A tumor‐specific microenvironment is characterized by hypoxia, in which oxygen tension is considerably lower than in normal tissues. The hypoxic status of various solid tumors has been attributed as ...an indicator of adverse prognosis due to tumor progression toward a more malignant phenotype with increased metastatic potential and resistance to treatment. Various exogenous and endogenous markers for hypoxia are currently available and studied in relation to each other, tumor architecture, and tumor microenvironment. Over the last few decades, various methods have been suggested to assess the level of oxygenation in solid tumors. Among them, nitroimidazole compounds have provided promising information on tumor hypoxia. To quantify the extent of hypoxia requires that nitroimidazole binding be primarily dependent on oxygen concentration as well as nitroreductase levels in the tumor cells. Furthermore, recent progress in molecular biology has highlighted a transcription factor, hypoxia‐inducible factor (HIF)‐1, whose activity is induced by hypoxia. HIF‐1 plays a central role in malignant progression by inducing the expression of various genes, whose functions are strongly associated with malignant alteration of the entire tumor. The cellular changes induced by HIF‐1 are extremely important therapeutic targets of cancer therapy, particularly in the therapy against refractory cancers. In this review, we will discuss the significance of pimonidazole and HIF‐1 as exogenous and endogenous hypoxia markers, respectively, as well as their evaluation and imaging of tumor hypoxia. (Cancer Sci 2009)
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable ...luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized. In the mouse brain, genetic labeling with neural activity sensors allowed tracking of small clusters of hippocampal neurons activated by novel environments. In a marmoset, we recorded video-rate bioluminescence from neurons in the striatum, a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light source to spur unprecedented scientific, medical, and industrial applications.
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BFBNIB, NMLJ, NUK, ODKLJ, PNG, SAZU, UL, UM, UPUK
Tumor hypoxia has been considered to be a potential therapeutic problem because it renders solid tumors more resistant to sparsely ionizing radiation (IR) and chemotherapeutic drugs. Moreover, recent ...laboratory and clinical data have shown that tumor hypoxia is also associated with a more malignant phenotype and poor survival in patients suffering from various solid tumors. Therefore, selective targeting of hypoxic tumor cells has been explored, and since severe hypoxia (pO2<0.33%, 2.5 mmHg) does not occur in normal tissue, tumor hypoxia could be exploited for therapeutic advantage. However, the following three characteristics of hypoxic tumor regions present obstacles in targeting hypoxic cells. First, it is difficult to deliver a sufficient amount of drug to a region that is remote from blood vessels. Second, one must specifically target hypoxic tumor cells while sparing normal well‐oxygenated tissue from damage. Finally, the severely hypoxic tumor cells to be attacked have often stopped dividing. Therefore, high delivery efficiency, high specificity and selective cytotoxicity are all necessary to target and combat hypoxic tumor cells. The current review describes progress on the biological aspects of tumor hypoxia and provides a compilation of the recent molecular approaches used to target hypoxic tumors. These approaches include our work with a unique hypoxia‐targeting protein drug, TOP3, with which we have sought to address the above three difficulties.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Lung metastasis is a major cause of mortality in patients with osteosarcoma (OS). A better understanding of the molecular mechanism of OS lung metastasis may facilitate development of new therapeutic ...strategies to prevent the metastasis. We have established high‐ and low‐metastatic sublines (LM8‐H and LM8‐L, respectively) from Dunn OS cell line LM8 by using in vivo image‐guided screening. Among the genes whose expression was significantly increased in LM8‐H compared to LM8‐L, the transcription factor lymphoid enhancer‐binding factor 1 (LEF1) was identified as a factor that promotes LM8‐H cell extravasation into the lungs. To identify downstream effectors of LEF1 that are involved in OS lung metastasis, 13 genes were selected based on LM8 microarray data and genomewide meta‐analysis of a public database for OS patients. Among them, the cytoglobin (Cygb) gene was identified as a key effector in promoting OS extravasation into the lungs. CYGB overexpression increased the extravasation ability of LM8‐L cells, whereas knocking out the Cygb gene in LM8‐H cells reduced this ability. Our results showed a novel LEF1‐CYGB axis in OS lung metastasis and may provide a new way of developing therapeutic strategies to prevent OS lung metastasis.
