Intravital Förster resonance energy transfer imaging reveals osteopontin-mediated polymorphonuclear leukocyte activation by tumor cell emboli.

Myeloid-derived suppressor cells (MDSCs) cause paraneoplastic leukemoid reactions and facilitate tumor cell metastasis. However, the interaction of MDSCs with tumor cells in live tissue has not been adequately visualized. To accomplish this task, we developed an intravital imaging protocol to observe metastasized tumor cells in mouse lungs. For visualization of the activation of MDSCs, bone marrow cells derived from transgenic mice expressing a Förster resonance energy transfer biosensor for ERK were implanted into host mice. Under a two-photon excitation microscope, numerous polymorphonuclear cells (PMNs) were found to infiltrate the lungs of tumor-bearing mice in which 4T1 mammary tumor cells were implanted into the footpads. By Förster resonance energy transfer imaging, we found ERK activation in PMNs around the 4T1 tumor emboli in the lungs. Because antibody array analysis implied the involvement of osteopontin (OPN) in the metastasis of 4T1 cells, we further analyzed the effect of OPN knockdown. The OPN knockdown in 4T1 cells did not affect the cell growth, but markedly suppressed lung metastasis of 4T1 cells and ERK activation in PMNs in the lung. Intravenous injection of recombinant OPN restored the lung metastasis of OPN-deficient 4T1 cells, suggesting that OPN functioned in a paracrine manner. It has been reported that ERK activation of neutrophils causes NETosis and that PMNs promote metastasis of tumor cells by NETosis. In agreement with previous reports, the NETosis inhibitor DNase I inhibited lung metastasis of 4T1 cells. These observations suggest that OPN promotes metastasis of 4T1 cells by activating PMNs and inducing NETosis.

Live imaging of transforming growth factor-ß activated kinase 1 activation in Lewis lung carcinoma 3LL cells implanted into syngeneic mice and treated with polyinosinic:polycytidylic acid.

Transforming growth factor-ß activated kinase 1 (TAK1) has been shown to play a crucial role in cell death, differentiation, and inflammation. Here, we live-imaged robust TAK1 activation in Lewis lung carcinoma 3LL cells implanted into the s.c. tissue of syngeneic C57BL/6 mice and treated with polyinosinic:polycytidylic acid (PolyI:C). First, we developed and characterized a Förster resonance energy transfer-based biosensor for TAK1 activity. The TAK1 biosensor, named Eevee-TAK1, responded to stress-inducing reagents such as anisomycin, tumor necrosis factor-α, and interleukin1-ß. The anisomycin-induced increase in Förster resonance energy transfer was abolished by the TAK1 inhibitor (5z)-7-oxozeaenol. Activity of TAK1 in 3LL cells was markedly increased by PolyI:C in the presence of macrophages. 3LL cells expressing Eevee-TAK1 were implanted into mice and observed through imaging window by two-photon excitation microscopy. During the growth of tumor, the 3LL cells at the periphery of the tumor showed higher TAK1 activity than the 3LL cells located at the center of the tumor, suggesting that cells at the periphery of the tumor mass were under stronger stress. Injection of PolyI:C, which is known to induce regression of the implanted tumors, induced marked and homogenous TAK1 activation within the tumor tissues. The effect of PolyI:C faded within 4 days. These observations suggest that Eevee-TAK1 is a versatile tool to monitor cellular stress in cancer tissues.

Calcium transients trigger switch-like discharge of prostaglandin E2 in an extracellular signal-regulated kinase-dependent manner.

Prostaglandin E2 (PGE2) is a key player in a plethora of physiological and pathological events. Nevertheless, little is known about the dynamics of PGE2 secretion from a single cell and its effect on the neighboring cells. Here, by observing confluent Madin-Darby canine kidney (MDCK) epithelial cells expressing fluorescent biosensors, we demonstrate that calcium transients in a single cell cause PGE2-mediated radial spread of PKA activation (RSPA) in neighboring cells. By in vivo imaging, RSPA was also observed in the basal layer of the mouse epidermis. Experiments with an optogenetic tool revealed a switch-like PGE2 discharge in response to the increasing cytoplasmic Ca2+ concentrations. The cell density of MDCK cells correlated with the frequencies of calcium transients and the following RSPA. The extracellular signal-regulated kinase (ERK) activation also enhanced the frequency of RSPA in MDCK and in vivo. Thus, the PGE2 discharge is regulated temporally by calcium transients and ERK activity.

Generation of transgenic mice expressing a FRET biosensor, SMART, that responds to necroptosis.

