Epitope binning for multiple antibodies simultaneously using mammalian cell display and DNA sequencing.

Epitope binning, an approach for grouping antibodies based on epitope similarities, is a critical step in antibody drug discovery. However, conventional methods are complex, involving individual antibody production. Here, we established Epitope Binning-seq, an epitope binning platform for simultaneously analyzing multiple antibodies.

Secretory GFP reconstitution labeling of neighboring cells interrogates cell-cell interactions in metastatic niches.

Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell-cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues.

Identification of Surface Markers and Functional Characterization of Myeloid Derived Suppressor Cell-Like Adherent Cells.

Myeloid-derived suppressor cell (MDSC)-like adherent cells (MLACs) are a recently identified CD11b F4/80 myeloid cell subset that can infiltrate tumors early in development and promote their growth. Because of these functions, MLACs play an important role in establishing an immunosuppressive tumor microenvironment (TME). However, the lack of MLAC-specific markers has hampered further characterization of this cell type.

In vivo optical imaging of tumor stromal cells with hypoxia-inducible factor activity.

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).

Tissue-resident macrophages are major tumor-associated macrophage resources, contributing to early TNBC development, recurrence, and metastases.

Triple-negative breast cancer (TNBC) is an aggressive and highly heterogenous disease with no well-defined therapeutic targets. Treatment options are thus limited and mortality is significantly higher compared with other breast cancer subtypes. Mammary gland tissue-resident macrophages (MGTRMs) are found to be the most abundant stromal cells in early TNBC before angiogenesis.

In Vivo Imaging of Oxidative and Hypoxic Stresses in Mice Model of Amyotrophic Lateral Sclerosis.

Oxidative and hypoxic stresses are associated with the degeneration of both motor neurons and skeletal muscles in amyotrophic lateral sclerosis (ALS). In vivo bioluminescent imaging is used to monitor cellular responses to oxidative and hypoxic stresses in living ALS model mice bearing G93A-human Cu/Zn superoxide dismutase (SOD1) longitudinally using the IVIS spectrum imaging system. Double transgenic mice bearing both Keap1-dependent oxidative stress detector No-48 (OKD48) and G93A-SOD1 are useful for in vivo imaging of oxidative stress in ALS.

In Vivo Label-Free Observation of Tumor-Related Blood Vessels in Small Animals Using a Newly Designed Photoacoustic 3D Imaging System.

Photoacoustic (PA) technology can be used for non-invasive imaging of blood vessels. In this paper, we report on our prototype PA imaging system with a newly designed ultrasound sensor and its visualization performance of microvascular in animal. We fabricated an experimental system for animals using a high-frequency sensor.

Droplet-based valveless microfluidic system for phage-display screening against spheroids.

In this study, we proposed a droplet-based valveless microfluidic system that has the necessary functions to perform the binding, washing, eluting, and collecting processes of phage-display screening against spheroids, which can be expected to present a similar repertoire and number of membrane proteins as . Although spheroids have much larger sizes than single cells, spheroids are difficult to manipulate through manual operation. The proposed microfluidic system actively controls the position and velocity of droplets using a camera, three air pumps, and three liquid pumps to perform the processes for phage-display screening.

Antibody-guided design and identification of CD25-binding small antibody mimetics using mammalian cell surface display.

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.

A Murine Bone Metastasis Model Using Caudal Artery Injection and Bioluminescence Imaging.

The current standard murine model of bone metastasis by using intracardiac injection (IC) has some limitations despite the great utility of this model. This fact emphasizes the need for a new murine model to accelerate basic research of bone metastasis. The present protocol provides instructions on caudal artery (CA) injection that is an easy-to-use method to reliably construct a murine bone metastasis model with a variety type of cancer cell lines.

Reconstitution of an Anti-HER2 Antibody Paratope by Grafting Dual CDR-Derived Peptides onto a Small Protein Scaffold.

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.

Slicing Spheroids in Microfluidic Devices for Morphological and Immunohistochemical Analysis.

Microfluidic devices utilizing spheroids play important roles in in vitro experimental systems to closely simulate morphological and biochemical characteristics of the in vivo tumor microenvironment. For the observation and analysis of the inner structure of spheroids, sectioning is an efficient approach. However, conventional microfluidic devices are difficult for sectioning, and therefore, spheroids inside the microfluidic channels have not been sliced well.

Strategic design to create HER2-targeting proteins with target-binding peptides immobilized on a fibronectin type III domain scaffold.

