Forfatterne vil gerne takke UC Davis Mouse Biologi Program til avl af mus. Denne forskning blev støttet af en bevilling fra Merck KGaA, Darmstadt, Tyskland.

Alle dyreforsøg og eksperimenter blev udført under en protokol godkendt af University of California, Davis Institutional Animal Care og brug administrative rådgivende udvalg. 1.. MUC1.Tg Mouse Avl og opformering <ol><li> UC Davis Mouse Biologi Program (MBP) racer vildtype C57BL / 6 hanmus med heterozygot MUC1.Tg C57BL / 6 hunmus at etablere vores opdræt koloni. MUC1.Tg afkom leveres til undersøgelser efter behov. </li><li> MBP personale klippe tæerne af afkommet i en defineret mønst...

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Den vellykkede induktion af invasive overgangs-og planocellulært blærecarcinom i menneskelige MUC1.Tg mus tilbyder en præklinisk model for Immunterapi udvikling. Immunterapeutiske undersøgelser kræver brug af en spontan, immun intakt model for at vurdere den inflammatoriske respons på tumorprogression over tid samt immunresponset over for immunterapi. I en spontan tumor udvikling model forbliver tumormikromiljøet intakt og tumorer udvikler sig en mere repræsentativ vækstrate, der giver mulighed for vurdering af...

