Tetsuya Kadonosono

2-Nitroimidazole-Tricarbocyanine Conjugate as a Near-Infrared Fluorescent Probe for in Vivo Imaging of Tumor Hypoxia

We developed a novel near-infrared (NIR) fluorescent probe, GPU-167, for in vivo imaging of tumor hypoxia. GPU-167 comprises a tricarbocyanine dye as an NIR fluorophore and two 2-nitroimidazole moieties as exogenous hypoxia markers that undergo bioreductive activation and then selective entrapment in hypoxic cells. After treatment with GPU-167, tumor cells contained significantly higher levels of fluorescence in hypoxia than in normoxia. In vivo fluorescence imaging specifically detected GPU-167 in tumors 24 h after administration. Ex vivo analysis revealed that fluorescence showed a strong correlation with hypoxia inducible factor (HIF)-1 active hypoxic regions. These data suggest that GPU-167 is a promising in vivo optical imaging probe for tumor hypoxia.

A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging

In preclinical cancer research, bioluminescence imaging with firefly luciferase and D-luciferin has become a standard to monitor biological processes both in vitro and in vivo. However, the emission maximum (λmax) of bioluminescence produced by D-luciferin is 562 nm where light is not highly penetrable in biological tissues. This emphasizes a need for developing a red-shifted bioluminescence imaging system to improve detection sensitivity of targets in deep tissue. Here we characterize the bioluminescent properties of the newly synthesized luciferin analogue, AkaLumine-HCl. The bioluminescence produced by AkaLumine-HCl in reactions with native firefly luciferase is in the near-infrared wavelength ranges (λmax=677 nm), and yields significantly increased target-detection sensitivity from deep tissues with maximal signals attained at very low concentrations, as compared with D-luciferin and emerging synthetic luciferin CycLuc1. These characteristics offer a more sensitive and accurate method for non-invasive bioluminescence imaging with native firefly luciferase in various animal models.

High resolution imaging of intracellular oxygen concentration by phosphorescence lifetime

Optical methods using phosphorescence quenching by oxygen are suitable for sequential monitoring and non-invasive measurements for oxygen concentration (OC) imaging within cells. Phosphorescence intensity measurement is widely used with phosphorescent dyes. These dyes are ubiquitously but heterogeneously distributed inside the whole cell. The distribution of phosphorescent dye is a major disadvantage in phosphorescence intensity measurement. We established OC imaging system for a single cell using phosphorescence lifetime and a laser scanning confocal microscope. This system had improved spatial resolution and reduced the measurement time with the high repetition rate of the laser. By the combination of ubiquitously distributed phosphorescent dye with this lifetime imaging microscope, we can visualize the OC inside the whole cell and spheroid. This system uses reversible phosphorescence quenching by oxygen, so it can measure successive OC changes from normoxia to anoxia. Lower regions of OC inside the cell colocalized with mitochondria. The time-dependent OC change in an insulin-producing cell line MIN6 by the glucose stimulation was successfully visualized. Assessing the detailed distribution and dynamics of OC inside cells achieved by the presented system will be useful to understanding a physiological and pathological oxygen metabolism.

In vivo imaging of HIF-active tumors by an oxygen-dependent degradation protein probe with an interchangeable labeling system.

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.

Cell penetrating peptides improve tumor delivery of cargos through neuropilin-1-dependent extravasation

Cell-penetrating peptides (CPPs), also referred to as protein transduction domains (PTDs), can mediate the cellular uptake of a wide range of macromolecules including peptides, proteins, oligonucleotides, and nanoparticles, and thus have received considerable attention as a promising method for drug delivery in vivo. Here, we report that CPP/PTDs facilitate the extravasation of fused proteins by binding to neuropilin-1 (NRP1), a vascular endothelial growth factor (VEGF) co-receptor expressed on the surface of endothelial and some tumor cells. In this study, we examined the capacity of the amphipathic and cationic CPP/PTDs, PTD-3 and TAT-PTD, respectively, to bind cells in vitro and accumulate in xenograft tumors in vivo. Notably, these functions were significantly suppressed by pre-treatment with NRP1-neutralizing Ab. Furthermore, co-injection of iRGD, a cyclic peptide known to increase NRP1-dependent vascular permeability, significantly reduced CPP/PTD tumor delivery. This data demonstrates a mechanism by which NRP1 promotes the extravasation of CPP/PTDs that may open new avenues for the development of more efficient CPP/PTD delivery systems.

VEGF-A/NRP1 stimulates GIPC1 and Syx complex formation to promote RhoA activation and proliferation in skin cancer cells

ABSTRACT Neuropilin-1 (NRP1) has been identified as a VEGF-A receptor. DJM-1, a human skin cancer cell line, expresses endogenous VEGF-A and NRP1. In the present study, the RNA interference of VEGF-A or NRP1 suppressed DJM-1 cell proliferation. Furthermore, the overexpression of the NRP1 wild type restored shNRP1-treated DJM-1 cell proliferation, whereas NRP1 cytoplasmic deletion mutants did not. A co-immunoprecipitation analysis revealed that VEGF-A induced interactions between NRP1 and GIPC1, a scaffold protein, and complex formation between GIPC1 and Syx, a RhoGEF. The knockdown of GIPC1 or Syx reduced active RhoA and DJM-1 cell proliferation without affecting the MAPK or Akt pathway. C3 exoenzyme or Y27632 inhibited the VEGF-A-induced proliferation of DJM-1 cells. Conversely, the overexpression of the constitutively active form of RhoA restored the proliferation of siVEGF-A-treated DJM-1 cells. Furthermore, the inhibition of VEGF-A/NRP1 signaling upregulated p27, a CDK inhibitor. A cell-penetrating oligopeptide that targeted GIPC1/Syx complex formation inhibited the VEGF-A-induced activation of RhoA and suppressed DJM-1 cell proliferation. In conclusion, this new signaling pathway of VEGF-A/NRP1 induced cancer cell proliferation by forming a GIPC1/Syx complex that activated RhoA to degrade the p27 protein.

