Targeting placental growth factor/neuropilin 1 pathway inhibits growth and spread of medulloblastoma.

Medulloblastoma is the most common pediatric malignant brain tumor. Although current therapies improve survival, these regimens are highly toxic and are associated with significant morbidity. Here, we report that placental growth factor (PlGF) is expressed in the majority of medulloblastomas, independent of their subtype. Moreover, high expression of PlGF receptor neuropilin 1 (Nrp1) correlates with poor overall survival in patients. We demonstrate that PlGF and Nrp1 are required for the growth and spread of medulloblastoma: PlGF/Nrp1 blockade results in direct antitumor effects in vivo, resulting in medulloblastoma regression, decreased metastasis, and increased mouse survival. We reveal that PlGF is produced in the cerebellar stroma via tumor-derived Sonic hedgehog (Shh) and show that PlGF acts through Nrp1-and not vascular endothelial growth factor receptor 1-to promote tumor cell survival. This critical tumor-stroma interaction-mediated by Shh, PlGF, and Nrp1 across medulloblastoma subtypes-supports the development of therapies targeting PlGF/Nrp1 pathway.

Fast volumetric imaging with line-scan confocal microscopy by electrically tunable lens at resonant frequency.

In microscopic imaging of biological tissues, particularly real-time visualization of neuronal activities, rapid acquisition of volumetric images poses a prominent challenge. Typically, two-dimensional (2D) microscopy can be devised into an imaging system with 3D capability using any varifocal lens. Despite the conceptual simplicity, such an upgrade yet requires additional, complicated device components and usually suffers from a reduced acquisition rate, which is critical to properly document rapid neurophysiological dynamics. In this study, we implemented an electrically tunable lens (ETL) in the line-scan confocal microscopy (LSCM), enabling the volumetric acquisition at the rate of 20 frames per second with a maximum volume of interest of 315 × 315 × 80 µm3. The axial extent of point-spread-function (PSF) was 17.6 ± 1.6 µm and 90.4 ± 2.1 µm with the ETL operating in either stationary or resonant mode, respectively, revealing significant depth axial penetration by the resonant mode ETL microscopy. We further demonstrated the utilities of the ETL system by volume imaging of both cleared mouse brain ex vivo samples and in vivo brains. The current study showed a successful application of resonant ETL for constructing a high-performance 3D axially scanning LSCM (asLSCM) system. Such advances in rapid volumetric imaging would significantly enhance our understanding of various dynamic biological processes.

Experimental and computational analyses reveal dynamics of tumor vessel cooption and optimal treatment strategies.

Cooption of the host vasculature is a strategy that some cancers use to sustain tumor progression without-or before-angiogenesis or in response to antiangiogenic therapy. Facilitated by certain growth factors, cooption can mediate tumor infiltration and confer resistance to antiangiogenic drugs. Unfortunately, this mode of tumor progression is difficult to target because the underlying mechanisms are not fully understood. Here, we analyzed the dynamics of vessel cooption during tumor progression and in response to antiangiogenic treatment in gliomas and brain metastases. We followed tumor evolution during escape from antiangiogenic treatment as cancer cells coopted, and apparently mechanically compressed, host vessels. To gain deeper understanding, we developed a mathematical model, which incorporated compression of coopted vessels, resulting in hypoxia and formation of new vessels by angiogenesis. Even if antiangiogenic therapy can block such secondary angiogenesis, the tumor can sustain itself by coopting existing vessels. Hence, tumor progression can only be stopped by combination therapies that judiciously block both angiogenesis and cooption. Furthermore, the model suggests that sequential blockade is likely to be more beneficial than simultaneous blockade.

Optical Assessment of Tear Glucose by Smart Biosensor Based on Nanoparticle Embedded Contact Lens.