Blærekræft er den fjerde mest almindelige form for kræft og den ottende hyppigste årsag til kræftdødsfald i amerikanske mænd. I USA, er en anslået 72.500 nye tilfælde og 15.000 dødsfald som følge blærekræft forventes blandt mænd og kvinder samlet i 2013 1. Forekomsten af ​​blærekræft er cirka tre gange så højt hos mænd end hos kvinder. I USA tegner transitional cell carcinomer (TCC) for over 90% af tilfældene, mens pladecellecarcinomer (SCC) har en hyppighed på mindre end 2% 2. Den generelle relative 5-års overlevelse for papillary TCC er 91,5% sammenlignet med kun 30,9% for SCC 2.. Selvom noninvasive papillary TCCs tegner sig for cirka 75% af tilfældene på tidspunktet for diagnosen, selv med behandling mere end 50% af patienterne vil opleve en gentagelse inden for 5 år, med op til 30% af disse patienter udvikler sig til muskler invasiv sygdom 3,4 . Typiske behandlingsregimer for ikke-muskel-invasive sygdom omfatter transuretral resektion (TUR) efterfulgt af intravesikal kemoterapi. Hos patienter med high-grade Ta eller T1 tumorer kan en gentagelse TUR udføres før kemoterapi 3,4. For de patienter med lav-grade Ta tilbagefald eller high-grade Ta eller T1 læsioner, efterfulgt TUR af adjuvans kemoterapi eller immunterapi i form af Bacillus Calmette-Guerin (BCG), kan anvendes 3,4. Intravesikal BCG har vist sig at være overlegen i forhold til intravesikal mitomycin C med hensyn til tiden til tilbagefald 5. For T2 muskel invasiv sygdom, er den radikale cystectomy med eller uden neoadjuverende kemoterapi den anbefalede behandlingsforløb 3.. Hos patienter med SCC, synes radikal cystektomi at være den mest effektive behandling 6.. I betragtning af de meget høje tilbagefald på trods af de bedste behandlinger til rådighed, er der et klart behov for nye og mere effektive behandlinger for blærekræft. Udvidelse nye immunterapier til bladdis kræft er en mulig tilgang, der kan holde løftet til at udvide sygdomsfri overlevelse. Historisk har BCG været den eneste effektive immunterapi for blærekræft. Dens virkningsmekanisme menes at involvere den uspecifikke induktion af en T-hjælper 1 (Th1) typen immunrespons via stigende niveauer af interleukin-2 (IL-2) og interferon gamma (IFN-γ) 4.. Cellulære eller Th1 immunitet, er kritisk i cancer-immunterapi som humorale eller Th2, er immuniteten aldrig vist sig at være effektiv mod solide tumorer, med undtagelse af antistoffer rettet mod vækstfaktorreceptorer 7. I et forsøg på at forbedre på fordelene ved BCG monoterapi blev IFN-α 2B/BCG kombinationen immunterapi evalueret i et fase II klinisk studie med overbevisende resultater 8.. En alternativ metode til immunterapi for blærekræft kan være at målrette tumor-associerede antigener (TAA&#39;er), identifikation af som har gjort kræft immunterapi mere specifik 7 </sup&gt;. En sådan TAA mucin 1 (MUC1), som er et celleoverfladeglycoprotein overudtrykt i mange epitelceller kræftformer, såsom blære, bryst, lunge, og bugspytkirtelkræft 9,10. Ekspression og modificering af MUC1 også ændres væsentligt under carcinogenese, sådan at underglycosylation udsætter antigene sekvenser af aminosyrer kendt som variabelt antal tandemgentagelser (VNTR) på peptidet kerne. Mens MUC1 er en selvstændig molekyle, er disse immundominante VNTR regioner normalt ikke udsættes på grund af omfattende glycosylering, og dermed de opfattes af immunsystemet som fremmede 11,12. Cytotoksiske T-lymfocytter (CTL&#39;er), som specifikt genkender MUC1 epitoper er blevet isoleret fra de tumor-drænende lymfeknuder fra brystkræftpatienter 13, samt blod og knoglemarv af myelomatosepatienter 14,15, hvilket gør MUC1 et potentielt mål for en cellulært immunrespons. De immundominante VNTRs af underglycosylated form af MUC1 genkendes af CTL&#39;er, hvilket resulterer i ødelæggelse af tumorceller 16-19. Native cellulære og / eller humorale immunresponser på kræft MUC1 er dog ikke stærk nok til at fjerne tumorer. At forøge den allerede eksisterende svag immunrespons til MUC1, kan syntetiske immundominante peptider indføres gennem vaccination at generere et CTL-respons stærk nok til at være af klinisk fordel 18,20. En MUC1 liposomal vaccine er allerede vist sig at øge overlevelsen i patienter med lungecancer 21,22, generere CTL&#39;er kan dræbe MUC1-positive tumorceller, og producere en Th1-polariseret cytokin respons 23,24. Med en høj grad af MUC1 ekspression 9,11,25, er blærekræft en logisk kandidat til test MUC1-rettet immunterapi 26,27. Desuden MUC1 har potentiale som en prognostisk faktor i blærekræft 28, MUC1 ekspression i TCC er signifikant associeret med scenen og kvalitet, og metastatisk TCChar vist sig at fortsætte med at udtrykke MUC1 29.. For at vurdere den potentielle anvendelighed af MUC1-rettet immunterapi i blærekræft, udviklede vi en immun intakt humant MUC1 (hMUC1)-udtrykkende transgene (MUC1.Tg) musemodel af blærekræft congene på C57BL / 6 baggrund 30. Human MUC1 udtrykkes som en selvstændig protein under kontrol af sin egen promotor, hvilket resulterer i en vævsekspression mønster svarende observeret hos mennesker 30,31. Musene blev induceret med den kendte blære kræftfremkaldende N-butyl-N-(4-hydroxybutyl) nitrosamin (OH-BBN) 32, og derefter de resulterende tumorer blev vurderet for hMUC1 ekspression og tumor type og kvalitet. For at vurdere effekten af ​​kræftfremkaldende på Th1/Th2 cytokinniveauer under tumor udvikling blev serumprøver indsamlet jævnligt for multiplex analyse. Mus blev derefter behandlet med en MUC1 målrettet peptidvaccine, og serummet cytokin og immunreaktioner blev evalueindikeret med multiplex fluorometrisk microbead immunoassay og ELISpot.

