Overcoming retinoic acid resistance in HER2-enriched breast cancers: role of MYC.

HER2-enriched (HER2+) breast cancers express high levels of the growth-promoting HER2 protein. Although these cancers are treated with the HER2-targeted drug, trastuzumab, resistance to treatment is common. Retinoic acid (RA) is an anti-cancer agent that has been successfully used for the treatment of leukemia and holds promise for the treatment of solid cancers, including breast cancer. The HER2 gene is frequently co-amplified with RARA, a key determinant of RA sensitivity in breast cancers. It seems surprising, therefore, that HER2+ breast cancers are refractory to RA treatment. Here, we show that MYC mediates RA resistance by suppressing the expression of cellular retinoic acid binding protein 2 (CRABP2), resulting in RARα inactivation. CRABP2 is an intracellular RA transporter that delivers RA to the nuclear receptor RARα for its activation. Our results indicate that response to RA is enhanced by MYC depletion in HER2+ breast cancer cells and that RA treatment enhances trastuzumab responsiveness. Our findings support the use of RA and trastuzumab for the treatment of subsets of patients with breast cancers that are HER2-RARα co-amplified and have low levels of MYC.

The ultra-thin, minimally invasive surface electrode array NeuroWeb for probing neural activity.

Electrophysiological recording technologies can provide valuable insights into the functioning of the central and peripheral nervous systems. Surface electrode arrays made of soft materials or implantable multi-electrode arrays with high electrode density have been widely utilized as neural probes. However, neither of these probe types can simultaneously achieve minimal invasiveness and robust neural signal detection. Here, we present an ultra-thin, minimally invasive neural probe (the "NeuroWeb") consisting of hexagonal boron nitride and graphene, which leverages the strengths of both surface electrode array and implantable multi-electrode array. The NeuroWeb open lattice structure with a total thickness of 100 nm demonstrates high flexibility and strong adhesion, establishing a conformal and tight interface with the uneven mouse brain surface. In vivo electrophysiological recordings show that NeuroWeb detects stable single-unit activity of neurons with high signal-to-noise ratios. Furthermore, we investigate neural interactions between the somatosensory cortex and the cerebellum using transparent dual NeuroWebs and optical stimulation, and measure the times of neural signal transmission between the brain regions depending on the pathway. Therefore, NeuroWeb can be expected to pave the way for understanding complex brain networks with optical and electrophysiological mapping of the brain.

Tracing multiple scattering trajectories for deep optical imaging in scattering media.

Multiple light scattering hampers imaging objects in complex scattering media. Approaches used in real practices mainly aim to filter out multiple scattering obscuring the ballistic waves that travel straight through the scattering medium. Here, we propose a method that makes the deterministic use of multiple scattering for microscopic imaging of an object embedded deep within scattering media. The proposed method finds a stack of multiple complex phase plates that generate similar light trajectories as the original scattering medium. By implementing the inverse scattering using the identified phase plates, our method rectifies multiple scattering and amplifies ballistic waves by almost 600 times. This leads to a significant increase in imaging depth-more than three times the scattering mean free path-as well as the correction of image distortions. Our study marks an important milestone in solving the long-standing high-order inverse scattering problems.

Label-free adaptive optics single-molecule localization microscopy for whole zebrafish.

Specimen-induced aberration has been a major factor limiting the imaging depth of single-molecule localization microscopy (SMLM). Here, we report the application of label-free wavefront sensing adaptive optics to SMLM for deep-tissue super-resolution imaging. The proposed system measures complex tissue aberrations from intrinsic reflectance rather than fluorescence emission and physically corrects the wavefront distortion more than three-fold stronger than the previous limit. This enables us to resolve sub-diffraction morphologies of cilia and oligodendrocytes in whole zebrafish as well as dendritic spines in thick mouse brain tissues at the depth of up to 102 µm with localization number enhancement by up to 37 times and localization precision comparable to aberration-free samples. The proposed approach can expand the application range of SMLM to whole zebrafish that cause the loss of localization number owing to severe tissue aberrations.

Stationary-to-migratory transition in glioblastoma stem-like cells driven by a fatty acid-binding protein 7-RXRα neurogenic pathway.

BACKGROUND: Glioblastoma (GBM) stem-like cells (GSCs) are crucial drivers of treatment resistance and tumor recurrence. While the concept of "migrating" cancer stem cells was proposed a decade ago, the roles and underlying mechanisms of the heterogeneous populations of GSCs remain poorly defined. METHODS: Cell migration using GBM cell lines and patient-derived GSCs was examined using Transwell inserts and the scratch assay. Single-cell RNA sequencing data analysis were used to map GSC drivers to specific GBM cell populations. Xenografted mice were used to model the role of brain-type fatty acid-binding protein 7 (FABP7) in GBM infiltration and expansion. The mechanism by which FABP7 and its fatty acid ligands promote GSC migration was examined by gel shift and luciferase gene reporter assays. RESULTS: A subpopulation of FABP7-expressing migratory GSCs was identified, with FABP7 upregulating SOX2, a key modulator for GBM stemness and plasticity, and ZEB1, a prominent factor in GBM epithelial-mesenchymal transition and invasiveness. Our data indicate that GSC migration is driven by nuclear FABP7 through activation of RXRα, a nuclear receptor activated by polyunsaturated fatty acids (PUFAs). CONCLUSION: Infiltrative progression in GBM is driven by migratory GSCs through activation of a PUFA-FABP7-RXRα neurogenic pathway.

