Abstract 5240: Influence of cell permeable property of fluorescent dye on the POH in vivo imaging probe specific to HIF-active cancer cells

Tumor hypoxia plays a central role in malignant progression and is resistant to both radiotherapy and chemotherapy. Hypoxia-inducible factor (HIF) is a master transcriptional regulator for adaptation to hypoxia by inducing adaptive changes in gene expression for regulation of angiogenesis, proliferation, and metastasis in cancers. Protein stability of the alpha-subunit of HIF (HIFα) is strictly regulated by the oxygen sensor HIF prolyl hydroxylases (HPHDs). While HPHDs are inactive in hypoxic cells, they hydroxylate the Oxygen-Dependent Degradation (ODD) domain of HIFα in well-oxygenized cells and the hydroxylated HIFα is rapidly degraded through the ubiquitin-proteasome system. We recently developed PTD-ODD-HaloTag (POH) probes, which penetrated cell membrane by the role of PTD and specifically stabilized in HIF-active cells as HIFα. To evaluate the usefulness of the POH for an in vivo imaging probe, POH protein was covalently bound to HaloTag ligand (HL) labeled with AlexaFluora750 (AF750) or IR800, which have excitation and emission wavelengths in the near infrared (NIR) spectral range (700-800 nm) where non-specific background fluorescence is considerably reduced. The resultant POH-HL-AF750 (POH-A) and POH-HL-IR800 (POH-I) were then examined their target specificity by using cultured cancer cell lines in terms of the cell membrane permeability and the stability in HIF-active cells. When the cells were treated with POH-A or POH-I significantly higher levels of the POH protein and fluorescence intensity were detected in cells cultured under hypoxic or hypoxia-mimic conditions, where HPHDs were suppressed, than normoxic conditions. These findings revealed that both POH-A and POH-I probe were efficiently transduced into cells at the similar extent and specifically stabilized in HIF-active cells. Although POH-A and POH-I showed similar cell membrane permeability, HL-AF750 alone penetrated cell membrane about 16 times less than HL-IR800. Furthermore, POH-I-treated cells showed shorter fluorescence retention time than POH-A-treated cells, indicating that POH-I and/or its pieces diffused away from the cell faster than POH-A. When POH-A and POH-I were applied for in vivo optical imaging of HIF-active hypoxic cells in a subcutaneous xenograft model, POH-I showed significantly higher fluorescent intensity ratio in tumor versus background (T/B) than POH-A and the distribution in the liver and gastrointestinal tract were different between mice administrated with PO