Diabetes is a disease condition characterized by a prolonged, high blood glucose level, which may lead to devastating outcomes unless properly managed. Here, we introduce a simple camera-based optical monitoring system (OMS) utilizing the nanoparticle embedded contact lens that produces color changes matching the tear glucose level without any complicated electronic components. Additionally, we propose an image processing algorithm that automatically optimizes the measurement accuracy even in the presence of image blurring, possibly caused by breathing, subtle movements, and eye blinking. As a result, using in vivo mouse models and human tear samples we successfully demonstrated robust correlations across the glucose concentrations measured by three different independent techniques, validating the quantitative efficacy of the proposed OMS. For its methodological simplicity and accessibility, our findings strongly support that the innovation offered by the OMS and processing algorithm would greatly facilitate the glucose monitoring procedure and improve the overall welfare of diabetes patients.

Targeting CXCR4-dependent immunosuppressive Ly6Clow monocytes improves antiangiogenic therapy in colorectal cancer.

Antiangiogenic therapy with antibodies against VEGF (bevacizumab) or VEGFR2 (ramucirumab) has been proven efficacious in colorectal cancer (CRC) patients. However, the improvement in overall survival is modest and only in combination with chemotherapy. Thus, there is an urgent need to identify potential underlying mechanisms of resistance specific to antiangiogenic therapy and develop strategies to overcome them. Here we found that anti-VEGFR2 therapy up-regulates both C-X-C chemokine ligand 12 (CXCL12) and C-X-C chemokine receptor 4 (CXCR4) in orthotopic murine CRC models, including SL4 and CT26. Blockade of CXCR4 signaling significantly enhanced treatment efficacy of anti-VEGFR2 treatment in both CRC models. CXCR4 was predominantly expressed in immunosuppressive innate immune cells, which are recruited to CRCs upon anti-VEGFR2 treatment. Blockade of CXCR4 abrogated the recruitment of these innate immune cells. Importantly, these myeloid cells were mostly Ly6Clow monocytes and not Ly6Chigh monocytes. To selectively deplete individual innate immune cell populations, we targeted key pathways in Ly6Clow monocytes (Cx3cr1-/- mice), Ly6Chigh monocytes (CCR2-/- mice), and neutrophils (anti-Ly6G antibody) in combination with CXCR4 blockade in SL4 CRCs. Depletion of Ly6Clow monocytes or neutrophils improved anti-VEGFR2-induced SL4 tumor growth delay similar to the CXCR4 blockade. In CT26 CRCs, highly resistant to anti-VEGFR2 therapy, CXCR4 blockade enhanced anti-VEGFR2-induced tumor growth delay but specific depletion of Ly6G+ neutrophils did not. The discovery of CXCR4-dependent recruitment of Ly6Clow monocytes in tumors unveiled a heretofore unknown mechanism of resistance to anti-VEGF therapies. Our findings also provide a rapidly translatable strategy to enhance the outcome of anti-VEGF cancer therapies.

High-speed dual-layer scanning photoacoustic microscopy using focus tunable lens modulation at resonant frequency.

The range of imaging depth in optical resolution photoacoustic microscopy (PAM) is limited by the short depth of focus of high-numerical aperture objective lenses. In this paper, focus tunable lens modulation has been employed at the resonant frequency of focus tunable lenses in order to enhance both the range of imaging depth and the scanning speed. By electrically controlling the focal length in the axial direction of the sample, the range of imaging depth was extended approximately 1.22 times and the scanning speed was enhanced by approximately 7.40 times, in comparison to corresponding values of conventional PAM systems.

Image-based quantitative analysis of tear film lipid layer thickness for meibomian gland evaluation.

