Fluorescence labeled human serum albumin as an imaging agent for a SPARC(secreted protein acidic and rich in cysteine) expressing glioblastoma

Objectives: Human serum albumin(HSA) is used as a drug carrier or radioisotope labeled theranostic agent for clinical application. Though the mechanism of HSA uptake in tumor is still unclear, it has been known that albumin targets tumor based on the leaky blood vessel-related 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. We investigated the role of SPARC for HSA uptake in glioblastoma.

Methods: Fluorescence dye FNR648-N₃ and Cy5-N₃ were bound to label 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 (SPARC KD). After treatment with FNR648 or Cy5-HSA in U87MG and SPARC KD U87MG, fluorescent signals were measured with confocal microscopy. U87MG and SPARC KD U87MG cells were subcutaneously injected to generate xenograft model. After intravenous injection of FNR648-HSA in tumor-xenografted mouse, fluorescent signals were detected with IVIS Lumina II. Immuno-fluorescence staining with tumor frozen section was proceeded. Blood vessel was stained by CD31 antibody, and cell nucleus was stained with DAPI.

Results: SPARC mRNAs and proteins were highly expressed in U87MG, but not in SPARC KD U87MG. In vitro HSA uptake test showed that more Cy5-HSA was accumulated in U87MG than SPARC KD cells. SPARC treatment successfully recovers the uptake of fluorescent HSA in SPARC KD cells. In xenograft model, more FNR648-HSA was accumulated in U87MG than SPARC KD U87MG tumor. HSA accumulation in U87MG tumor region was maximized at 4 hour after injection in U87MG tumor, showing 4 times higher uptake than SPARC KD U87MG. FITC-dextran was used for vessel permeability test, and U87MG tumor showed relatively more permeable to vessel than SPARC KD. Though FITC-dextran was gradually excreted from both tumors, FNR648-HSA was accumulated in U87MG tumor. In confocal imaging of tumor section, albumin was more infiltrated into tumor stroma in U87MG tumor than SPARC KD U87MG. On the other hand, SPARC KD U87MG tumor showed retained albumin in blood vessel.

Conclusion: We visualized SPARC-dependent tumor targeting of fluorescence labeled HSA in glioblastoma. Our results demonstrate that fluorescence labeled HSA is an efficient imaging agent to target SPARC over expressing glioblastoma with enormous potential. Research Support: .