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.

A non-invasive system to monitor in vivo neural graft activity after spinal cord injury.

Expectations for neural stem/progenitor cell (NS/PC) transplantation as a treatment for spinal cord injury (SCI) are increasing. However, whether and how grafted cells are incorporated into the host neural circuit and contribute to motor function recovery remain unknown. The aim of this project was to establish a novel non-invasive in vivo imaging system to visualize the activity of neural grafts by which we can simultaneously demonstrate the circuit-level integration between the graft and host and the contribution of graft neuronal activity to host behaviour. We introduced Akaluc, a newly engineered luciferase, under the control of enhanced synaptic activity-responsive element (E-SARE), a potent neuronal activity-dependent synthetic promoter, into NS/PCs and engrafted the cells into SCI model mice. Through the use of this system, we found that the activity of grafted cells was integrated with host behaviour and driven by host neural circuit inputs. This non-invasive system is expected to help elucidate the therapeutic mechanism of cell transplantation treatment for SCI.

Functional visualization of NK cell-mediated killing of metastatic single tumor cells.

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 hr 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 hr after tumor cell arrival, but after 24 hr of arrival, nearly 100% of the interactions result in the survival of the tumor cell. During this 24-hr 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.

DHODH inhibition synergizes with DNA-demethylating agents in the treatment of myelodysplastic syndromes.

Dihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine nucleotide synthesis. DHODH inhibition has recently been recognized as a potential new approach for treating acute myeloid leukemia (AML) by inducing differentiation. We investigated the efficacy of PTC299, a novel DHODH inhibitor, for myelodysplastic syndrome (MDS). PTC299 inhibited the proliferation of MDS cell lines, and this was rescued by exogenous uridine, which bypasses de novo pyrimidine synthesis. In contrast to AML cells, PTC299 was inefficient at inhibiting growth and inducing the differentiation of MDS cells, but synergized with hypomethylating agents, such as decitabine, to inhibit the growth of MDS cells. This synergistic effect was confirmed in primary MDS samples. As a single agent, PTC299 prolonged the survival of mice in xenograft models using MDS cell lines, and was more potent in combination with decitabine. Mechanistically, a treatment with PTC299 induced intra-S-phase arrest followed by apoptotic cell death. Of interest, PTC299 enhanced the incorporation of decitabine, an analog of cytidine, into DNA by inhibiting pyrimidine production, thereby enhancing the cytotoxic effects of decitabine. RNA-seq data revealed the marked downregulation of MYC target gene sets with PTC299 exposure. Transfection of MDS cell lines with MYC largely attenuated the growth inhibitory effects of PTC299, suggesting MYC as one of the major targets of PTC299. Our results indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells and acts in a synergistic manner with decitabine. This combination therapy may be a new therapeutic option for the treatment of MDS.

Efficacy of the novel tubulin polymerization inhibitor PTC-028 for myelodysplastic syndrome.

Monomer tubulin polymerize into microtubules, which are highly dynamic and play a critical role in mitosis. Therefore, microtubule dynamics are an important target for anticancer drugs. The inhibition of tubulin polymerization or depolymerization was previously targeted and exhibited efficacy against solid tumors. The novel small molecule PTC596 directly binds tubulin, inhibits microtubule polymerization, downregulates MCL-1, and induces p53-independent apoptosis in acute myeloid leukemia cells. We herein investigated the efficacy of PTC-028, a structural analog of PTC596, for myelodysplastic syndrome (MDS). PTC-028 suppressed growth and induced apoptosis in MDS cell lines. The efficacy of PTC028 in primary MDS samples was confirmed using cell proliferation assays. PTC-028 synergized with hypomethylating agents, such as decitabine and azacitidine, to inhibit growth and induce apoptosis in MDS cells. Mechanistically, a treatment with PTC-028 induced G2/M arrest followed by apoptotic cell death. We also assessed the efficacy of PTC-028 in a xenograft mouse model of MDS using the MDS cell line, MDS-L, and the AkaBLI bioluminescence imaging system, which is composed of AkaLumine-HCl and Akaluc. PTC-028 prolonged the survival of mice in xenograft models. The present results suggest a chemotherapeutic strategy for MDS through the disruption of microtubule dynamics in combination with DNA hypomethylating agents.

Single-cell bioluminescence imaging of deep tissue in freely moving animals.

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.

A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging.

In preclinical cancer research, bioluminescence imaging with firefly luciferase and D-luciferin has become a standard to monitor biological processes both in vitro and in vivo. However, the emission maximum (λmax) of bioluminescence produced by D-luciferin is 562 nm where light is not highly penetrable in biological tissues. This emphasizes a need for developing a red-shifted bioluminescence imaging system to improve detection sensitivity of targets in deep tissue. Here we characterize the bioluminescent properties of the newly synthesized luciferin analogue, AkaLumine-HCl. The bioluminescence produced by AkaLumine-HCl in reactions with native firefly luciferase is in the near-infrared wavelength ranges (λmax=677 nm), and yields significantly increased target-detection sensitivity from deep tissues with maximal signals attained at very low concentrations, as compared with D-luciferin and emerging synthetic luciferin CycLuc1. These characteristics offer a more sensitive and accurate method for non-invasive bioluminescence imaging with native firefly luciferase in various animal models.

Synthesis of Firefly Luciferin Analogues and Evaluation of the Luminescent Properties.

Five new firefly luciferin (1) analogues were synthesized and their light emission properties were examined. Modifications of the thiazoline moiety in 1 were employed to produce analogues containing acyclic amino acid side chains (2-4) and heterocyclic rings derived from amino acids (5 and 6) linked to the benzothiazole moiety. Although methyl esters of all of the synthetic derivatives exhibited chemiluminescence activity, only carboluciferin (6), possessing a pyrroline-substituted benzothiazole structure, had bioluminescence (BL) activity (λmax =547 nm). Results of bioluminescence studies with AMP-carboluciferin (AMP=adenosine monophosphate) and AMP-firefly luciferin showed that the nature of the thiazoline mimicking moiety affected the adenylation step of the luciferin-luciferase reaction required for production of potent BL. In addition, BL of 6 in living mice differed from that of 1 in that its luminescence decay rate was slower.

Multicolor Bioluminescence Obtained Using Firefly Luciferin.

Firefly bioluminescence is widely used in life science research as a useful analysis tool. For example, the adenosine-5`-triphosphate (ATP)-dependent enzymatic firefly bioluminescence reaction has long been utilized as a microbial monitoring tool. Rapid and sensitive firefly luciferin-luciferase combinations are used not only to measure cell viability but also for reporter-gene assays. Recently, bioluminescence was utilized as a noninvasive, real-time imaging tool for living subjects to monitor cells and biological events. However, the number of commercialized luciferase genes is limited and tissue-permeable near-infrared (NIR) region emitting light is required for in vivo imaging. In this review, recent studies describing synthetic luciferin analogues predicted to have red-shifted bioluminescence are summarized. Luciferase substrates emitting red, green, and blue light that were designed and developed in our laboratory are presented. The longest emission wavelength of the synthesized luciferin analogues was recorded at 675 nm, which is within the NIR region. This compound is now commercially available as "Aka Lumine®".