BACKGROUND: Dry eye syndrome is one of the most common ocular diseases, and meibomian gland dysfunction (MGD) is the leading cause of evaporative dry eye syndrome. When the tear film lipid layer becomes thin due to obstructive or hyposecretory meibomian gland dysfunction, the excessive evaporation of the aqueous layer can occur, and this causes evaporative dry eye syndrome. Thus, measuring the lipid layer thickness (LLT) is essential for accurate diagnosis and proper treatment of evaporative dry eye syndrome. METHODS: We used a white LED panel with a slit lamp microscope to obtain videos of the lipid layer interference patterns on the cornea. To quantitatively analyze the LLT from interference colors, we developed a novel algorithm that can automatically perform the following processes on an image frame: determining the radius of the iris, locating the center of the pupil, defining region of interest (ROI), tracking the ROI, compensating for the color of iris and illumination, and producing comprehensive analysis output. A group of dry eye syndrome patients with hyposecretory MGD, dry eye syndrome without MGD, hypersecretory MGD, and healthy volunteers were recruited. Their LLTs were analyzed and statistical information-mean and standard deviation, the relative frequency of LLT at each time point, and graphical LLT visualization-were produced. RESULTS: Using our algorithm, we processed the lipid layer interference pattern and automatically analyzed the LLT distribution of images from patients. The LLT of hyposecretory MGD was thinner (45.2 ± 11.6 nm) than that of dry eye syndrome without MGD (69.0 ± 9.4 nm) and healthy volunteers (68.3 ± 13.7 nm) while the LLT of hypersecretory MGD was thicker (93.5 ± 12.6 nm) than that of dry eye syndrome without MGD. Patients' LLTs were statistically analyzed over time, visualized with 3D surface plots, and displayed using 3D scatter plots of image pixel data for comprehensive assessment. CONCLUSIONS: We developed an image-based algorithm for quantitative measurement as well as statistical analysis of the LLT despite fluctuation and eye movement. This pilot study demonstrates that the quantitative LLT analysis of patients is consistent with the functions of meibomian glands clinically evaluated by an ophthalmologist. This approach is a significant step forward in developing a fully automated instrument for evaluating dry eye syndrome and for providing proper guidance of treatment.

Imaging of the Finger Vein and Blood Flow for Anti-Spoofing Authentication Using a Laser and a MEMS Scanner.

A new authentication method employing a laser and a scanner is proposed to improve image contrast of the finger vein and to extract blood flow pattern for liveness detection. A micromirror reflects a laser beam and performs a uniform raster scan. Transmissive vein images were obtained, and compared with those of an LED. Blood flow patterns were also obtained based on speckle images in perfusion and occlusion. Curvature ratios of the finger vein and blood flow intensities were found to be nearly constant, regardless of the vein size, which validated the high repeatability of this scheme for identity authentication with anti-spoofing.

Holistic random encoding for imaging through multimode fibers.

The input numerical aperture (NA) of multimode fiber (MMF) can be effectively increased by placing turbid media at the input end of the MMF. This provides the potential for high-resolution imaging through the MMF. While the input NA is increased, the number of propagation modes in the MMF and hence the output NA remains the same. This makes the image reconstruction process underdetermined and may limit the quality of the image reconstruction. In this paper, we aim to improve the signal to noise ratio (SNR) of the image reconstruction in imaging through MMF. We notice that turbid media placed in the input of the MMF transforms the incoming waves into a better format for information transmission and information extraction. We call this transformation as holistic random (HR) encoding of turbid media. By exploiting the HR encoding, we make a considerable improvement on the SNR of the image reconstruction. For efficient utilization of the HR encoding, we employ sparse representation (SR), a relatively new signal reconstruction framework when it is provided with a HR encoded signal. This study shows for the first time to our knowledge the benefit of utilizing the HR encoding of turbid media for recovery in the optically underdetermined systems where the output NA of it is smaller than the input NA for imaging through MMF.

Combined targeting of HER2 and VEGFR2 for effective treatment of HER2-amplified breast cancer brain metastases.

Brain metastases are a serious obstacle in the treatment of patients with human epidermal growth factor receptor-2 (HER2)-amplified breast cancer. Although extracranial disease is controlled with HER2 inhibitors in the majority of patients, brain metastases often develop. Because these brain metastases do not respond to therapy, they are frequently the reason for treatment failure. We developed a mouse model of HER2-amplified breast cancer brain metastasis using an orthotopic xenograft of BT474 cells. As seen in patients, the HER2 inhibitors trastuzumab and lapatinib controlled tumor progression in the breast but failed to contain tumor growth in the brain. We observed that the combination of a HER2 inhibitor with an anti-VEGF receptor-2 (VEGFR2) antibody significantly slows tumor growth in the brain, resulting in a striking survival benefit. This benefit appears largely due to an enhanced antiangiogenic effect: Combination therapy reduced both the total and functional microvascular density in the brain xenografts. In addition, the combination therapy led to a marked increase in necrosis of the brain lesions. Moreover, we observed even better antitumor activity after combining both trastuzumab and lapatinib with the anti-VEGFR2 antibody. This triple-drug combination prolonged the median overall survival fivefold compared with the control-treated group and twofold compared with either two-drug regimen. These findings support the clinical development of this three-drug regimen for the treatment of HER2-amplified breast cancer brain metastases.

