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. In this system, epitope similarity between the query antibodies (qAbs) displayed on antigen-expressing cells and a fluorescently labeled reference antibody (rAb) targeting a desired epitope is analyzed by flow cytometry. The qAbs with epitope similar to the rAb can be identified by next-generation sequencing analysis of fluorescence-negative cells. Sensitivity and reliability of this system are confirmed using rAbs, pertuzumab and trastuzumab, which target human epidermal growth factor receptor 2. Epitope Binning-seq enables simultaneous epitope evaluation of 14 qAbs at various abundances in libraries, grouping them into respective epitope bins. This versatile platform is applicable to diverse antibodies and antigens, potentially expediting the identification of clinically useful antibodies.

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. This study identifies the gene signature of MLACs by analyzing RNA-sequencing (RNA-seq) and public single-cell RNA-seq data, revealing that MLACs are an independent cell population that are distinct from other intratumoral myeloid cells. After combining proteome analysis of membrane proteins with RNA-seq data, H2-Ab1 and CD11c are indicated as marker proteins that can support the isolation of MLAC subsets from CD11b+ F4/80- myeloid cells by fluorescence-activated cell sorting. The CD11b+ F4/80- H2-Ab1+ and CD11b+ F4/80- CD11c+ MLAC subsets represent approximately half of the MLAC population that is isolated based on their adhesion properties and possess gene signatures and functional properties similar to those of the MLAC population. Additionally, membrane proteome analysis suggests that MLACs express highly heterogeneous surface proteins. This study facilitates an integrated understanding of heterogeneous intratumoral myeloid cells, as well as the molecular and cellular details of the development of an immunosuppressive TME.

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. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.

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). 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.

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. We therefore aimed to explore novel therapeutic approaches for TNBC by focusing on MGTRMs. Local depletion of MGTRMs in mammary gland fat pads the day before TNBC cell transplantation significantly reduced tumor growth and tumor-associated macrophage (TAM) infiltration in mice. Furthermore, local depletion of MGTRMs at the site of TNBC resection markedly reduced recurrence and distant metastases, and improved chemotherapy outcomes. This study demonstrates that MGTRMs are a major TAM resource and play pivotal roles in the growth and malignant progression of TNBC. The results highlight a possible novel anti-cancer approach targeting tissue-resident macrophages.

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. We developed a bioluminescence resonance energy transfer (BRET) probe that is regulated by HIF-1α-specific ubiquitin-proteasome system. G93A-SOD1 mice injected with the BRET probe are useful to investigate the spatiotemporal responses to hypoxic stress in ALS. In this chapter, we introduce a practical protocol of in vivo imaging of both oxidative and hypoxic stress in ALS model mice.

AGE/RAGE axis regulates reversible transition to quiescent states of ALK-rearranged NSCLC and pancreatic cancer cells in monolayer cultures.

Cancer recurrence due to tumor cell quiescence after therapy and long-term remission is associated with cancer-related death. Previous studies have used cell models that are unable to return to a proliferative state; thus, the transition between quiescent and proliferative states is not well understood. Here, we report monolayer cancer cell models wherein the human non-small cell lung carcinoma cell line H2228 and pancreatic cancer cell line AsPC-1 can be reversibly induced to a quiescent state under hypoxic and serum-starved (HSS) conditions. Transcriptome and metabolome dual-omics profiles of these cells were compared with those of the human lung adenocarcinoma cell line A549, which was unable to enter a quiescent state under HSS conditions. The quiescence-inducible cells had substantially lower intracellular pyruvate and ATP levels in the quiescent state than in the proliferative state, and their response to sudden demand for energy was dramatically reduced. Furthermore, in quiescence-inducible cells, the transition between quiescent and proliferative states of these cells was regulated by the balance between the proliferation-promoting Ras and Rap1 signaling and the suppressive AGE/RAGE signaling. These cell models elucidate the transition between quiescent and proliferative states, allowing the development of drug-screening systems for quiescent tumor cells.

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. The system has two modes: still image mode by wide scanning and moving image mode by small rotation of sensor array. Optical test target, euthanized mice and rats, and live mice were used as objects. The results of optical test target showed that the spatial resolution was about two times higher than that of our conventional prototype. The image performance in vivo was evaluated in euthanized healthy mice and rats, allowing visualization of detailed blood vessels in the liver and kidneys. In tumor-bearing mice, different results of vascular induction were shown depending on the type of tumor and the method of transplantation. By utilizing the video imaging function, we were able to observe the movement of blood vessels around the tumor. We have demonstrated the feasibility of the system as a less invasive animal experimental device, as it can acquire vascular images in animals in a non-contrast and non-invasive manner.

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 in vivo. 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. The cross section of the microchannel is large in width and height for the passage of spheroids. Valves that can close such a large cross-sectional microchannel are not readily available. Thus, we proposed valveless flow control using liquid pumps. In addition, the proposed microfluidic system involves complex flow channels with airflow subchannels to perform phage-display screening. For washing, nonspecific-binding phages remaining in the flow channels must be minimized. The proposed microfluidic system can perform selective blocking and flush washing. Selective blocking can prevent the airflow channels from becoming hydrophilic with blocking liquid, and flush washing can flush phages remaining in the flow channel. We experimentally verified the functions of the developed microfluidic device based on the proposed system.