Bone resorption facilitates osteoblastic bone metastatic colonization by cooperation of insulin‐like growth factor and hypoxia

Bone metastasis is a multistep process that includes cancer cell dissemination, colonization, and metastatic growth. Furthermore, this process involves complex, reciprocal interactions between cancer cells and the bone microenvironment. Bone resorption is known to be involved in both osteolytic and osteoblastic bone metastasis. However, the precise roles of the bone resorption in the multistep process of osteoblastic bone metastasis remain unidentified. In this study, we show that bone resorption plays important roles in cancer cell colonization during the initial stage of osteoblastic bone metastasis. We applied bioluminescence/X‐ray computed tomography multimodal imaging that allows us to spatiotemporally analyze metastasized cancer cells and bone status in osteoblastic bone metastasis models. We found that treatment with receptor activator of factor‐κB ligand (RANKL) increased osteoblastic bone metastasis when given at the same time as intracardiac injection of cancer cells, but failed to increase metastasis when given 4 days after cancer cell injection, suggesting that RANKL‐induced bone resorption facilitates growth of cancer cells colonized in the bone. We show that insulin‐like growth factor‐1 released from the bone during bone resorption and hypoxia‐inducible factor activity in cancer cells cooperatively promoted survival and proliferation of cancer cells in bone marrow. These results suggest a mechanism that bone resorption and hypoxic stress in the bone microenvironment cooperatively play an important role in establishing osteoblastic metastasis.

In Vivo Imaging of Brain Ischemia Using an Oxygen-Dependent Degradative Fusion Protein Probe

Within the ischemic penumbra, blood flow is sufficiently reduced that it results in hypoxia severe enough to arrest physiological function. Nevertheless, it has been shown that cells present within this region can be rescued and resuscitated by restoring perfusion and through other protective therapies. Thus, the early detection of the ischemic penumbra can be exploited to improve outcomes after focal ischemia. Hypoxia-inducible factor (HIF)-1 is a transcription factor induced by a reduction in molecular oxygen levels. Although the role of HIF-1 in the ischemic penumbra remains unknown, there is a strong correlation between areas with HIF-1 activity and the ischemic penumbra. We recently developed a near-infrared fluorescently labeled-fusion protein, POH-N, with an oxygen-dependent degradation property identical to the alpha subunit of HIF-1. Here, we conduct in vivo imaging of HIF-active regions using POH-N in ischemic brains after transient focal cerebral ischemia induced using the intraluminal middle cerebral artery occlusion technique in mice. The results demonstrate that POH-N enables the in vivo monitoring and ex vivo detection of HIF-1-active regions after ischemic brain injury and suggest its potential in imaging and drug delivery to HIF-1-active areas in ischemic brains.

Detection of the onset of ischemia and carcinogenesis by hypoxia-inducible transcription factor-based in vivo bioluminescence imaging.

An animal model for the early detection of common fatal diseases such as ischemic diseases and cancer is desirable for the development of new drugs and treatment strategies. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates oxygen homeostasis and plays key roles in a number of diseases, including cancer. Here, we established transgenic (Tg) mice that carry HRE/ODD-luciferase (HOL) gene, which generates bioluminescence in an HIF-1-dependent manner and was successfully used in this study to monitor HIF-1 activity in ischemic tissues. To monitor carcinogenesis in vivo, we mated HOL mice with rasH2 Tg mice, which are highly sensitive to carcinogens and are used for short-term carcinogenicity assessments. After rasH2-HOL Tg mice were treated with N-methyl-N-nitrosourea, bioluminescence was detected noninvasively as early as 9 weeks in tissues that contained papillomas and malignant lesions. These results suggest that the Tg mouse lines we established hold significant potential for monitoring the early onset of both ischemia and carcinogenesis and that these lines will be useful for screening chemicals for carcinogenic potential.

Hypoxia‐inducible factor‐targeting prodrug TOP3 combined with gemcitabine or TS‐1 improves pancreatic cancer survival in an orthotopic model

Pancreatic cancer is one of the most lethal digestive system cancers with a 5‐year survival rate of 4–7%. Despite extensive efforts, recent chemotherapeutic regimens have provided only limited benefits to pancreatic cancer patients. Gemcitabine and TS‐1, the current standard‐of‐care chemotherapeutic drugs for treatment of this severe cancer, have a low response rate. Hypoxia is one of the factors contributing to treatment resistance. Specifically, overexpression of hypoxia‐inducible factor, a master transcriptional regulator of cell adaption to hypoxia, is strongly correlated with poor prognosis in many human cancers. TAT‐ODD‐procaspase‐3 (TOP3) is a protein prodrug that is specifically processed and activated in hypoxia‐inducible factor‐active cells in cancers, leading to cell death. Here, we report combination therapies in which TOP3 was combined with gemcitabine or TS‐1. As monotherapy, gemcitabine and TS‐1 showed a limited effect on hypoxic and starved pancreatic cancer cells, whereas co‐treatment with TOP3 successfully overcame this limitation in vitro. Furthermore, combination therapies of TOP3 with these drugs resulted in a significant improvement in survival of orthotopic pancreatic cancer models involving the human pancreatic cancer cell line SUIT‐2. Overall, our study indicates that the combination of TOP3 with current chemotherapeutic drugs can significantly improve treatment outcome, offering a promising new therapeutic option for patients with pancreatic cancer.