Secreted Protein Acidic and Rich in Cysteine (SPARC) mediated fluorescence labeled human serum albumin uptake in glioblastoma

Abstract

ABSTRACT

Jung Hwan Jo Interdisciplinary Program in Cancer Biology The Graduate School Seoul National University

Objective: Human serum albumin (HSA) is used as a drug carrier for clinical application. Though the mechanism of HSA uptake in tumor is still unclear, it has been known that albumin targets tumor through the leaky blood vessel by enhanced permeability and retention (EPR) effect, indicating that albumin accumulates in tumor by simple infiltration, not by specific uptake. However, it has been suggested that secreted protein acidic and rich in cysteine (SPARC) could sequester albumin in tumor stroma and partly relate to the tumor specific uptake of albumin. For evaluating possible use of HSA as a specific targeting agent of SPARC-expressing tumor, I visualized SPARC-dependent HSA uptake in glioblastomas.

Methods: Fluorescence labeling, FNR648-N3 was conjugated to HSA by click chemistry reaction. Human glioblastoma cell line, U87MG, was used for evaluating SPARC-dependant HSA uptake. SPARC expression was down-regulated with shSPARC in U87MG cells. After treatment with FNR648-HSA in U87MG and shSPARC-U87MG, fluorescent signals were measured with confocal microscopy. Colocalization of Cy3-SPARC and FNR648-HSA was observed with confocal FRET analysis. For in vivo study, U87MG and shSPARC-U87MG cells were subcutaneously injected to the thighs of a mouse to generate xenograft model. After intravenous injection of FNR648-HSA in a tumor-xenografted mouse, fluorescent signals were detected with IVIS Lumina II. FITC-dextran was used for vessel permeability test. Immuno-fluorescence staining for tumor frozen section was proceeded with CD31 antibody (for blood vessel) and DAPI (for nucleus). Confocal tile scanning and high resolution imaging were used for detecting fluorescence signal in overall tumor tissue.

Results: SPARC proteins were highly expressed in U87MG, but not in shSPARC-U87MG. In vitro HSA uptake test showed that more FNR648-HSA was accumulated in U87MG than shSPARC-U87MG cells. Exogenous SPARC treatment successfully recovered the uptake of FNR648-HSA in shSPARC-U87MG cells. Furthermore, SPARC and HSA were colocalized in U87MG cells. In xenograft model, FNR648-HSA was accumulated 4 times more in U87MG than shSPARC-U87MG. When tumors were detached and compared the difference between them, though vascular permeability of FITC-dextran in U87MG tumor was similar to shSPARC-U87MG tumor regardless of SPARC expression, more FNR648-HSA was accumulated in U87MG tumor than shSPARC-U87MG tumor. In confocal imaging of tumor section, HSA was escaped from blood vessel and internalized into U87MG tumor cells. On the other hand, HSA was retained in the blood vessel of shSPARC-U87MG tumor.

Conclusion: SPARC have an impact on HSA uptake in glioblastoma. An expression of SPARC increases HSA uptake in a glioblastoma xenografted mouse model. Thus, HSA has a potential for a drug delivery system in SPARC expressing glioblastomas.