In Vivo Imaging of HIF-Active Tumors by an Oxygen-Dependent Degradation Protein Probe with an Interchangeable Labeling System

Abstract

Hypoxia-inducible factor (HIF) functions as a master transcriptional regulator for adaptation to hypoxia by inducing adaptive changes in gene expression for regulation of proliferation, angiogenesis, apoptosis and energy metabolism. Cancers with high expression of the alpha subunit of HIF (HIFα) are often malignant and treatment-resistant. Therefore, the development of a molecular probe that can detect HIF activity has great potential value for monitoring tumor hypoxia. HIF prolyl hydroxylases (HPHDs) act as oxygen sensors that regulate the fate of HIFα protein through its oxygen-dependent degradation (ODD) domain. We constructed a recombinant protein PTD-ODD-HaloTag (POH) that is under the same ODD regulation as HIFα and contains protein transduction domain (PTD) and an interchangeable labeling system. Administration of near-infrared fluorescently labeled POH (POH-N) to mouse models of cancers allowed successful monitoring of HIF-active regions. Immunohistochemical analysis for intratumoral localization of POH probe revealed its specificity to HIF-active cells. Furthermore, lack of the PTD domain or a point mutation in the ODD domain abrogated the specificity of POH-N to HIF-active cells. Overall results indicate that POH is a practical probe specific to HIF-active cell in cancers and suggest its large potential for imaging and targeting of HIF-related diseases.

Importance of the physicochemical properties of fluorescent dyes for obtaining target-specific in vivo images by membrane-permeable macromolecular imaging probes

“…POH protein (46 kDa) was prepared as described previously [15]. Briefly, the plasmid POH/pGEX-6P-3 encodes PTD-ODDHaloTag7 protein (POH), which consists of polypeptides indicated in (Supplement figure S1).…”

“…HaloTag ligand-Alexa Fluor 750 (HL-A) (Molecular weight: ~1800) was prepared as described previously [15]. Synthesis procedure of HL-P was indicated in the supporting information.…”

“…We previously constructed POH proteins labeled with IRDye800 (I) or Alexa Fluor750 (A), namely POH-I, or POH-A [15]. NIRF dyes are covalently bound to POH through HT and its ligand (HL) reaction, that is each POH-N contains one NIRF dye at the specific site ( Figure 1A).…”

“…A previous study indicates that most POH protein is degraded in and excreted from non-target cells 3 h after entering the cells [15] and that PTD-ODD fusion protein probe quickly disappeared from blood flow (only 15% in blood flow 1 h after injection and then half-life in blood was approximately 2 h) [19]. Therefore, we investigated whether the differential off-target images after injection of POH-Ns in vivo are caused by intact POH-Ns or HL-Ns derived from degraded POH-Ns.…”

“…We recently developed a functional MPM, PTD-ODD-HaloTagdoi:10.7243/2050-120X-2-2 -1α (548-603), and a HaloTag (HT) that is easily conjugated with any chemical through covalent binding of HT and its ligand. The PTD of POH efficiently enables it to enter cells and the ODD allows it to be selectively stabilized in HIF-active cells [15], which are responsible for treatment resistance and malignant progression of tumor hypoxia [16]. POH-N, a NIRF dye-conjugated POH successfully imaged HIF-active cells in vitro and in vivo.…”