Speckle suppression via sparse representation for wide-field imaging through turbid media.

Speckle suppression is one of the most important tasks in the image transmission through turbid media. Insufficient speckle suppression requires an additional procedure such as temporal ensemble averaging over multiple exposures. In this paper, we consider the image recovery process based on the so-called transmission matrix (TM) of turbid media for the image transmission through the media. We show that the speckle left unremoved in the TM-based image recovery can be suppressed effectively via sparse representation (SR). SR is a relatively new signal reconstruction framework which works well even for ill-conditioned problems. This is the first study to show the benefit of using the SR as compared to the phase conjugation (PC) a de facto standard method to date for TM-based imaging through turbid media including a live cell through tissue slice.

Gradient index lens based combined two-photon microscopy and optical coherence tomography.

We report a miniaturized probe-based combined two-photon microscopy (TPM) and optical coherence tomography (OCT) system. This system is to study the colorectal cancer in mouse models by visualizing both cellular and structural information of the colon in 3D with TPM and OCT respectively. The probe consisted of gradient index (GRIN) lenses and a 90° reflecting prism at its distal end for side-viewing, and it was added onto an objective lens-based TPM and OCT system. The probe was 2.2 mm in diameter and 60 mm in length. TPM imaging was performed by raster scanning of the excitation focus at the imaging speed of 15.4 frames/s. OCT imaging was performed by combining the linear sample translation and probe rotation along its axis. This miniaturized probe based dual-modal system was characterized with tissue phantoms containing fluorescent microspheres, and applied to image mouse colonic tissues ex vivo as a demonstration. As OCT and TPM provided structural and cellular information of the tissues respectively, this probe based multi-modal imaging system can be helpful for in vivo studies of preclinical animal models such as mouse colonic tumorigenesis.

Toward a miniature endomicroscope: pixelation-free and diffraction-limited imaging through a fiber bundle.

A fiber bundle is widely used for endoscopic imaging due to its direct image delivery capability. However, there exists an inevitable pixelation artifact, which limits spatial resolution to the diameter of individual fibers. In this Letter, we present a method that can eliminate this artifact and achieve diffraction-limited spatial resolution. We exploited the binary control of a digital micromirror device to measure a transmission matrix of a fiber bundle and to subsequently control mode mixing among individual fibers. In doing so, we achieved a 22 kHz scanning rate of a diffraction-limited focused spot and obtained fluorescence endoscope imaging (58 µm × 58 µm) with near video-rate (10.3 Hz) acquisition. Our study lays a foundation for developing an ultrathin and high-resolution microendoscope.

Advances in laser speckle imaging: From qualitative to quantitative hemodynamic assessment.

Laser speckle imaging (LSI) techniques have emerged as a promising method for visualizing functional blood vessels and tissue perfusion by analyzing the speckle patterns generated by coherent light interacting with living biological tissue. These patterns carry important biophysical tissue information including blood flow dynamics. The noninvasive, label-free, and wide-field attributes along with relatively simple instrumental schematics make it an appealing imaging modality in preclinical and clinical applications. The review outlines the fundamentals of speckle physics and the three categories of LSI techniques based on their degree of quantification: qualitative, semi-quantitative and quantitative. Qualitative LSI produces microvascular maps by capturing speckle contrast variations between blood vessels containing moving red blood cells and the surrounding static tissue. Semi-quantitative techniques provide a more accurate analysis of blood flow dynamics by accounting for the effect of static scattering on spatiotemporal parameters. Quantitative LSI such as optical speckle image velocimetry provides quantitative flow velocity measurements, which is inspired by the particle image velocimetry in fluid mechanics. Additionally, discussions regarding the prospects of future innovations in LSI techniques for optimizing the vascular flow quantification with associated clinical outlook are presented.