Necroptosis is a regulated form of cell death involved in various pathological conditions, including ischemic reperfusion injuries, virus infections, and drug-induced tissue injuries. However, it is not fully understood when and where necroptosis occurs in vivo. We previously generated a Forster resonance energy transfer (FRET) biosensor, termed SMART (the sensor for MLKL activation by RIPK3 based on FRET), which monitors conformational changes of MLKL along with progression of necroptosis in human and murine cell lines in vitro. Here, we generate transgenic (Tg) mice that express the SMART biosensor in various tissues. The FRET ratio is increased in necroptosis, but not apoptosis or pyroptosis, in primary cells. Moreover, the FRET signals are elevated in renal tubular cells of cisplatin-treated SMART Tg mice compared to untreated SMART Tg mice. Together, SMART Tg mice may provide a valuable tool for monitoring necroptosis in different types of cells in vitro and in vivo.

Intravital imaging reveals cell cycle-dependent myogenic cell migration during muscle regeneration.

During muscle regeneration, extracellular signal-regulated kinase (ERK) promotes both proliferation and migration. However, the relationship between proliferation and migration is poorly understood in this context. To elucidate this complex relationship on a physiological level, we established an intravital imaging system for measuring ERK activity, migration speed, and cell-cycle phases in mouse muscle satellite cell-derived myogenic cells. We found that in vivo, ERK is maximally activated in myogenic cells two days after injury, and this is then followed by increases in cell number and motility. With limited effects of ERK activity on migration on an acute timescale, we hypothesized that ERK increases migration speed in the later phase by promoting cell-cycle progression. Our cell-cycle analysis further revealed that in myogenic cells, ERK activity is critical for G1/S transition, and cells migrate more rapidly in S/G2 phase 3 days after injury. Finally, migration speed of myogenic cells was suppressed after CDK1/2-but not CDK1-inhibitor treatment, demonstrating a critical role of CDK2 in myogenic cell migration. Overall, our study demonstrates that in myogenic cells, the ERK-CDK2 axis promotes not only G1/S transition but also migration, thus providing a novel mechanism for efficient muscle regeneration.

A Highly Sensitive FRET Biosensor for AMPK Exhibits Heterogeneous AMPK Responses among Cells and Organs.

AMP-activated protein kinase (AMPK), a master regulator of cellular metabolism, is a potential target for type 2 diabetes. Although extensive in vitro studies have revealed the complex regulation of AMPK, much remains unknown about the regulation in vivo. We therefore developed transgenic mice expressing a highly sensitive fluorescence resonance energy transfer (FRET)-based biosensor for AMPK, called AMPKAR-EV. AMPKAR-EV allowed us to readily examine the role of LKB1, a canonical stimulator of AMPK, in drug-induced activation and inactivation of AMPK in vitro. In transgenic mice expressing AMPKAR-EV, the AMP analog AICAR activated AMPK in muscle. In contrast, the antidiabetic drug metformin activated AMPK in liver, highlighting the organ-specific action of AMPK stimulators. Moreover, we found that AMPK was activated primarily in fast-twitch muscle fibers after tetanic contraction and exercise. These observations suggest that the AMPKAR-EV mouse will pave a way to understanding the heterogeneous responses of AMPK among cell types in vivo.

[Cricoarytenoid arthritis diagnosed after tracheostomy in a rheumatoid arthritis patient].

A 65-year-old woman was scheduled for total knee replacement. She had been suffering from rheumatoid arthritis for 22 years. She also had a history of occasional acute dyspnea, which had been diagnosed as asthmatic bronchitis. Preoperative examinations of the airway revealed limited neck flexion, a small jaw, and normal mouth opening. After epidural catheterization, anesthesia was induced with propofol, and a #3 laryngeal mask airway (LMA) was inserted. However, her lungs could not be ventilated through the LMA. Despite repeated attempts, proper placement of the LMA could not be achieved. Hence, a 7.0 mm ID armored endotracheal tube was inserted through an intubating LMA. Anesthesia was maintained with nitrous oxide and sevoflurane in oxygen. The surgery proceeded uneventfully. Five minutes after extubation, inspiratory dyspnea occurred. The patient's trachea was re-intubated nasally with a bronchofiberscope. Since the bronchofiberscopy revealed remarkable laryngeal edema, percutaneous tracheostomy was performed. On the 3 rd postoperative day, cricoarytenoid arthritis that had caused occasional airway obstruction was diagnosed, although her laryngeal edema disappeared. She went home with a permanent tracheostomy. Although cricoarytenoid arthritis is a common occurrence in patients with rheumatoid arthritis, the diagnosis can be difficult. A scrupulous preoperative evaluation and awareness of cricoarytenoid arthritis are necessary for optimal anesthetic management.