Abstract 3412: Quadruple immunofluorescence protocol for identification of the metastatic potential of prostate CTCs
Authors: Gakhar Gunjan , Liu He , Bander Neil , Nanus David 
Published: August 2012

Abstract
Background: Prostate cancer (PCa) is the leading cause of cancer in men and second to lung cancer in cancer-associated mortality in United States. The prognosis for 5-year survival is &gt;90% if detected at an early stage while it decreases to 31% for patients diagnosed with metastases. Circulating tumor cells (CTCs) are being studied to develop new anti-metastatic therapies. Our immunofluorescence protocol will help in identification of the metastatic potential of prostate CTCs. Methods: We have developed and optimized quadruple immunofluorescence protocol for the characterization of PCa CTCs. Cells were seeded on cell-tak coated coverslips, fixed with 2% HCHO and washed with PBS containing glycine. Triton-X (0.1%) for 10 min was used to permeabilize the cells followed by three times wash with PBS containing 0.5% BSA (PBB). Cells were incubated with 2% BSA for 1 hr at room temperature following which cells were washed three times with PBB. Anti-rat HECA-452 and anti-rabbit CXCR4 primary unconjugated antibodies were added for 1 hr and washed with PBB three times. Cells were incubated with highly cross-adsorbed secondary antibodies for goat anti-rabbit 405 and goat anti-rat 594 for 1 hr at room temperature. Cells were washed with PBB and incubated with two primary conjugated antibodies for PSMA (AF 488) and EpCAM (AF 647) for 1 hr. Cells were washed, mounted and analyzed using Zeiss LSM 700 confocal microscope. Results: We have been successfully able to visualize four markers in a single cell. Different cell lines were used as negative and positive control for different markers. U937(PSMA-, HECA452+, EpCAM-, CXCR4+), MDAPCa2b(PSMA+, HECA452+, EpCAM+, CXCR4+), PC3(PSMA-, HECA452-, EpCAM+, CXCR4+), KG1(PSMA-, HECA452α, EpCAM-, CXCR4-). Isotype and secondary antibody controls were used for unconjugated antibodies to determine the specificity of primary and secondary antibodies, respectively. We chose surface markers involved in the adherence of PCa CTCs to the blood vessel and homing to the bone. Previous studies have shown that hematopoietic progenitor cells utilize the coordinated expression of CXCR4 and E-selectin to migrate to the bone marrow. CXCR4 is a chemokine receptor, which binds to SDF-1α present on the human bone marrow while HECA-452 is an epitope present on the selectins and is associated with E-selectin ligand activity. EpCAM and PSMA were used to identify prostate epithelial CTCs.Images were taken under the transmitted light to observe the cell. Conclusion: The multi-marker immunofluorescence (MMI) is challenging because of limited species of specific antibodies and simultaneous usage of limited lasers for the excitation. However, MMI is important in assessing different cell markers in CTCs where the sample size is very limited. Our protocol can be used for the detection and characterization of prostate CTCs.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3412. doi:1538-7445.AM2012-3412