<table border="1">
	<tbody>
		<tr>
			<td>Reagent&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN)</td>
			<td>TCI America</td>
			<td>B0938</td>
			<td></td>
		</tr>
		<tr>
			<td>20 G Gavage Needles</td>
			<td>Popper &#38; Sons, Inc.</td>
			<td>7921</td>
			<td>Stainless steel</td>
		</tr>
		<tr>
			<td>Peptide Vaccine</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>investigational agent</td>
		</tr>
		<tr>
			<td>BD Microtainers</td>
			<td>BD</td>
			<td>365957</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Cassettes</td>
			<td>Simport</td>
			<td>M490-12</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>Fisher Scientific</td>
			<td>SF100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysis Buffer</td>
			<td>Pierce</td>
			<td>87787</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease &#38; Phosphatase inhibitor cocktail</td>
			<td>Thermo Scientific</td>
			<td>78444</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Cytokine 20plex Kit</td>
			<td>Invitrogen</td>
			<td>LMC006</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Microsphere Beads</td>
			<td>Luminex</td>
			<td>MC100xx-01</td>
			<td>xx is the bead region</td>
		</tr>
		<tr>
			<td>Anti-mouse TNF- Capture Antibody</td>
			<td>BD Pharmingen</td>
			<td>551225</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse TNF- Detection Antibody</td>
			<td>BD Pharmingen</td>
			<td>554415</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IFN- Capture Antibody</td>
			<td>Abcam</td>
			<td>ab10742</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IFN- Detection Antibody</td>
			<td>Abcam</td>
			<td>ab83136</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Sigma</td>
			<td>P3813-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay Buffer</td>
			<td>Millipore</td>
			<td>L-MAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokine Standard</td>
			<td>Millipore</td>
			<td>MXM8070</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-screen HTS 96well filter plates</td>
			<td>Millipore</td>
			<td>MSBVN1210</td>
			<td></td>
		</tr>
		<tr>
			<td>SA-PE</td>
			<td>Invitrogen</td>
			<td>SA10044</td>
			<td></td>
		</tr>
		<tr>
			<td>100 m Nylon Tissue Sieves</td>
			<td>BD</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Splenocyte Separation Media</td>
			<td>Lonza</td>
			<td>17-829E</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF- /IL-4 ELISpot plates</td>
			<td>R&#38;D Systems</td>
			<td>ELD5217</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-MUC1 monoclonal antibody</td>
			<td>Epitomics</td>
			<td>2900-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-actin monoclonal antibody</td>
			<td>Sigma</td>
			<td>A1978</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit HRP antibody</td>
			<td>Promega</td>
			<td>W401B</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse HRP antibody</td>
			<td>Santa Cruz Biotechnology, Inc.</td>
			<td>SC-2005</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>BioRad</td>
			<td>162-0174</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Protean TGX Precast Gels</td>
			<td>BioRad</td>
			<td>456-1083</td>
			<td></td>
		</tr>
		<tr>
			<td>Muse Count &#38; Viability Kit</td>
			<td>Millipore</td>
			<td>MCH100104</td>
			<td></td>
		</tr>
		<tr>
			<td>MUC1 Antibody</td>
			<td>BD Pharmingen</td>
			<td>550486</td>
			<td>IHC antibody</td>
		</tr>
		<tr>
			<td>Animal Research Peroxidase Kit</td>
			<td>Dako</td>
			<td>K3954</td>
			<td>IHC staining</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Equipment and Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore plate vaccum apparatus</td>
			<td>Millipore</td>
			<td>MSVMHTS00</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminex Lx200</td>
			<td>Millipore / Luminex</td>
			<td>40-013</td>
			<td>Manufactured by Luminex, distributed by Millipore</td>
		</tr>
		<tr>
			<td>Luminex Xponent Software</td>
			<td>Millipore / Luminex</td>
			<td>N/A</td>
			<td>Version 3.1; included with Luminex Lx200</td>
		</tr>
		<tr>
			<td>Milliple Analyst Software</td>
			<td>Milliplex / VigeneTech</td>
			<td>40-086</td>
			<td>Version 5.1</td>
		</tr>
		<tr>
			<td>Muse Cell Analyzer</td>
			<td>Millipore</td>
			<td>0500-3115</td>
			<td></td>
		</tr>
		<tr>
			<td>Muse Software</td>
			<td>Millipore</td>
			<td>N/A</td>
			<td>Version 1.1.0.0; included with Analyzer</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Unitron</td>
			<td>Z730</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism Software</td>
			<td>Graphpad Software Inc.</td>
			<td>N/A</td>
			<td>Version 5.1</td>
		</tr>
		<tr>
			<td>Mini Protean Tetra Cell Gel apparatus</td>
			<td>BioRad</td>
			<td>165-8001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans Blot SD Cell and PowerPac</td>
			<td>BioRad</td>
			<td>170-3849</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of invasive transitional cell bladder carcinoma in immune intact human muc1 transgenic mice:  a model for immunotherapy development

