Figure 5.

Z_PDGFRβ affibody-mediated binding of hTRAIL to pericytes. (A) Binding of Z-hTRAIL and hTRAIL to pericytes. For the protein binding assay, cells were incubated with FAM-labeled proteins followed by flow cytometry. To determine the PDGFRβ-dependent binding, cells were preincubated with anti-PDGFRβ antibody prior to incubation with Z-hTRAIL. (B) Cytotoxicity of Z-hTRAIL bound to pericytes in colorectal tumor cells. Pericytes were first incubated with Z-hTRAIL or hTRAIL. After being washed with PBS, different amounts of pericytes were mixed to a constant number of tumor cells (1.5×10⁴ cells for LS174T and HCT116 or 2×10⁴ cells for COLO205) and further incubated overnight. The surviving cells were measured using CCK8. Pericytes that were not preincubated with proteins were used as controls. The survival rates of tumor cells mixed with pericytes preincubated without proteins were considered 100 %. (C) Apoptosis of tumor cells co-cultured with pericytes loaded with Z-hTRAIL or hTRAIL. Apoptotic cells were visualized by Annexin V staining. Pericytes attached to the apoptotic cells (green) are indicated by arrows. (D) Dynamic distribution of Z-hTRAIL in LS174T tumor xenografts. Mice bearing tumor grafts were intravenously injected with FAM-labeled Z-hTRAIL or an equal molar amount of hTRAIL (green). At 5, 30, and 60 min post-injection, the tumor grafts were collected and sectioned under frozen conditions. Subsequently, the slides were stained with anti-PDGFRβ antibody (red). The nuclei of cells were visualized using DAPI (blue). Original magnification, ×200.