Brug Genetic Mosaics

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

Udvikling af en menneskelig prækliniske Model af Osteoclastogenesis fra perifert blod monocytter Co kulturperler med bryst kræft cellelinjer

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

Etablering af en primær kultur af Patient-afledte bløddelssarkom

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

Berigelse og karakterisering af Tumor immun- og ikke-immune Microenvironments i etablerede subkutane Murine tumorer

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

Murine model af metastatisk levertumorer i fastsættelsen af ischemia reperfusion skade

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

Opnåelse kræft stamceller kugler fra gynækologiske og brystkræft tumorer

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

Ortotopisk transplantation af brysttumorer som prækliniske modeller for brystkræft

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

Quail Chorioallantoic membran - et værktøj til fotodynamisk diagnose og terapi

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

Højopløsnings ultralyd til analyse af ortopiske ATC-tumorer i en genetisk manipuleret musemodel

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