口丸 高弘

Abstract 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.

Abstract 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.

Abstract 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.

Ras genes are frequently mutated in many cancer types; however, there are currently no conclusively effective anticancer drugs against Ras-induced cancer. Therefore, the downstream effectors of Ras signaling need to be identified for the development of promising novel therapeutic approaches. We previously reported that oncogenic Ras induced the expression of NF-HEV/IL-33, a member of the interleukin-1 family, and showed that intracellular IL-33 was required for oncogenic Ras-induced cellular transformation. In the present study, we demonstrated that the c-Mer proto-oncogene tyrosine kinase (MerTK), a receptor tyrosine kinase, played essential roles in oncogenic Ras/IL-33 signaling. The expression of MerTK was enhanced in transformed NIH-3T3 cells by the expression of oncogenic Ras, H-Ras (G12V), in an IL-33-dependent manner. In human colorectal cancer tissues, MerTK expression also correlated with IL-33 expression. The knockdown of IL-33 or MerTK effectively attenuated the migration of NIH-3T3 cells transformed by H-Ras (G12V) and A549, LoVo, and HCT116 cells harboring an oncogenic K-Ras mutation. Furthermore, the suppression of Ras-induced cell migration by the knockdown of IL-33 was rescued by the enforced expression of MerTK. The present results also revealed that MerTK was effectively phosphorylated in NIH-3T3 cells transformed by Ras (G12V). Ras signaling was essential for the tyrosine phosphorylation of MerTK, and the kinase activity of MerTK was indispensable for accelerating cell migration. Collectively, the present results reveal a novel role for MerTK in cancer malignancy, which may be utilized to develop novel therapeutic strategies that target Ras-transformed cells.

近年、飛躍的な進歩を遂げた1細胞オミクス技術によって、個々の細胞の遺伝子発現プロファイルの測定が可能になり、実験動物やヒトの組織を構成するヘテロな細胞種やその表現系がこれまでにない解像度で明らかにされつつある。それと同時に、現行の1細胞オミクス解析をさらに発展させるための課題も表出している。例えば、生体組織を構成する無数の細胞の種別・表現系が明らかになれば、それらの相互作用が、組織恒常性やその破綻において果たす役割の解明が期待される。本研究では、分割型緑色蛍光タンパク質(分割型GFP)を用いた遺伝子コード型の細胞間相互作用の可視化技術と1細胞オミクス解析を組み合わせることで、転移過程におけるがん細胞と組織構成細胞の相互作用機構を解明する新規アプローチを提案する。(著者抄録)

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.

During the development of analgesic tolerance to morphine, the V1b vasopressin receptor has been proposed to bind to β-arrestin 2 and the µ-opioid receptor to enable their interaction. However, direct evidence of such a high-order complex is lacking. Using bioluminescent resonance energy transfer between a split Nanoluciferase and the Venus fluorescent protein, the NanoBit-NanoBRET system, we found that β-arrestin 2 closely located near the heteromer µ-V1b receptor in the absence of an agonist and moved closer to the receptor carboxyl-termini upon agonist stimulation. An additive effect of the two agonists for opioid and vasopressin receptors was detected on the NanoBRET between the µ-V1b heteromer and β-arrestin 2. To increase the agonist response of NanoBRET, the ratio of the donor luminophore to the acceptor fluorophore was decreased to the detection limit of luminescence. In the first phase of access, β-arrestin 2 was likely to bind to the unstimulated V1b receptor in both its phosphorylated and unphosphorylated forms. In contrast, the second-phase access of β-arrestin 2 was agonist dependent, indicating a possible pharmacological intervention strategy. Therefore, our efficient method should be useful for evaluating chemicals that directly target the vasopressin binding site in the µ-V1b heteromer to reduce the second-phase access of β-arrestin 2 and thereby to alleviate tolerance to morphine analgesia.

The western pattern diet is rich not only in fat and calorie but also in phosphate. Negative impacts of excessive fat and calorie intake on health are widely accepted, whereas potential harms of excessive phosphate intake are poorly recognized. Here we show the mechanism by which dietary phosphate damages the kidney. When phosphate intake was excessive relative to the functioning nephron number, circulating fibroblast growth factor-23 (FGF23), a hormone that increases phosphate excretion per nephron, was increased to maintain phosphate homeostasis. FGF23 suppressed phosphate reabsorption in renal tubules and thus raised the phosphate concentration in the tubular fluid. Once it exceeded a threshold, microscopic particles containing calcium phosphate crystals appeared in the tubular lumen, which damaged tubular cells through binding to Toll-like receptor-4 expressed on them. Persistent tubular damage induced interstitial fibrosis, reduced the nephron number, and further boosted FGF23 to trigger a deterioration spiral leading to progressive nephron loss. In humans, progression of chronic kidney disease (CKD) ensued when the serum FGF23 level exceeded 53 pg/mL. The present study identified the calcium phosphate particles in the renal tubular fluid as an effective therapeutic target to decelerate nephron loss during the course of aging and CKD progression.

Depolarization of circularly polarized light scattered from biological tissues depends on structural changes in cell nuclei, which can provide valuable information for differentiating cancer tissues concealed in healthy tissues. In this study, we experimentally verified the possibility of cancer identification using scattering of circularly polarized light. We investigated the polarization of light scattered from a sliced biological tissue with various optical configurations. A significant difference between circular polarizations of light scattered from cancerous and healthy tissues is observed, which is sufficient to distinguish a cancerous region. The line-scanning experiments along a region incorporating healthy and cancerous parts indicate step-like behaviors in the degree of circular polarization corresponding to the state of tissues, whether cancerous or normal. An oblique and perpendicular incidence induces different resolutions for identifying cancerous tissues, which indicates that the optical arrangement can be selected according to the priority of resolution.

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.

This journal is © 2020 The Royal Society of Chemistry. 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 (KD) 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.

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.

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.

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.

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.

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. These events were clearly negated by the addition of mevalonate or geranylgeranyl pyrophosphate, indicating that the protein geranylgeranylation is implicated in the apoptotic resistance to MEK inhibitors. Furthermore, mechanistically, the combined treatment of CH5126766 with statins upregulated TNF-related apoptosis-inducing ligand (TRAIL), which was dependent on inhibition of the mevalonate pathway and is involved in apoptosis induction in human breast cancer MDA-MB-231 cells. The present study not only revealed that the mevalonate pathway could be targetable to enhance the efficacy of MEK inhibitors, but also proposes that combinatorial treatment of MEK inhibitors with statins may be a promising therapeutic strategy to sensitize cancer cells to apoptosis.

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.

現在,ホタルルシフェラーゼ(Fluc)とその天然基質D-ルシフェリンを用いた生物発光システムが,マウスなど小動物の非侵襲的生体イメージングに広く利用されている。そして最近,D-ルシフェリンよりも生体発光イメージングに適した合成基質の開発などにより,生体発光イメージングの更なる高感度化と多機能化が実現している。本稿では,生体発光イメージングのための新技術を既説し,今後の展望について紹介する。(著者抄録)