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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.

Calcium transients drive cells to discharge prostaglandin E₂ (PGE₂). We visualized PGE₂-induced protein kinase A (PKA) activation and quantitated PGE₂ secreted from a single cell by combining fluorescence microscopy and a simulation model. For this purpose, we first prepared PGE₂-producer cells that express either an optogenetic or a chemogenetic calcium channel stimulator: OptoSTIM1 or Gq-DREADD, respectively. Second, we prepared reporter cells expressing the Gs-coupled PGE₂ reporter EP2 and the PKA biosensor Booster-PKA, which is based on the principle of Förster resonance energy transfer (FRET). Upon the stimulation-induced triggering of calcium transients, a single producer cell discharges PGE₂ to stimulate PKA in the surrounding reporter cells. Due to the flow of the medium, the PKA-activated area exhibited a comet-like smear when HeLa cells were used. In contrast, radial PKA activation was observed when confluent MDCK cells were used, indicating that PGE₂ diffusion was restricted to the basolateral space. By fitting the radius of the PKA-activated area to a simulation model based on simple diffusion, we estimated that a single HeLa cell secretes 0.25 fmol PGE₂ upon a single calcium transient to activate PKA in more than 1000 neighboring cells. This model also predicts that the PGE₂ discharge rate is comparable to the diffusion rate. Thus, our method quantitatively envisions that a single calcium transient affects more than 1000 neighboring cells via PGE₂.

The ErbB-family receptors play pivotal roles in the proliferation, migration, and survival of epithelial cells. Because our knowledge on the ErbB-family receptors was obtained largely by the exogenous application of their ligands, it remains unknown to which extent each of the ErbB contributes to these outputs. We here knocked out each ErbB gene, various combinations of ErbB genes, or all in Madin-Darby canine kidney cells to delineate the contribution of each gene. ERK activation waves during collective cell migration were mediated primarily by ErbB1 and secondarily by the ErbB2/ErbB3 heterodimer. Either ErbB1 or the ErbB2/ErbB3 complex was sufficient for the G1/S progression. The saturation cell density was markedly reduced in cells deficient in all ErbB-proteins, but not cells retaining only ErbB2, which cannot bind to ligands. Thus, the ligand-independent ErbB2 activity is sufficient for preventing apoptosis at high cell density. In short, systematic knockout of ErbB-family genes delineated the roles of each ErbB receptor.

Inflammation can contribute to the development and progression of cancer. The inflammatory responses in the tumor microenvironment are shaped by complex sequences of dynamic intercellular cross-talks among diverse types of cells, and recapitulation of these dynamic events in vitro has yet to be achieved. Today, intravital microscopy with two-photon excitation microscopes (2P-IVM) is the mainstay technique for observing intercellular cross-talks in situ, unraveling cellular and molecular mechanisms in the context of their spatiotemporal dynamics. In this review, we summarize the current state of 2P-IVM with fluorescent indicators of signal transduction to reveal the cross-talks between cancer cells and surrounding cells including both immune and non-immune cells. We also discuss the potential application of red-shifted indicators along with optogenetic tools to 2P-IVM. In an era of single-cell transcriptomics and data-driven research, 2P-IVM will remain a key advantage in delivering the missing spatiotemporal context in the field of cancer research.

Upon the initiation of collective cell migration, the cells at the free edge are specified as leader cells; however, the mechanism underlying the leader cell specification remains elusive. Here, we show that lamellipodial extension after the release from mechanical confinement causes sustained extracellular signal-regulated kinase (ERK) activation and underlies the leader cell specification. Live-imaging of Madin-Darby canine kidney (MDCK) cells and mouse epidermis through the use of Förster resonance energy transfer (FRET)-based biosensors showed that leader cells exhibit sustained ERK activation in a hepatocyte growth factor (HGF)-dependent manner. Meanwhile, follower cells exhibit oscillatory ERK activation waves in an epidermal growth factor (EGF) signaling-dependent manner. Lamellipodial extension at the free edge increases the cellular sensitivity to HGF. The HGF-dependent ERK activation, in turn, promotes lamellipodial extension, thereby forming a positive feedback loop between cell extension and ERK activation and specifying the cells at the free edge as the leader cells. Our findings show that the integration of physical and biochemical cues underlies the leader cell specification during collective cell migration.

Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is unknown. Here we have combined ultra-sensitive bioluminescence imaging with intravital two-photon microscopy involving genetically-encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hrs of arrival, but not thereafter. Intravital dynamic imaging revealed that 50% of NK-tumor cell encounters lead to tumor cell death in the first 4 hrs after tumor cell arrival, but after 24 hrs of arrival, nearly 100% of the interactions result in the survival of the tumor cell. During this 24 hrs period, the probability of ERK activation in NK cells upon encountering the tumor cells was decreased from 68% to 8%, which correlated with the loss of the activating ligand CD155/PVR/Necl5 from the tumor cell surface. Thus, by quantitatively visualizing the NK-tumor cell interaction at the early stage of metastasis, we have revealed the crucial parameters of NK cell immune surveillance in the lung.

Prostaglandin E₂ (PGE₂) promotes tumor progression through evasion of anti-tumor immunity. In stark contrast to cyclooxygenase-dependent production of PGE₂, little is known whether or not PGE₂ secretion is regulated within tumor tissues. Here, we show that VEGF-dependent release of thromboxane A₂ (TXA₂) triggers Ca²⁺ transients in tumor cells, culminating in PGE₂ secretion and subsequent immune evasion in the early stages of tumorigenesis. Ca²⁺ transients caused cPLA2 activation and triggered the arachidonic acid cascade. Ca²⁺ transients were monitored as the surrogate marker of PGE₂ secretion. Intravital imaging of BrafV600E mouse melanoma cells revealed that the proportion of cells exhibiting Ca²⁺ transients is markedly higher in vivo than in vitro. The TXA₂ receptor was indispensable for the Ca²⁺ transients in vivo, high intra-tumoral PGE₂ concentration, and evasion of anti-tumor immunity. Notably, treatment with a vascular endothelial growth factor (VEGF) receptor antagonist and an anti-VEGF antibody rapidly suppressed Ca²⁺ transients and reduced TXA₂ and PGE₂ concentrations in tumor tissues. These results identify the VEGF-TXA₂ axis as a critical promoter of PGE₂-dependent tumor immune evasion, providing a molecular basis underlying the immunomodulatory effect of anti-VEGF therapies.

In vertebrates, retinal rod and cone photoreceptor cells rely significantly on glycolysis. Lactate released from photoreceptor cells fuels neighboring retinal pigment epithelium cells and Müller glial cells through oxidative phosphorylation. To understand this highly heterogeneous metabolic environment around photoreceptor cells, single-cell analysis is needed. Here, we visualized cellular AMP-activated protein kinase (AMPK) activity and ATP levels in the retina by two-photon microscopy. Transgenic mice expressing a hyBRET-AMPK biosensor were used for measuring the AMPK activity. GO-ATeam2 transgenic mice were used for measuring the ATP level. Temporal metabolic responses were successfully detected in the live retinal explants upon drug perfusion. A glycolysis inhibitor, 2-deoxy-d-glucose (2-DG), activated AMPK and reduced ATP. These effects were clearly stronger in rods than in cones. Notably, rod AMPK and ATP started to recover at 30 min from the onset of 2-DG perfusion. Consistent with these findings, ex vivo electroretinogram recordings showed a transient slowdown in rod dim flash responses during a 60-min 2-DG perfusion, whereas cone responses were not affected. Based on these results, we propose that cones surrounded by highly glycolytic rods become less dependent on glycolysis, and rods also become less dependent on glycolysis within 60 min upon the glycolysis inhibition.