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)
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.
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.
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.
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.
Tumors contain various stromal cells, such as immune cells, endothelial cells, and fibroblasts, which contribute to the development of a tumor-specific microenvironment characterized by hypoxia and ...inflammation, and are associated with malignant progression. In this study, we investigated the activity of intratumoral hypoxia-inducible factor (HIF), which functions as a master regulator of the cellular response to hypoxia and inflammation. We constructed the HIF activity-monitoring reporter gene hypoxia-response element-Venus-Akaluc (HVA) that expresses the green fluorescent protein Venus and modified firefly luciferase Akaluc in a HIF activity-dependent manner, and created transgenic mice harboring HVA transgene (HVA-Tg). In HVA-Tg, HIF-active cells can be visualized using AkaBLI, an ultra-sensitive in vivo bioluminescence imaging technology that produces an intense near-infrared light upon reaction of Akaluc with the D-luciferin analog AkaLumine-HCl. By orthotopic transplantation of E0771, a mouse triple negative breast cancer cell line without a reporter gene, into HVA-Tg, we succeeded in noninvasively monitoring bioluminescence signals from HIF-active stromal cells as early as 8 days after transplantation. The HIF-active stromal cells initially clustered locally and then spread throughout the tumors with growth. Immunohistochemistry and flow cytometry analyses revealed that CD11b
F4/80
macrophages were the predominant HIF-active stromal cells in E0771 tumors. These results indicate that HVA-Tg is a useful tool for spatiotemporal analysis of HIF-active tumor stromal cells, facilitating investigation of the roles of HIF-active tumor stromal cells in tumor growth and malignant progression.
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.
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.
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.
Single-walled carbon nanotubes (SWCNTs) show strong fluorescence in the 1000–1700 nm second near-infrared (NIR-II) wavelength range and are considered promising candidates for angiographic imaging ...probes. Oxygen-doped SWCNTs coated with phospholipid-polyethylene glycol (o-SWCNT-PEG) show exceptional potential, as they emit fluorescence at ∼1300 nm through excitation with 980 nm light. Here, with the aim of putting o-SWCNTs to practical use as an angiographic agent in animal experiments, the retention time after intravenous administration in the vasculature of mice and the biodistribution were studied. To provide bio affinity, the o-SWCNTs were coated with phospholipid polyethylene glycol. The intravenously injected o-SWCNT-PEG circulated within the vasculature for 3 h and cleared within 1 day. There was prominent fluorescence and Raman signals from the SWCNTs in the liver and spleen early in the experiment; the signals remained for 1 month. No apparent abnormalities in weight or appearance were observed after 2 months, suggesting low toxicity of o-SWCNT-PEG. These characteristics of o-SWCNT-PEG would make it useful as an angiographic imaging probe in the NIR-II wavelength range.
PDF
