Immunohistokemisk farvning af B7-H1 (PD-L1) på paraffinindlejret lysbilleder af adenocarcinom i pancreas Tissue
Summary
B7-H1 (PD-L1) og dens binding til PD-1 udgør en væsentlig tumorinduceret immunosuppressive signal i tumorens mikromiljø. En immunohistokemisk farvning teknik til karakterisering af ekspression og lokalisering af B7-H1 i pancreas adenocarcinom er beskrevet her.
Abstract
B7-H1/PD-L1, et medlem af B7-familien af immun-regulerende celleoverfladeproteiner, spiller en vigtig rolle i den negative regulering af cellemedierede immunresponser gennem dens interaktion med dets receptor, programmeret død-1 (PD -1) 1,2. Overekspression af B7-H1 af tumorceller er blevet bemærket i et antal humane cancere, herunder melanom, glioblastom, og carcinomer i lunge, bryst, colon, ovarie og nyreceller, og har vist sig at hæmme anti-tumor T- celle immunitet 3-8.
For nylig er B7-H1 ekspression af pancreas adenocarcinom væv blevet identificeret som et potentielt prognostisk markør 9,10. Endvidere har blokering af B7-H1 i en musemodel af kræft i bugspytkirtlen vist sig at producere en anti-tumorrespons 11. Disse data antyder vigtigheden af B7-H1 som et potentielt terapeutisk mål. Anti-B7-H1 blokade antistoffer er derfor ved at blive afprøvet i kliniske forsøg med henblik på mulmultiplum humane faste tumorer, herunder melanom og kræft i lunge, colon, nyre, mave og pancreas 12.
For at blive i stand til at identificere de patienter, der vil få gavn af B7-H1 målretning terapier, er det vigtigt at undersøge sammenhængen mellem udtryk og lokalisering af B7-H1 og patientens respons på behandling med B7-H1 blokade antistoffer. Undersøgelse af ekspressionen af B7-H1 i menneskets bugspytkirtel adenocarcinom væv gennem immunhistokemi vil give en bedre forståelse af, hvordan dette samarbejde hæmmende signalmolekyle bidrager til undertrykkelse af antitumor immunitet i tumor mikromiljø. Anti-B7-H1 monoklonalt antistof (klon 5H1) udviklet af Chen og medarbejdere har vist sig at give pålidelige farvningsresultaterne i kryosektioner af flere typer af humane neoplastiske væv 4,8, men farvning af paraffinindlejrede objektglas havde været en udfordring indtil nylig 13-18. Vi har devløbet bort B7-H1 farvningsprotokol for paraffinindlejrede lysbilleder af adenocarcinom i pancreas væv. B7-H1 farvningsprotokol beskrevet her producerer konsekvent membranøs og cytoplasmatisk farvning af B7-H1 med lidt baggrund.
Protocol
Representative Results
Discussion
Immunohistokemisk farvning af B7-H1 på kryosektioner er blevet rapporteret i tidligere undersøgelser af forskellige cancere 3,4. Men kliniske tumorprøver normalt kun er tilgængelige i form af paraffinindstøbte blokke. Den 5H1 monoklonale antistof er for nylig blevet anvendt med succes til farvning af paraffin-indlejrede tumorvæv, herunder renalcellecarcinom, cervixkarcinom, blærecancer og melanoma 13-18. B7-H1 farvning af paraffin-indlejrede 9,19 eller kryostat 20 dele …
Declarações
The authors have nothing to disclose.
Acknowledgements
EB, KMB, og HX bidrager ligeligt. Denne undersøgelse blev støttet af NCI SPORE i Mave Cancers P50 CA062924, NIH K23 CA148964, Lustgarten Foundation, Viragh Foundation, ASCO Young Investigator Award, NIH 5T32 CA0090701-28 Training Grant, Sol Goldman bugspytkirtlen Cancer Center, og National Bugspytkirtel Foundation.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| CSA System | Dako | K1500 | Includes the following reagents mentioned in the protocol: peroxidase block, streptavidin-biotin complex, amplification reagent, streptavidin-HRP, and DAB substrate and chromogen |
| Avidin/Biotin Blocking Kit | Vector | SP-2001 | |
| Block ACE | Serotec | BUF029 | |
| Biotin anti-mouse IgG1 | BD | 553441 | |
| Mouse IgG1 K Isotype Control | eBioscience | 14-4714-85 | |
| TBS(Tris-buffered saline), 10X | Cellgro | 46-012-CM | 10xTBS contains 80 g/L NaCl and 24.2 g/L Tirs; Diluted to 1X with ddH2O for washes; |
| Tween 20 | Sigma-Aldrich | P1379 | 1 ml added to 1 L of 1X TBS to make TBST |
| Target Retrieval, 20x | Celerus Wave | 014-3000-000 | Diluted to 1x with ddH2O |
| 190 Proof Ethyl Alcohol | Pharmco-Aaper | 111000190 | |
| 200 Proof Ethyl Alcohol | Pharmo-Aaper | 111000200 | |
| Xylene | VWR | 95057-827 | |
| Hematoxylin | Richard-Allan Scientific | 72804 | |
| Cytoseal 60 | Richard-Allan Scientific | 8310-4 | |
| Coverslips | Corning | 2940-245 | |
| Humidity chamber | Sigma | H6644 | |
| Table 1. Reagents and equipment. |
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