Forfatterne ønsker å takke UC Davis Mouse Biology Program for avl musene. Denne forskningen ble støttet av et stipend fra Merck KGaA, Darmstadt, Tyskland.

Alle dyrestudier og eksperimenter ble utført under en protokoll godkjent av University of California, Davis Institutional Animal Care og bruk Administrative Advisory Committee. En. MUC1.Tg Mouse Avl og forplantning <ol><li> UC Davis Mouse Biology Program (MBP) raser villtype C57BL / 6 mannlige mus med heterozygot MUC1.Tg C57BL / 6 hunnmus å etablere vår kolonien. MUC1.Tg avkom blir levert for studier etter behov. </li><li> MBP personell klippe tærne av avkommet i et definert mønster (...

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Den vellykkede induksjon av invasiv overgangsordning og plateepitelkreft blære carcinoma in human MUC1.Tg mus tilbyr en preklinisk modell for Immunterapi utvikling. Immunoterapeutiske studier krever bruk av en spontan, intakt immun-modell for å evaluere den inflammatoriske responsen til tumorprogresjon over tid, så vel som av immunresponsen mot immunterapi. I en spontan svulst utvikling modell, forblir svulst microenvironment intakt og svulster utvikle seg på et mer representativt vekstrate som gjør det mulig for v...

Blærekreft er den fjerde vanligste formen for kreft og den åttende største årsaken til kreft dødsfall i amerikanske menn. I USA, er anslagsvis 72 500 nye tilfeller og 15 000 dødsfall av blærekreft forventet blant menn og kvinner samlet i 2013 en. Forekomsten av blærekreft er omtrent tre ganger så høy hos menn sammenlignet med kvinner. I USA, overgangsstønad karsinomer (TCC) står for over 90% av tilfellene, mens plateepitelkreft karsinom (SCC) har en insidens på mindre enn 2% 2. Den samlede relative 5-års overlevelse for papillær TCC er 91,5% sammenlignet med bare 30,9% for SCC to. Selv om ikke-invasiv papillær TCCs stå for ca 75% av tilfellene på tidspunktet for diagnosen, selv med behandling mer enn 50% av pasientene vil oppleve et tilbakefall innen fem år, med opp til 30% av disse pasientene utvikler seg til muskel invasiv sykdom 3,4 . Typiske behandlingsregimer for ikke-muskel invasive sykdom inkluderer transurethral reseksjon (TUR) etterfulgt av intravesikal kjemoterapi. Hos pasienter med høyverdig Ta eller T1 svulster, kan en gjenta TUR utføres før kjemoterapi 3,4. For pasienter med lavgradige Ta tilbakefall eller høyverdig Ta eller T1 lesjoner, etterfulgt TUR etter adjuvant kjemoterapi eller immunterapi i form av Bacillus Calmette-Guerin (BCG) kan brukes 3,4. Intravesikal BCG har vist seg å være overlegen i forhold til intravesikal mitomycin C med hensyn til tiden for tilbakefall 5.. For T2 muskel invasiv sykdom, er radikal cystektomi med eller uten neoadjuvant kjemoterapi den anbefalte behandlingen tre. Hos pasienter med SCC, synes radikal cystektomi å være den mest effektive behandlingen seks. Gitt de svært høy forekomst av tilbakefall til tross for de beste behandlinger tilgjengelig, er det et klart behov for nye og mer effektive behandlingsformer for blærekreft. Utvide nye immunotherapies for bladdeh kreft er en mulig tilnærming som kan holde løftet for å forlenge sykdomsfri overlevelse. Historisk har BCG vært den eneste effektive immunterapi for blærekreft. Virkningsmekanismen er antatt å involvere den ikke spesifikke induksjon av en T-hjelper 1 (Th1) type immunrespons via økende nivåer av interleukin-2 (IL-2) og interferon gamma (IFN-γ) 4.. Cellular eller Th1 immunitet, er kritisk i cancer immunoterapi som humoral eller Th2 har immunitet aldri vist seg å være effektiv mot faste tumorer, med unntak av antistoffer rettet mot vekstfaktorreseptorer 7. I et forsøk på å forbedre fordelene med BCG monoterapi, ble IFN-α 2B/BCG kombinasjon immunterapi evaluert i en fase II klinisk studie med noe svar åtte. En alternativ tilnærming til immunterapi for blærekreft kan være å målrette tumor-assosierte antigener (TAA), identifisering av noe som har gjort cancer immunoterapi mer spesifikk 7 </sopp&gt;. En slik TAA er mucin 1 (MUC1), som er et celleoverflate-glykoprotein overuttrykt i mange epitelcelle kreftformer slik som blære-, bryst-, lunge-og bukspyttkjertelkreft 9,10. Uttrykket og modifikasjon av MUC1 er også vesentlig endret i løpet av karsinogenese, eksponerer slik at underglycosylation antigene sekvenser av aminosyrer er kjent som variabelt antall tandem gjentar (VNTR) på peptid kjerne. Mens MUC1 er en selv-molekyl, er disse immunodominante VNTR regionene normalt ikke utsatt på grunn av omfattende glykosylering, og dermed de blir sett av immunsystemet som utenlandske 11,12. Cytotoksiske T-lymfocytter (CTLs) som spesifikt gjenkjenner MUC1 epitoper har blitt isolert fra de tumor-drenerende lymfeknutene brystkreftpasienter 13, så vel som blod og benmarg av myelom 14,15, slik MUC1 et potensielt mål for en cellulær immunrespons. De immunodominante VNTRs av underglycosylated form av MUC1 er anerkjent av CTLs, noe som resulterer i ødeleggelse av kreftceller 16-19. Native cellulære og / eller humorale immunresponser mot cancerøse MUC1 er imidlertid ikke sterk nok til å eliminere svulster. Å forsterke den allerede eksisterende svake immunforsvar mot MUC1, kan syntetiske immunodominante peptider bli introdusert gjennom vaksinering for å generere en CTL respons sterk nok til å være av klinisk nytte 18,20. En MUC1 liposomale vaksine har allerede vist seg å øke overlevelse i lungekreftpasienter 21,22, generere CTLs stand til å drepe MUC1-positive kreftceller, og produsere en Th1-polarisert cytokin respons 23,24. Med et høyt nivå av MUC1 uttrykk 9,11,25, er blærekreft en logisk kandidat for testing MUC1-rettet immunterapi 26,27. Videre har MUC1 potensial som en prognostisk faktor i blæren 28 kreft, MUC1 uttrykk i TCC er signifikant assosiert med scene og karakter, og metastatisk TCChar vist seg å fortsette å uttrykke MUC1 29.. For å evaluere den potensielle nytten av MUC1-rettet immunterapi i blærekreft, utviklet vi en immun intakt menneskelig MUC1 (hMUC1)-uttrykke transgen (MUC1.Tg) mus modell av blærekreft congenic på C57BL / 6 bakgrunnen 30. Humant MUC1 er uttrykt som en selv-protein under kontroll av sin egen promoter, noe som resulterer i en vev uttrykk mønster i overensstemmelse med det som for mennesker 30,31. Musene ble indusert med den kjente blære karsinogen N-butyl-N-(4-hydroksybutyl) nitrosamin (OH-BBN) 32, og deretter ble det resulterende tumorer ble vurdert med hensyn hMUC1 ekspresjon og tumor type, grad. For å vurdere effekten av kreftfremkallende på Th1/Th2 cytokin nivåer under tumor utvikling, ble serumprøver samlet periodisk for multiplex analyse. Mus ble deretter behandlet med en MUC1-rettet peptid vaksine og serum cytokin og immunresponser ble evaluted ved 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 preklinisk modell av blærekreft ble utviklet i human mucin 1 (MUC1) transgene (MUC1.Tg) mus for det formål å evaluere immunterapi og / eller cytotoksisk kjemoterapi. Ved induksjon av blærekreft, C57BL / 6 mus (MUC1.Tg og villtype) ble behandlet oralt med carcinogen N-butyl-N-(4-hydroksybutyl) nitrosamin (OH-BBN) ved 3,0 mg / dag, 5 dager / uke i 12 uker. For å vurdere effekten av OH-BBN på serum cytokin profil under tumor utvikling, ble fullblod samlet inn via submandibular blør før behandlingen og hver fjerde uke. I tillegg ble en MUC1-målrettet peptid vaksine og placebo administrert til grupper av mus ukentlig i åtte uker. Multiplex fluorometriske microbead immunoanalyses av serumcytokinene under tumor utvikling og etter vaksinasjon ble utført. Ved opphør, interferon gamma (IFN-γ) / interleukin-4 (IL-4) elispot analyse for MUC1 spesifikke T-celle immunrespons og histopatologiske evalueringer av tumor typeog karakteren ble utført. Resultatene viste at: (1) forekomst av blærekreft i både MUC1.Tg og vill type mus var 67%, (2) overgangsreglene celle carcinoma (TCC) utviklet ved forholdet 2:1 i forhold til plateepitelkreft karsinom (SCC) , (3) inflammatoriske cytokiner økte med tiden i løpet av tumorutvikling, og (4) administrering av peptidet vaksine induserer en Th1-polariserte cytokin serum profil og en MUC1 spesifikk T-celle-respons. Alle svulster i MUC1.Tg mus var positive for MUC1 uttrykk, og halvparten av alle svulster i MUC1.Tg og vill type mus var invasiv. I konklusjonen, ved hjelp av et team tilnærming gjennom samordning av innsatsen til pharmacologists, immunologer, patologer og molekylærbiologer, har vi utviklet en immun intakt transgen musemodell av blærekreft som uttrykker hMUC1.