Tonic excitation by astrocytic GABA causes neuropathic pain by augmenting neuronal activity and glucose metabolism.

Neuropathic pain is a debilitating condition caused by the hyperexcitability of spinal dorsal horn neurons and is often characterized by allodynia. Although neuron-independent mechanisms of hyperexcitability have been investigated, the contribution of astrocyte-neuron interactions remains unclear. Here, we show evidence of reactive astrocytes and their excessive GABA release in the spinal dorsal horn, which paradoxically leads to the tonic excitation of neighboring neurons in a neuropathic pain model. Using multiple electrophysiological methods, we demonstrated that neuronal hyperexcitability is attributed to both increased astrocytic GABA synthesis via monoamine oxidase B (MAOB) and the depolarized reversal potential of GABA-mediated currents (EGABA) via the downregulation of the neuronal K+/Cl- cotransporter KCC2. Furthermore, longitudinal 2-deoxy-2-[18F]-fluoro-D-glucose microPET imaging demonstrated increased regional glucose metabolism in the ipsilateral dorsal horn, reflecting neuronal hyperexcitability. Importantly, inhibiting MAOB restored the entire astrocytic GABA-mediated cascade and abrogated the increased glucose metabolism and mechanical allodynia. Overall, astrocytic GABA-mediated tonic excitation is critical for neuronal hyperexcitability, leading to mechanical allodynia and neuropathic pain.

In vivo wide-area cellular imaging by side-view endomicroscopy.

In vivo imaging of small animals offers several possibilities for studying normal and disease biology, but visualizing organs with single-cell resolution is challenging. We describe rotational side-view confocal endomicroscopy, which enables cellular imaging of gastrointestinal and respiratory tracts in mice and may be extensible to imaging organ parenchyma such as cerebral cortex. We monitored cell infiltration, vascular changes and tumor progression during inflammation and tumorigenesis in colon over several months.

Orthotopic Mouse Models of Urinary Bladder Cancer.

BACKGROUND/AIM: Urinary bladder cancer has various etiologies and tends to recur and then progress to a higher grade. When muscles are invaded, the response to conventional therapy is poor and the quality of life deteriorates rapidly. Here, we summarize and compare two representative methods used to create the syngeneic mouse models required for immunological research. MATERIALS AND METHODS: In this study, we utilized six-week-old female C3H/HeNCrl mice and the mouse bladder tumor cell line MBT-2. The first method involved transurethral catheterization with poly-L-lysine pretreatment (catheter group), while the second method involved transperitoneal incision and direct injection of tumor cells into the bladder wall (open group). Mouse postoperative status was monitored on a weekly basis using magnetic resonance imaging (MRI). RESULTS: The catheter group had a tumor development rate of 47% (7 out of 15 mice), with only 1 mouse developing an intravesical tumor. In contrast, the open group had a higher tumor formation rate of 69% (47 out of 68 mice), with 27 mice showing intravesical tumor formation. Notably, with a lower cell count, urinary obstruction events were observed 2 weeks post-inoculation, which is one week later than the higher cell count group. CONCLUSION: In this study, we conducted a comparative analysis between the transurethral catheterization method and the transperitoneal incision and direct injection method in animal bladder tumor models. Our findings provide evidence of the consistent effectiveness in constructing a stable model within the open group. Well-designed orthotopic animal models are essential.

Photobiomodulation Therapy Restores Olfactory Function Impaired by Photothrombosis in Mouse Olfactory Bulb.