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. 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.

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. Bioluminescence imaging visualized that cancer cells injected via the caudal artery in the tail were efficiently delivered to a hind limb bone, where it is a common site affected with bone metastasis in mice. CA injection rarely causes stress-induced acute death in mice and enables us to inject a large number of cancer cells, thereby greatly increasing the frequency of bone metastasis in hind limb bones. Importantly, CA injection is technically as easy as tail vein injection and causes no lethal stress, indicating that it is a model that also contributes to animal welfare. CA injection model, therefore, could represent a powerful tool for many researchers to study molecular mechanisms of bone metastasis in mice.

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. 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.

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. We proposed a microfluidic device created from embedding resin for sectioning. Spheroids were cultured, embedded by resin, and sectioned in the microfluidic device. Slices of the sectioned spheroids yielded clear images at the cellular level. According to morphological and immunohistochemical analyses of the slices of the spheroid, specific protein distribution was observed.

[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. To apply this finding to the development of chemically synthesizable small antibody mimetics, we have established an efficient method of creating such proteins, named fluctuation-regulated affinity proteins (FLAPs). To identify desirable scaffolds, first, 13 human proteins (46-104 aa) were selected from the Protein Data Bank. Then, thirteen graft acceptor (GA) sites that efficiently immobilize the grafted peptide structure were identified from six small protein scaffolds using molecular dynamics simulation. To assess the designed antibody mimetics in vitro, human epidermal growth factor receptor 2 (HER2)-binding peptides were selected from the anti-HER2 antibody drugs trastuzumab and pertuzumab by calculating the binding energy, and these were then grafted into the GA sites of scaffolds to create 65 FLAP candidates. The FLAP candidates were expressed in bacteria as fusion proteins with Renilla luciferase (Rluc), and their relative binding affinity to HER2 was easily determined by measuring the Rluc bioluminescence intensity without protein purification. Finally, four out of the 65 showed specific binding to HER2 with a dissociation constant (KD) of 24-65 nM, and these were used for the detection of HER2-expressing cancer cells. Our design strategy will promote the development of antibody mimetics for the effective treatment of cancers and other diseases.

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). Thirteen graft acceptor (GA) sites that efficiently immobilise the grafted peptide structure were initially selected from six small protein scaffolds by computational identification. Five CDR peptides extracted by binding energy calculations from mAbs against breast cancer marker human epithelial growth factor receptor type 2 (HER2) were then grafted to the selected scaffolds. The combination of five CDR peptides and 13 GA sites in six scaffolds revealed that three of the 65 combinations showed specific binding to HER2 with dissociation constants (KD) of 270-350 nM in biolayer interferometry and 24-65 nM in ELISA. The FLAPs specifically detected HER2-overexpressing cancer cells. Thus, the present strategy is a promising and practical method for developing small antibody mimetics.

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 vivo. 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. HBPs were selected by phage-library screening and grafted onto FN3 to create FN3-HBPs, and the HBP-FLAP with the highest affinity (HBP sequence: YCAHNM) was identified after affinity maturation of the grafted HBP. HBP-FLAP containing the YCAHNM peptide showed increased proteolysis-resistance, binding to HER2 with a dissociation constant (K D) of 58 nM in ELISA and 287 nM in biolayer interferometry and specifically detects HER2-expressing cancer cells. In addition, HBP-FLAP clearly delineated HER2-expressing tumors with a half-life of 6 h after intravenous injection into tumor-bearing mice. FN3-based FLAP is an excellent platform for developing target-binding small proteins for clinical applications.

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. In evaluations of the ability to inhibit the hypoxia-induced transcriptional activity of HIF-1, compound 3c showed a higher level compared with that of known inhibitor, YC-1. The compound 3c suppressed HIF-1α protein accumulation without affecting the levels of HIF-1α mRNA.

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. In the present study, we investigated hypoxic stress in ALS model mice bearing G93A-human Cu/Zn superoxide dismutase by in vivo HIF-1α imaging, and treated the ALS mice with DMOG. In vivo HIF-1α imaging analysis showed enhanced hypoxic stress in both the spinal cord and muscles of lower limbs of ALS mice, even at the pre-symptomatic stage. HIF-1α expression decreased as the disease progressed until 126 days of age. DMOG treatment significantly ameliorated the decrease in HIF-1α expression, the degeneration of both spinal motor neurons and myofibers in lower limbs, gliosis and apoptosis in the spinal cord. This was accompanied by prolonged survival. The present study suggests that in vivo bioluminescence resonance energy transfer (BRET) HIF-1α imaging is useful for evaluating hypoxic stress in ALS, and that the enhancement of HIF-1α is a therapeutic target for ALS patients.

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. 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.

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. In order to find high-migratory cells in cell populations while suppressing artifacts and to collect these cells while minimizing damages, we developed a microfluidic high-migratory cell collector with the ability to sort cancer cells according to cellular migration and mechanical detachment. High-migratory cancer cells travel further from the starting line when all of the cells are seeded on the same starting line. The high-migratory cells are detached using a stretch of cell adhesive surface using a water-driven balloon actuator. Using this cell collector, we selected high-migratory HeLa cells that migrated about 100m in 12 h and collected the cells.