De prekliniske vurdering av virkningene av nye immunterapi og kombinasjoner i blærekreft krever utvikling av en egnet dyremodell. I vår transgen musemodell, resulterte induksjon med kjemisk karsinogen OH-BBN i en høy rate av blærekreft forekomsten av overveiende TCC med noen SCC, som er lik blæren kreft hos mennesker. Å bestemme tumorhistologi, MUC1 uttrykk status og immunrespons til peptid vaksine behandling, ble 21 MUC1.Tg og 18 villtype mus avlives for innsamling av blod, blærer, og milt (figur 1)</str...

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

Et N-butyl-N-(4-hydroksybutyl) nitrosamin-indusert blærekreft modell ble utviklet i human mucin 1 (MUC1) transgene mus for å teste MUC1-rettet immunterapi. Etter administrering av en MUC1-målrettet peptid vaksine, ble en cytotoksisk T-lymfocytt respons på MUC1 bekreftet ved å måle serum cytokin nivåer og T-celle spesifikke aktivitet.

Induksjon av Invasive Transitional Cell blære carcinoma in Immune Intakt Menneskelige MUC1 transgene mus: En modell 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

JoVE Journal

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

Protokoll for lang varighet 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 ...

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Generering av Subkutan og Intrahepatisk menneskeleverkreft xenografts i immunodeficient 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 Stinnhet fremkalt Visceromotor Responses som en modell for blære Smerter i Mus

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

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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

Induksjon av akselerert aterosklerose hos mus: "Wire-Injury" Modell

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

Teknikken med minimal invasiv tverrgående aorta Constriction i mus for induksjon av venstre ventrikkel 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

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