Tumor-binding peptides such as human epidermal growth factor receptor 2 (HER2)-binding peptides are attractive therapeutic and diagnostic options for cancer. However, the HER2-binding peptides (HBPs) developed thus far are susceptible to proteolysis and lose their affinity to HER2 . In this report, a method to create a HER2-binding fluctuation-regulated affinity protein (HBP-FLAP) consisting of a fibronectin type III domain (FN3) scaffold with a structurally immobilized HBP is presented.

[Semi-rational Design of Target-binding Small Proteins for Cancer Treatment].

Small proteins that have a high affinity for cancer cell surface markers can be promising cheap alternatives to antibodies (antibody mimetics). Various types of antibody mimetics have thus been extensively developed. We recently found that a target-binding peptide binds to its target molecule more strongly when it is structurally constrained.

Design Strategy to Create Antibody Mimetics Harbouring Immobilised Complementarity Determining Region Peptides for Practical Use.

Monoclonal antibodies (mAbs) are attractive therapeutics for treating a wide range of human disorders, and bind to the antigen through their complementarity-determining regions (CDRs). Small stable proteins containing structurally retained CDRs are promising alternatives to mAbs. In this report, we present a method to create such proteins, named fluctuation-regulated affinity proteins (FLAPs).

Design, synthesis, and evaluation of indeno[2,1-c]pyrazolones for use as inhibitors against hypoxia-inducible factor (HIF)-1 transcriptional activity.

HIF-1 is regarded as a promising target for the drugs used in cancer chemotherapy, and creating readily accessible templates for the development of synthetic drug candidates that could inhibit HIF-1 transcriptional activity is an important pursuit. In this study, indeno[2,1-c]pyrazolones were designed as readily available synthetic inhibitors of HIF-1 transcriptional activity. Nine compounds were synthesized in 4-5 steps from commercially available starting materials.

Imaging Hypoxic Stress and the Treatment of Amyotrophic Lateral Sclerosis with Dimethyloxalylglycine in a Mice Model.

Hypoxia inducible factor-1α (HIF-1α) is a key transcription factor that maintains oxygen homeostasis. Hypoxic stress is related to the pathogenesis of amyotrophic lateral sclerosis (ALS), and impaired HIF-1α induces motor neuron degeneration in ALS. Dimethyloxalylglycine (DMOG) upregulates the stability of HIF-1α expression and shows neuroprotective effects, but has not been used in ALS as an anti-hypoxic stress treatment.

Characterization and Biodistribution Analysis of Oxygen-Doped Single-Walled Carbon Nanotubes Used as in Vivo Fluorescence Imaging Probes.

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.

Microfluidic High-Migratory Cell Collector Suppressing Artifacts Caused by Microstructures.

The small number of high-migratory cancer cells in a cell population make studies on high-migratory cancer cells difficult. For the development of migration assays for such cancer cells, several microfluidic devices have been developed. However, they measure migration that is influenced by microstructures and they collect not only high-migratory cells, but also surrounding cells.

Microfluidic Device for Screening for Target Cell-Specific Binding Molecules by Using Adherent Cells.

This paper proposes a microfluidic device for screening molecules such as aptamers, antibodies, proteins, etc. for target cell-specific binding molecules. The discovery of cancer cell-specific binding molecules was the goal of this study.

A reliable murine model of bone metastasis by injecting cancer cells through caudal arteries.

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.

Novel lymphoid enhancer-binding factor 1-cytoglobin axis promotes extravasation of osteosarcoma cells into the lungs.

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.

Blockage of the mevalonate pathway overcomes the apoptotic resistance to MEK inhibitors with suppressing the activation of Akt in cancer cells.

With increasing clinical demands for MEK inhibitors in cancer treatment, overcoming the resistance to MEK inhibitors is an urgent problem to be solved. Numerous reports have shown that MEK inhibition results in the activation of PI3K-Akt signaling, which may confer apoptotic resistance to MEK inhibitors. We here demonstrate that the blockade of the mevalonate pathway using the antilipidemic drug statins represses Akt activation following MEK inhibition and induces significant apoptosis when co-treated with CH5126766 or trametinib.

Novel adherent CD11b Gr-1 tumor-infiltrating cells initiate an immunosuppressive tumor microenvironment.

The immunosuppressive tumor microenvironment is a hallmark of cancer. Myeloid-derived suppressor cells (MDSCs) are CD11b Gr-1 tumor-infiltrating immature myeloid cells that strongly mediate tumor immunosuppression. The CD11b Gr-1 cells are a heterogeneous cell population, and the impacts of each subpopulation on tumor progression are not yet completely understood.