2165 POSTER Targeting of Interferon Gamma to Stromal Fibroblasts Using a PDGF Receptor Recognizing Carrier Reduces Tumour Growth in Vivo

Abstract

Background: Stromal fibroblasts are the key cell types in tumour stroma, that support angiogenesis, tumour cell proliferation and metastasis. Therefore, inhibition of stromal fibroblasts activity might inhibit tumour growth. Interferon gamma (IFNγ) is a potent cytokine and has been used for the treatment of experimental fibrosis. However, poor pharmacokinetics and severe side effects prevented its clinical application. In this study, we hypothesized that specific delivery of IFNγ to stromal fibroblasts may be beneficial to inhibit the tumour growth. Since Platelet-derived Growth Factor beta receptor (PDGFβR) is abundantly expressed on stromal fibroblasts, we developed a PDGFβR-specific drug carrier (PPB-HSA) by modifying albumin with a PDGFβR-recognizing cyclic peptide to deliver IFNγ. Materials and Methods: The IFNγ was conjugated to PPB-HSA carrier via a heterobifunctional PEG linker and characterized with Western blot analyses and nitric oxide release assay in RAW monocytes. In vitro, PPBHSA- IFNγ was examined for its effectivity in 3T3 fibroblasts using wound healing assay, immunocytochemistry and qRT-PCR. To simulate fibroblastsinduced angiogenesis process, tube formation assay was developed in which conditioned medium from 3T3 fibroblasts (incubated with TGFb and IFNγ or IFNγ constructs) was added to the endothelial cells (H5V) and tubes formed were counted. In vivo, the effects of the targeted PPB-HSAIFNγ on tumour growth were determined in subcutaneous B16 melanoma tumour model in mice. Treatments with vehicle, IFNγ, PPB-HSA-IFNγ, PPBHSA (n = 5 per group) at the equivalent doses (5 μg/dose/mouse) were administered intravenously. Results: PPB-HSA-IFNγ construct was successfully synthesized and the conjugated IFNγ retained its biological activity. The construct showed PDGFβR-specific binding in 3T3 cells which was blocked with anti-PDGFR antibody. The IFNγ construct significantly inhibited the proliferation and migration of 3T3 cells as determined with wound healing assay. Treatment with the targeted IFNγ drastically reduced TGFb-induced collagen-I, alpha smooth muscle actin and fibronectin expression in staining and gene expression. Furthermore, the PPB-HSA-IFNγ inhibited the 3T3 fibroblastsinduced angiogenesis as determined with the tube formation assay in H5V cells. In vivo, the targeted IFNγ construct attenuated the tumour growth by 60% (p < 0.01) compared to vehicle whereas untargeted IFNγ and PPBHSA carrier did not induce any reduction in the tumour growth. Conclusions: These data demonstrate that specific targeting of IFNγ to the stromal fibroblasts using PPB-HSA carrier is a potential therapeutic strategy to inhibit tumour growth.