An ischemic stroke typically accompanies numerous disorders ranging from somatosensory dysfunction to cognitive impairments, inflicting patients with various neurologic symptoms. Among pathologic outcomes, post-stroke olfactory dysfunctions are frequently observed. Despite the well-known prevalence, therapy options for such compromised olfaction are limited, likely due to the complexity of olfactory bulb architecture, which encompasses both the peripheral and central nervous systems. As photobiomodulation (PBM) emerged for treating ischemia-associated symptoms, the effectiveness of PBM on stroke-induced impairment of olfactory function was explored. Novel mouse models with olfactory dysfunctions were prepared using photothrombosis (PT) in the olfactory bulb on day 0. The post-PT PBM was performed daily from day 2 to day 7 by irradiating the olfactory bulb via an 808 nm laser with a fluence of 40 J/cm2 (325 mW/cm2 for 2  smin per day). The buried food test (BFT) was used to score behavioral acuity in food-deprived mice to assess the olfactory function before PT, after PT, and after PBM. Histopathological examinations and cytokine assays were performed on the mouse brains harvested on day 8. The results from BFT were specific to an individual, with positive correlations between the baseline latency time measured before PT and its alteration at the ensuing stages for both the PT and PT + PBM groups. Also, the correlation analysis in both groups showed highly similar, significant positive relationships between the early and late latency time change independent of PBM, implicating a common recovery mechanism. Particularly, PBM treatment accelerated the recovery of impaired olfaction following PT by suppressing inflammatory cytokines and enhancing both glial and vascular factors (e.g., GFAP, IBA-1, and CD31). PBM therapy during the acute phase of ischemia improves the compromised olfactory function by modulating microenvironments and inflammation status of the affected tissue.

Nanozyme-based colorimetric biosensor with a systemic quantification algorithm for noninvasive glucose monitoring.

Diabetes mellitus accompanies an abnormally high glucose level in the bloodstream. Early diagnosis and proper glycemic management of blood glucose are essential to prevent further progression and complications. Biosensor-based colorimetric detection has progressed and shown potential in portable and inexpensive daily assessment of glucose levels because of its simplicity, low-cost, and convenient operation without sophisticated instrumentation. Colorimetric glucose biosensors commonly use natural enzymes that recognize glucose and chromophores that detect enzymatic reaction products. However, many natural enzymes have inherent defects, limiting their extensive application. Recently, nanozyme-based colorimetric detection has drawn attention due to its merits including high sensitivity, stability under strict reaction conditions, flexible structural design with low-cost materials, and adjustable catalytic activities. This review discusses various nanozyme materials, colorimetric analytic methods and mechanisms, recent machine learning based analytic methods, quantification systems, applications and future directions for monitoring and managing diabetes.

Cranial and Spinal Window Preparation for in vivo Optical Neuroimaging in Rodents and Related Experimental Techniques.

Optical neuroimaging provides an effective neuroscience tool for multi-scale investigation of the neural structures and functions, ranging from molecular, cellular activities to the inter-regional connectivity assessment. Amongst experimental preparations, the implementation of an artificial window to the central nervous system (CNS) is primarily required for optical visualization of the CNS and associated brain activities through the opaque skin and bone. Either thinning down or removing portions of the skull or spine is necessary for unobstructed long-term in vivo observations, for which types of the cranial and spinal window and applied materials vary depending on the study objectives. As diversely useful, a window can be designed to accommodate other experimental methods such as electrophysiology or optogenetics. Moreover, auxiliary apparatuses would allow the recording in synchrony with behavior of large-scale brain connectivity signals across the CNS, such as olfactory bulb, cerebral cortex, cerebellum, and spinal cord. Such advancements in the cranial and spinal window have resulted in a paradigm shift in neuroscience, enabling in vivo investigation of the brain function and dysfunction at the microscopic, cellular level. This Review addresses the types and classifications of windows used in optical neuroimaging while describing how to perform in vivo studies using rodent models in combination with other experimental modalities during behavioral tests. The cranial and spinal window has enabled longitudinal examination of evolving neural mechanisms via in situ visualization of the brain. We expect transformable and multi-functional cranial and spinal windows to become commonplace in neuroscience laboratories, further facilitating advances in optical neuroimaging systems.