En præklinisk model for invasiv blærekræft blev udviklet i human mucin 1 (MUC1) transgene (MUC1.Tg) mus med henblik på evaluering af immunterapi og / eller kemoterapi. At fremkalde blærekræft, C57BL / 6 mus (MUC1.Tg og vildtype) blev behandlet oralt med det kræftfremkaldende N-butyl-N-(4-hydroxybutyl) nitrosamin (OH-BBN) på 3,0 mg / dag, 5 dage / uge i 12 uger. For at vurdere effekten af ​​OH-BBN på serum-cytokinprofil under tumor udvikling blev fuldblod indsamlet via submandibulære blødninger før behandling og hver fjerde uge. Derudover blev en MUC1 målrettet peptidvaccine og placebo administreret til grupper af mus ugentligt i otte uger. Multiplex fluorometriske microbead immunoanalyses af serumcytokiner under tumor udvikling og efter vaccination blev udført. Ved forsøgets afslutning, interferon gamma (IFN-γ) / interleukin-4 (IL-4) ELISpot analyse for MUC1 specifik T-celle-immunreaktion og histopatologiske evalueringer af tumortypeog klasse blev udført. Resultaterne viste, at: (1) forekomsten af ​​blærekræft i både MUC1.Tg og vildtypemus var 67%, (2) transitional cell carcinomer (TCC) udviklet på en 2:1 sammenlignet med pladecellecarcinomer (SCC) , (3) inflammatoriske cytokiner steg med tiden under tumor udvikling, og (4) indgivelse af peptidet Vaccinen fremkalder et Th1-polariseret serum cytokinprofil og et MUC1 specifik T-celle-respons. Alle tumorer i MUC1.Tg mus var positive for MUC1 ekspression, og halvdelen af ​​alle tumorer i MUC1.Tg og vildtypemus var invasiv. Konklusionen er, at bruge en team tilgang gennem koordinering af indsatsen fra farmakologer, immunologer, patologer og molekylærbiologer har vi udviklet en immun intakt transgen musemodel af blærekræft, der udtrykker hMUC1.

Den prækliniske vurdering af virkningerne af nye immunterapier og kombinationer i blærekræft kræver udvikling af en passende dyremodel. I vores transgene musemodel, resulterede induktion med kemiske kræftfremkaldende OH-BBN i en høj blærekræft forekomsten af ​​overvejende TCC med nogle SCC, der svarer til blærekræft hos mennesker. At bestemme tumorhistologien, MUC1 ekspression status og immunresponset på peptidvaccine behandling blev 21 MUC1.Tg og 18 vildtypemus aflivet til indsamling af blod, blærer, og...

Watch this Scientific Journal Video about Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development at JoVE.com

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

En N-butyl-N-(4-hydroxybutyl) nitrosamin-induceret blærekræft model blev udviklet i human mucin 1 (MUC1) transgene mus med henblik på prøvning MUC1-rettet immunterapi. Efter indgivelse af et MUC1 målrettet peptidvaccine, blev en cytotoksisk T-lymfocyt respons på MUC1 bekræftet ved måling af serum cytokinniveauer og T-celle specifik aktivitet.

Induktion af Invasive Transitional Cell blærecarcinom i Immune intakt humant MUC1 transgene mus: En model for immunterapi Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Research

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Medicine

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

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 ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Protokol for Long Duration Whole Body Hypertermi i mus

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

Generering af Subkutan og Intrahepatisk Menneskelige hepatocellulært carcinom xenotransplantater i immunsvækkede mus

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Urinblæren Udspilning Evoked Visceromotor Responses som en model for blæresmerter i mus

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

Facial Nerve aksotomi i mus: en model til at studere motoneuron Reaktion på Skade

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Induktion af accelereret åreforkalkning i Mus: Den "Wire-Skade" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Teknik, minimalt Invasive tværgående aorta konstriktion i mus til induktion af venstre ventrikel hypertrofi

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

En minimalt invasiv Model til at analysere Endochondral fraktur Healing i mus Under standardiserede biomekaniske forhold

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...