We found that the cytoglobin (Cygb) gene is an important effector that promotes extravasation of osteosarcoma (OS) to the lungs. Our data showed a novel function of the LEF1‐CYGB axis in OS lung metastasis and may provide novel targets for drugs that prevent OS lung metastasis.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Small antibody mimetics that contain high-affinity target-binding peptides can be lower cost alternatives to monoclonal antibodies (mAbs). We have recently developed a method to create small antibody ...mimetics called FLuctuation-regulated Affinity Proteins (FLAPs), which consist of a small protein scaffold with a structurally immobilized target-binding peptide. In this study, to further develop this method, we established a novel screening system for FLAPs called monoclonal antibody-guided peptide identification and engineering (MAGPIE), in which a mAb guides selection in two manners. First, antibody-guided design allows construction of a peptide library that is relatively small in size, but sufficient to identify high-affinity binders in a single selection round. Second, in antibody-guided screening, the fluorescently labeled mAb is used to select mammalian cells that display FLAP candidates with high affinity for the target using fluorescence-activated cell sorting. We demonstrate the reliability and efficacy of MAGPIE using daclizumab, a mAb against human interleukin-2 receptor alpha chain (CD25). Three FLAPs identified by MAGPIE bound CD25 with dissociation constants of approximately 30 nM as measured by biolayer interferometry without undergoing affinity maturation. MAGPIE can be broadly adapted to any mAb to develop small antibody mimetics.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Target‐binding small proteins are promising alternatives to conventional monoclonal antibodies (mAbs), offering advantages in terms of tissue penetration and manufacturing costs. Recently, a design ...strategy to create small proteins called fluctuation‐regulated affinity proteins (FLAPs) consisting of a structurally immobilized peptide from the complementarity‐determining region (CDR) loops of mAbs (CDR‐derived peptide) and a protein scaffold was developed. Because mAb paratopes are usually composed of multiple CDRs, FLAPs with multiple binding peptides may have an enhanced target‐binding capability. Here, a strategy to create FLAPs bearing dual CDR‐derived peptides (D‐FLAPs) using the anti‐human epithelial growth factor receptor type 2 (HER2) mAb trastuzumab as a basis is developed. Computationally selected CDR‐derived peptides are first grafted onto two adjacent loops of the fibronectin type III domain (FN3) scaffold, yielding 80 D‐FLAP candidates. After computational screening based on their similarity to the parental mAb with regard to the conformation of paratope residues, two candidates are selected. After further evaluation with ELISA, one D‐FLAP with HYTTPP and GDGFYA peptides from CDR‐L3 and CDR‐H3 of the parental mAb, respectively, is found to bind HER2 with a dissociation constant of 58 nm. This method applies to various mAb drugs and allows the rational design of small protein alternatives.
Target‐binding small proteins are promising alternatives to monoclonal antibodies (mAbs). In this study, a computational strategy to design and screen mimetics called fluctuation‐regulated affinity proteins bearing dual complementarity‐determining region‐derived peptides (D‐FLAPs) is described. This strategy can be applied to various mAb drugs and allow the rational design of small protein alternatives. This article is part of an AFOB (Asian Federation of Biotechnology) Special issue. To learn more about the AFOB visit www.afob.org.
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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.
We recently developed a fusion protein drug TOP3, which is designed to be specifically stabilized and activated in hypoxia‐inducible factor (HIF)‐active cancers. HIF is strongly correlated with poor prognosis of pancreatic cancer patients. In this study, we reported 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.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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.
Although the current murine model of bone metastasis using intracardiac (IC) injection successfully recapitulates the process of bone metastasis, further progress in the study of bone metastasis ...requires a new model to circumvent some limitations of this model. Here, we present a new murine model of bone metastasis achieved by injecting cancer cells through the intra-caudal arterial (CA). This model does not require high technical proficiency, predominantly delivers cancer cells to bone marrow of hind limbs with much higher efficiency than IC injection, and greatly shortens the period of overt bone metastasis development. Moreover, CA injection barely causes acute death of mice, enabling us to inject a larger number of cancer cells to further accelerate the development of bone metastasis with a wide variety of cell lines. Our model may open a new avenue for understanding the bone metastatic processes and development of drugs preventing bone metastasis and recurrence.
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.
Bone resorption facilitates cancer cell colonization of osteoblastic bone metastasis. We revealed that insulin‐like growth factor‐1 released from the bone during bone resorption and hypoxia‐inducible factor activity cooperatively promote cancer cell colonization in hypoxic bone marrow.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK