Reinforced antimyeloma therapy via dual-lymphoid activation mediated by a panel of antibodies armed with bridging-BiTE.

Immunotherapy using bispecific antibodies including bispecific T-cell engager (BiTE) has the potential to enhance the efficacy of treatment for relapsed/refractory multiple myeloma. However, myeloma may still recur after treatment because of downregulation of a target antigen and/or myeloma cell heterogeneity. To strengthen immunotherapy for myeloma while overcoming its characteristics, we have newly developed a BiTE-based modality, referred to as bridging-BiTE (B-BiTE). B-BiTE was able to bind to both a human immunoglobulin G-Fc domain and the CD3 molecule. Clinically available monoclonal antibodies (mAbs) were bound with B-BiTE before administration, and the mAb/B-BiTE complex induced antitumor T-cell responses successfully while preserving and supporting natural killer cell reactivity, resulting in enhanced antimyeloma effects via dual-lymphoid activation. In contrast, any unwanted off-target immune-cell reactivity mediated by mAb/B-BiTE complexes or B-BiTE itself appeared not to be observed in vitro and in vivo. Importantly, sequential immunotherapy using 2 different mAb/B-BiTE complexes appeared to circumvent myeloma cell antigen escape, and further augmented immune responses to myeloma relative to those induced by mAb/B-BiTE monotherapy or sequential therapy with 2 mAbs in the absence of B-BiTE. Therefore, this modality facilitates easy and prompt generation of a broad panel of bispecific antibodies that can induce deep and durable antitumor responses in the presence of clinically available mAbs, supporting further advancement of reinforced immunotherapy for multiple myeloma and other refractory hematologic malignancies.

Extended Depth of Focus Two-Photon Light-Sheet Microscopy for In Vivo Fluorescence Imaging of Large Multicellular Organisms at Cellular Resolution.

Two-photon excitation in light-sheet microscopy advances applications to live imaging of multicellular organisms. In a previous study, we developed a two-photon Bessel beam light-sheet microscope with a nearly 1-mm field of view and less than 4-µm axial resolution, using a low magnification (10×), middle numerical aperture (NA 0.5) detection objective. In this study, we aimed to construct a light-sheet microscope with higher resolution imaging while maintaining the large field of view, using low magnification (16×) with a high NA 0.8 objective. To address potential illumination and detection mismatch, we investigated the use of a depth of focus (DOF) extension method. Specifically, we used a stair-step device composed of five-layer annular zones that extended DOF two-fold, enough to cover the light-sheet thickness. Resolution measurements using fluorescent beads showed that the reduction in resolutions was small. We then applied this system to in vivo imaging of medaka fish and found that image quality degradation at the distal site of the beam injection could be compensated. This demonstrates that the extended DOF system combined with wide-field two-photon light-sheet microscopy offers a simple and easy setup for live imaging application of large multicellular organism specimens with sub-cellular resolution.

Computer-Aided Detection of Quantitative Signatures for Breast Fibroepithelial Tumors Using Label-Free Multi-Photon Imaging.

Fibroadenomas (FAs) and phyllodes tumors (PTs) are major benign breast tumors, pathologically classified as fibroepithelial tumors. Although the clinical management of PTs differs from FAs, distinction by core needle biopsy diagnoses is still challenging. Here, a combined technique of label-free imaging with multi-photon microscopy and artificial intelligence was applied to detect quantitative signatures that differentiate fibroepithelial lesions. Multi-photon excited autofluorescence and second harmonic generation (SHG) signals were detected in tissue sections. A pixel-wise semantic segmentation method using a deep learning framework was used to separate epithelial and stromal regions automatically. The epithelial to stromal area ratio and the collagen SHG signal strength were investigated for their ability to distinguish fibroepithelial lesions. An image segmentation analysis with a pixel-wise semantic segmentation framework using a deep convolutional neural network showed the accurate separation of epithelial and stromal regions. A further investigation, to determine if scoring the epithelial to stromal area ratio and the SHG signal strength within the stromal area could be a marker for differentiating fibroepithelial tumors, showed accurate classification. Therefore, molecular and morphological changes, detected through the assistance of computational and label-free multi-photon imaging techniques, enable us to propose quantitative signatures for epithelial and stromal alterations in breast tissues.

In vivo optical imaging of cancer cell function and tumor microenvironment.

In vivo optical imaging using fluorescence and bioluminescence is superior to other methods in terms of spatiotemporal resolution and specificity, and represents a new technology for comprehensively studying living organisms in a less invasive way. Nowadays, it is an indispensable technology for studying many aspects of cancer biology, including dynamic invasion and metastasis. In observations of fluorescence or bioluminescence signals in a living body, various problems were caused by optical characteristics such as absorption and scattering and, therefore, observation of deep tissue was difficult. Recent developments in techniques for observation of the deep tissues of living animals overcame this difficulty by improving bioluminescent proteins, fluorescent proteins, and fluorescent dyes, as well as detection technologies such as two-photon excitation microscopy. In the present review, we introduce these technological developments and in vivo application of bioluminescence and fluorescence imaging, and discuss future perspectives on the use of in vivo optical imaging technology in cancer research.

Quantitative imaging with Fucci and mathematics to uncover temporal dynamics of cell cycle progression.

Cell cycle progression is strictly coordinated to ensure proper tissue growth, development, and regeneration of multicellular organisms. Spatiotemporal visualization of cell cycle phases directly helps us to obtain a deeper understanding of controlled, multicellular, cell cycle progression. The fluorescent ubiquitination-based cell cycle indicator (Fucci) system allows us to monitor, in living cells, the G1 and the S/G2/M phases of the cell cycle in red and green fluorescent colors, respectively. Since the discovery of Fucci technology, it has found numerous applications in the characterization of the timing of cell cycle phase transitions under diverse conditions and various biological processes. However, due to the complexity of cell cycle dynamics, understanding of specific patterns of cell cycle progression is still far from complete. In order to tackle this issue, quantitative approaches combined with mathematical modeling seem to be essential. Here, we review several studies that attempted to integrate Fucci technology and mathematical models to obtain quantitative information regarding cell cycle regulatory patterns. Focusing on the technological development of utilizing mathematics to retrieve meaningful information from the Fucci producing data, we discuss how the combined methods advance a quantitative understanding of cell cycle regulation.

Live imaging-based model selection reveals periodic regulation of the stochastic G1/S phase transition in vertebrate axial development.

In multicellular organism development, a stochastic cellular response is observed, even when a population of cells is exposed to the same environmental conditions. Retrieving the spatiotemporal regulatory mode hidden in the heterogeneous cellular behavior is a challenging task. The G1/S transition observed in cell cycle progression is a highly stochastic process. By taking advantage of a fluorescence cell cycle indicator, Fucci technology, we aimed to unveil a hidden regulatory mode of cell cycle progression in developing zebrafish. Fluorescence live imaging of Cecyil, a zebrafish line genetically expressing Fucci, demonstrated that newly formed notochordal cells from the posterior tip of the embryonic mesoderm exhibited the red (G1) fluorescence signal in the developing notochord. Prior to their initial vacuolation, these cells showed a fluorescence color switch from red to green, indicating G1/S transitions. This G1/S transition did not occur in a synchronous manner, but rather exhibited a stochastic process, since a mixed population of red and green cells was always inserted between newly formed red (G1) notochordal cells and vacuolating green cells. We termed this mixed population of notochordal cells, the G1/S transition window. We first performed quantitative analyses of live imaging data and a numerical estimation of the probability of the G1/S transition, which demonstrated the existence of a posteriorly traveling regulatory wave of the G1/S transition window. To obtain a better understanding of this regulatory mode, we constructed a mathematical model and performed a model selection by comparing the results obtained from the models with those from the experimental data. Our analyses demonstrated that the stochastic G1/S transition window in the notochord travels posteriorly in a periodic fashion, with doubled the periodicity of the neighboring paraxial mesoderm segmentation. This approach may have implications for the characterization of the pathophysiological tissue growth mode.

Lower lip reconstruction using a combined technique of the webster and johanson methods.

Oral reconstruction, especially with lower lip defects greater than 80% of the lip, is still challenging for plastic surgeons. Webster technique is mostly used for lower lip defects greater than 80% of the lip; however, resulting scars in the chin area (Schuchardt flap, a half-circle scar) are relatively conspicuous in Asian populations because of the trapdoor deformity. On the other hand, Johanson staircase flap technique, which is used to reconstruct lower lip defects of up to two thirds of the lip, results in relatively inconspicuous scarring and prevents trapdoor deformity. Thus, instead of Schuchardt flaps, we designed staircase flaps with a Webster technique. Two patients with lower lip carcinoma were operated on using this new technique. Large triangles of the skin and subcutaneous fat were removed from the nasolabial folds, and small staircases were removed from the lower lip to allow medial movement of the cheek tissues. The cosmetic and functional results were improved from the original Webster technique.

Administration of prosaposin-derived neurotrophic factor to neural tube defects facilitates regeneration and restores neurological functions.

Neural tube defects (NTDs) cause fetal and pediatric deaths or lifelong neurological disabilities. No effective treatment is currently available for NTDs. We attempted to elucidate the pathogenesis of NTDs and propose a therapeutic strategy. Intra-amniotic treatment with prosaposin-derived 18-mer peptide (PS18) protected the spinal cord from secondary damage and rescued neurological function in an established chicken model of spina bifida aperta (SBA), the severe type of NTDs. PS18 promoted the formation of a neuroectodermal covering over the defective neural tube within 24-h after treatment, enhanced the regeneration/restoration process, and decreased apoptotic activity in the developing spinal cord. PS18 reduced the SBA wound and almost completely formed the spinal cord. SBA chicks that received PS18 exhibited relatively normal walking and sensorimotor responses, and reduced pain-associated behavior in postnatal life. In conclusion, PS18 is a promising therapeutic agent for NTDs and may be useful for treating other types of spinal cord injuries.

Comparative analysis of Hmx expression and the distribution of neuronal somata in the trigeminal ganglion in lamprey and shark: insights into the homology of the trigeminal nerve branches and the evolutionary origin of the vertebrate jaw.

The evolutionary origin of the jaw remains one of the most enigmatic events in vertebrate evolution. The trigeminal nerve is a key component for understanding jaw evolution, as it plays a crucial role as a sensorimotor interface for the effective manipulation of the jaw. This nerve is also found in the lamprey, an extant jawless vertebrate. The trigeminal nerve has three major branches in both the lamprey and jawed vertebrates. Although each of these branches was classically thought to be homologous between these two taxa, this homology is now in doubt. In the present study, we compared expression patterns of Hmx, a candidate genetic marker of the mandibular nerve (rV3, the third branch of the trigeminal nerve in jawed vertebrates), and the distribution of neuronal somata of trigeminal nerve branches in the trigeminal ganglion in lamprey and shark. We first confirmed the conserved expression pattern of Hmx1 in the shark rV3 neuronal somata, which are distributed in the caudal part of the trigeminal ganglion. By contrast, lamprey Hmx genes showed peculiar expression patterns, with expression in the ventrocaudal part of the trigeminal ganglion similar to Hmx1 expression in jawed vertebrates, which labeled the neuronal somata of the second branch. Based on these results, we propose two alternative hypotheses regarding the homology of the trigeminal nerve branches, providing new insights into the evolutionary origin of the vertebrate jaw.

Mathematical modeling of invadopodia formation.

In invasive cancer cells, specialized sub-cellular membrane structures which carry out a pivotal process in cancer invasion, termed invadopodia, are observed. Invadopodia appear irregularly within the cytoplasm and their general shape is small punctuated finger-like protrusions with dimension up to several µm long. They may exist and persist on a timescale between several tens of minutes to one hour. The formation of invadopodia requires the integration of several processes that include actin reorganization, extracellular matrix (ECM) degradation, signaling processes through receptors such as the epidermal growth factor receptor (EGFR) and matrix metalloproteinase (MMP) synthesis and delivery to the location of the invading front. In this paper, we consider a mathematical model investigating the coupling of these fundamental processes, and we investigate how invadopodia appear in this model. We investigate the spatio-temporal dynamics of the model in two spatial dimensions by using numerical computational simulations. We show that in a special parameter region of the model, random fluctuations of ECM degradation and a positive feedback loop regarding the up-regulation of MMPs allow us to reproduce finger-like protrusions which have similar size and lifetime as invadopodia. This study provides a new insight into how invadopodia appear in cancer cells and why space and time scales exist for invadopodia.

Control and inhibition analysis of complex formation processes.

BACKGROUND: Proteolytic degradation of the extracellular matrix (ECM) is a key event in tumour metastasis and invasion. Matrix metalloproteinases (MMPs) are a family of endopeptidases that degrade most of the components of the ECM. Several broad-spectrum MMP inhibitors (MMPIs) have been developed, but have had little success due to side effects. Thus, it is important to develop mathematical methods to provide new drug treatment strategies. Matrix metalloproteinase 2 (MMP2) activation occurs via a mechanism involving complex formation that consists of membrane type 1 MMP (MT1-MMP), tissue inhibitor of matrix metalloproteinase 2 (TIMP2) and MMP2. Here, we focus on developing a method for analysing the complex formation process. RESULTS: We used control analysis to investigate inhibitor responses in complex formation processes. The essence of the analysis is to define the response coefficient which measures the inhibitory efficiency, a small fractional change of concentration of a targeting molecule in response to a small fractional change of concentration of an inhibitor. First, by using the response coefficient, we investigated models for general classes of complex formation processes: chain reaction systems composed of ordered steps, and chain reaction systems and site-binding reaction systems composed of unordered multi-branched steps. By analysing the ordered step models, we showed that parameter-independent inequalities between the response coefficients held. For the unordered multi-branched step models, we showed that independence of the response coefficients with respect to equilibrium constants held. As an application of our analysis, we discuss a mathematical model for the MMP2 activation process. By putting the experimentally derived parameter values into the model, we were able to conclude that the TIMP2 and MMP2 interaction is the most efficient interaction to consider in selecting inhibitors. CONCLUSIONS: Our result identifies a new drug target in the process of the MMP2 activation. Thus, our analysis will provide new insight into the design of more efficient drug strategies for cancer treatment.

Usefulness of Harmonic Focus during anterolateral thigh flap elevation.

Meticulous hemostasis and careful ligation of branches are necessary for pedicle dissection during flap elevation. The aim of this study was to evaluate the effectiveness of the Harmonic Focus handpiece (Ethicon Endo-Surgery, Inc., Blue Ash, Cincinnati, OH, USA) in reducing operation time, bleeding volume, and volume of postoperative drainage during anterolateral thigh flap elevation. Ten patients requiring flap elevation were divided into two groups: (1) Harmonic Focus group (three men, two women), and (2) control group (three men, two women). Operating time was found to be lower in the Harmonic Focus group than in the control group. Bleeding volume and postoperative drainage volume were nearly identical in the Harmonic Focus group and the control group, and the number of silk ligatures was significantly lower in the Harmonic Focus group compared with the control group. Although somewhat costly, the Harmonic Scalpel with the Harmonic Focus handpiece is advantageous for flap elevation, and it is likely that Harmonic Scalpel use will increase in plastic surgery.

Reduced dynamic loads due to hip dislocation induce acetabular cartilage degeneration by IL-6 and MMP3 via the STAT3/periostin/NF-κB axis.

Developmental dysplasia of the hip (DDH) is characterized by anatomical abnormalities of the hip joint, ranging from mild acetabular dysplasia to hip subluxation and eventually dislocation. The mechanism underlying the cartilage degeneration of the hip joints exposed to reduced dynamic loads due to hip dislocation remains unknown. We established a rodent hip dislocation (disarticulation; DA) model of DDH (DA-DDH rats and mice) by swaddling. Expression levels of periostin (Postn) and catabolic factors, such as interleukin-6 (IL-6) and matrix metalloproteinase 3 (Mmp3), increased and those of chondrogenic markers decreased in the acetabular cartilage of the DA-DDH models. Postn induced IL-6 and Mmp3 expression in chondrocytes through integrin αVß3, focal adhesion kinase, Src, and nuclear factor-κB (NF-κB) signaling. The microgravity environment created by a random positioning machine induced Postn expression in chondrocytes through signal transducer and activator of transcription 3 (STAT3) signaling. IL-6 stimulated Postn expression via STAT3 signaling. Furthermore, cartilage degeneration was suppressed in the acetabulum of Postn-/- DA-DDH mice compared with that in the acetabulum of wild type DA-DDH mice. In summary, reduced dynamic loads due to hip dislocation induced acetabular cartilage degeneration via IL-6 and MMP3 through STAT3/periostin/NF-κB signaling in the rodent DA-DDH models.

Wide field light-sheet microscopy with lens-axicon controlled two-photon Bessel beam illumination.

Two-photon excitation can lower phototoxicity and improve penetration depth, but its narrow excitation range restricts its applications in light-sheet microscopy. Here, we propose simple illumination optics, a lens-axicon triplet composed of an axicon and two convex lenses, to generate longer extent Bessel beams. This unit can stretch the beam full width at half maximum of 600-1000 µm with less than a 4-µm waist when using a 10× illumination lens. A two-photon excitation digital scanned light-sheet microscope possessing this range of field of view and ~2-3-µm axial resolution is constructed and used to analyze the cellular dynamics over the whole body of medaka fish. We demonstrate long-term time-lapse observations over several days and high-speed recording with ~3 mm3 volume per 4 s of the embryos. Our system is minimal and suppresses laser power loss, which can broaden applications of two-photon excitation in light-sheet microscopy.

Donor-acceptor-acceptor-type near-infrared fluorophores that contain dithienophosphole oxide and boryl groups: effect of the boryl group on the nonradiative decay.

The use of donor-π-acceptor (D-π-A) skeletons is an effective strategy for the design of fluorophores with red-shifted emission. In particular, the use of amino and boryl moieties as the electron-donating and -accepting groups, respectively, can produce dyes that exhibit high fluorescence and solvatochromism. Herein, we introduce a dithienophosphole P-oxide scaffold as an acceptor-spacer to produce a boryl- and amino-substituted donor-acceptor-acceptor (D-A-A) π-system. The thus obtained fluorophores exhibit emission in the near-infrared (NIR) region, while maintaining high fluorescence quantum yields even in polar solvents (e.g. λ em = 704 nm and Φ F = 0.69 in CH3CN). A comparison of these compounds with their formyl- or cyano-substituted counterparts demonstrated the importance of the boryl group for generating intense emission. The differences among these electron-accepting substituents were examined in detail using theoretical calculations, which revealed the crucial role of the boryl group in lowering the nonradiative decay rate constant by decreasing the non-adiabatic coupling in the internal conversion process. The D-A-A framework was further fine-tuned to improve the photostability. One of these D-A-A dyes was successfully used in bioimaging to visualize the blood vessels of Japanese medaka larvae and mouse brain.

A novel negative regulatory mechanism of Smurf2 in BMP/Smad signaling in bone.

Transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) play important roles in bone metabolism. Smad ubiquitination regulatory factors (Smurfs) regulate TGF-ß/BMP signaling via ubiquitination, resulting in degradation of signaling molecules to prevent excessive activation of TGF-ß/BMP signaling. Though Smurf2 has been shown to negatively regulate TGF-ß/Smad signaling, its involvement in BMP/Smad signaling in bone metabolism has not been thoroughly investigated. In the present study, we sought to evaluate the role of Smurf2 in BMP/Smad signaling in bone metabolism. Absorbable collagen sponges containing 3 µg of recombinant human BMP2 (rhBMP2) were implanted in the dorsal muscle pouches of wild type (WT) and Smurf2-/- mice. The rhBMP2-induced ectopic bone in Smurf2-/- mice showed greater bone mass, higher mineral apposition and bone formation rates, and greater osteoblast numbers than the ectopic bone in WT mice. In WT mice, the ectopic bone consisted of a thin discontinuous outer cortical shell and scant inner trabecular bone. In contrast, in Smurf2-/- mice, the induced bone consisted of a thick, continuous outer cortical shell and abundant inner trabecular bone. Additionally, rhBMP2-stimulated bone marrow stromal cells (BMSCs) from Smurf2-/- mice showed increased osteogenic differentiation. Smurf2 induced the ubiquitination of Smad1/5. BMP/Smad signaling was enhanced in Smurf2-/- BMSCs stimulated with rhBMP2, and the inhibition of BMP/Smad signaling suppressed osteogenic differentiation of these BMSCs. These findings demonstrate that Smurf2 negatively regulates BMP/Smad signaling, thereby identifying a new regulatory mechanism in bone metabolism.

Therapeutic effects of the PKR inhibitor C16 suppressing tumor proliferation and angiogenesis in hepatocellular carcinoma in vitro and in vivo.

The therapeutic effects of C16, which is an inhibitor of RNA-dependent protein kinase (PKR), on growth of hepatocellular carcinoma (HCC) cells and tumor progression in vitro and in vivo were evaluated. Huh7 cells, a human HCC cell line, were used. The effects of C16 on cell viability were evaluated with the MTT assay, and real-time RT-PCR was performed. Huh7 cells were grafted into immunodeficient mice, and the in vivo effects of C16 on tumorigenesis were examined. C16 suppressed proliferation of HCC cells in a dose-dependent manner in vitro. Mouse models with xenograft transplantation showed that the inhibitor suppressed the growth of HCC cells in vivo. Moreover, C16 decreased angiogenesis in HCC tissue in the xenograft model. Consistent with these results in mice, transcript levels of vascular endothelial growth factor-A and factor-B, platelet-derived growth factor-A and factor-B, fibroblast growth factor-2, epidermal growth factor, and hepatocyte growth factor, which are angiogenesis-related growth factors, were significantly decreased by C16 in vitro. In conclusion, the PKR inhibitor C16 blocked tumor cell growth and angiogenesis via a decrease in mRNA levels of several growth factors. C16 may be useful in the treatment of HCC.

Direct comparison of target-reactivity and cross-reactivity induced by CAR- and BiTE-redirected T cells for the development of antibody-based T-cell therapy.

The development of chimeric antigen receptor (CAR) and bispecific T-cell engager (BiTE) has led to the successful application of cancer immunotherapy. The potential reactivity mediated by CAR- and BiTE-redirected T cells needs to be assessed to facilitate the application of these treatment options to a broader range of patients. Here, we have generated CAR and BiTE possessing the same single chain fragment variable (scFv) specific for the HLA-A2/NY-ESO-1157-165 complex (A2/NY-ESO-1157). Using HLA-A2+NY-ESO-1+ myeloma cells and peptides presented by HLA-A2 molecules as a model, both sets of redirected T cells recognized and killed HLA-A2+NY-ESO-1+ myeloma cells in an A2/NY-ESO-1157-specific manner in vitro. Moreover, CAR- and BiTE-activated T cells showed similar functional avidity, as assessed by cytokine production and killing activity, both displaying antitumor reactivity against HLA-A2+NY-ESO-1+ myeloma cells in vivo. Interestingly, cross-reactivity for homologous peptides presented by HLA-A*02:01 and NY-ESO-1157 peptide presented by HLA-A2 alleles was not identical between CAR- and BiTE-redirected T cells, probably due to structural differences of modified antibodies. These results have demonstrated that both antitumor CAR- and BiTE-activated T cells have comparable potential to recognize tumors, while paying attention to unknown off-target reactivity that would differ for each antibody-based modality even if the same scFv was employed.

Visualization of epithelial-mesenchymal transition in an inflammatory microenvironment-colorectal cancer network.

Epithelial-mesenchymal transition (EMT) is a biological process by which epithelial cells acquire mesenchymal characteristics. In malignant tumors, EMT is crucial for acquisition of a mesenchymal phenotype with invasive and metastatic properties, leading to tumor progression. An inflammatory microenvironment is thought to be responsible for the development and progression of colorectal cancer (CRC); however, the precise role of inflammatory microenvironments in EMT-related CRC progression remains unclear. Here, we show the spatiotemporal visualization of CRC cells undergoing EMT using a fluorescence-guided EMT imaging system in which the mesenchymal vimentin promoter drives red fluorescent protein (RFP) expression. An inflammatory microenvironment including TNF-α, IL-1ß, and cytokine-secreting inflammatory macrophages induced RFP expression in association with the EMT phenotype in CRC cells. In vivo experiments further demonstrated the distribution of RFP-positive CRC cells in rectal and metastatic tumors. Our data suggest that the EMT imaging system described here is a powerful tool for monitoring EMT in inflammatory microenvironment-CRC networks.