Characterizing dry mass and volume changes in human multiple myeloma cells upon treatment with proteotoxic and genotoxic drugs.

Multiple myeloma (MM) is a cancer of terminally differentiated plasma cells. MM remains incurable, but overall survival of patients has progressively increased over the past two decades largely due to novel agents such as proteasome inhibitors (PI) and the immunomodulatory agents. While these therapies are highly effective, MM patients can be de novo resistant and acquired resistance with prolonged treatment is inevitable. There is growing interest in early, accurate identification of responsive versus non-responsive patients; however, limited sample availability and need for rapid assays are limiting factors. Here, we test dry mass and volume as label-free biomarkers to monitor early response of MM cells to treatment with bortezomib, doxorubicin, and ultraviolet light. For the dry mass measurement, we use two types of phase-sensitive optical microscopy techniques: digital holographic tomography and computationally enhanced quantitative phase microscopy. We show that human MM cell lines (RPMI8226, MM.1S, KMS20, and AMO1) increase dry mass upon bortezomib treatment. This dry mass increase after bortezomib treatment occurs as early as 1 h for sensitive cells and 4 h for all tested cells. We further confirm this observation using primary multiple myeloma cells derived from patients and show that a correlation exists between increase in dry mass and sensitivity to bortezomib, supporting the use of dry mass as a biomarker. The volume measurement using Coulter counter shows a more complex behavior; RPMI8226 cells increase the volume at an early stage of apoptosis, but MM.1S cells show the volume decrease typically observed with apoptotic cells. Altogether, this cell study presents complex kinetics of dry mass and volume at an early stage of apoptosis, which may serve as a basis for the detection and treatment of MM cells.

Fourier holographic endoscopy for imaging continuously moving objects.

Coherent fiber bundles are widely used for endoscopy, but conventional approaches require distal optics to form an object image and acquire pixelated information owing to the geometry of the fiber cores. Recently, holographic recording of a reflection matrix enables a bare fiber bundle to perform pixelation-free microscopic imaging as well as allows a flexible mode operation, because the random core-to-core phase retardations due to any fiber bending and twisting could be removed in situ from the recorded matrix. Despite its flexibility, the method is not suitable for a moving object because the fiber probe should remain stationary during the matrix recording to avoid the alteration of the phase retardations. Here, we acquire a reflection matrix of a Fourier holographic endoscope equipped with a fiber bundle and explore the effect of fiber bending on the recorded matrix. By removing the motion effect, we develop a method that can resolve the perturbation of the reflection matrix caused by a continuously moving fiber bundle. Thus, we demonstrate high-resolution endoscopic imaging through a fiber bundle, even when the fiber probe changes its shape along with the moving objects. The proposed method can be used for minimally invasive monitoring of behaving animals.

Exploiting volumetric wave correlation for enhanced depth imaging in scattering medium.

Imaging an object embedded within a scattering medium requires the correction of complex sample-induced wave distortions. Existing approaches have been designed to resolve them by optimizing signal waves recorded in each 2D image. Here, we present a volumetric image reconstruction framework that merges two fundamental degrees of freedom, the wavelength and propagation angles of light waves, based on the object momentum conservation principle. On this basis, we propose methods for exploiting the correlation of signal waves from volumetric images to better cope with multiple scattering. By constructing experimental systems scanning both wavelength and illumination angle of the light source, we demonstrated a 32-fold increase in the use of signal waves compared with that of existing 2D-based approaches and achieved ultrahigh volumetric resolution (lateral resolution: 0.41 [Formula: see text], axial resolution: 0.60 [Formula: see text]) even within complex scattering medium owing to the optimal coherent use of the broad spectral bandwidth (225 nm).

Computational conjugate adaptive optics microscopy for longitudinal through-skull imaging of cortical myelin.

Myelination processes are closely related to higher brain functions such as learning and memory. While their longitudinal observation has been crucial to understanding myelin-related physiology and various brain disorders, skull opening or thinning has been required to secure clear optical access. Here we present a high-speed reflection matrix microscope using a light source with a wavelength of 1.3 µm to reduce tissue scattering and aberration. Furthermore, we develop a computational conjugate adaptive optics algorithm designed for the recorded reflection matrix to optimally compensate for the skull aberrations. These developments allow us to realize label-free longitudinal imaging of cortical myelin through an intact mouse skull. The myelination processes of the same mice were observed from 3 to 10 postnatal weeks to the depth of cortical layer 4 with a spatial resolution of 0.79 µm. Our system will expedite the investigations on the role of myelination in learning, memory, and brain disorders.

Flexible-type ultrathin holographic endoscope for microscopic imaging of unstained biological tissues.

Ultrathin lensless fibre endoscopes offer minimally invasive investigation, but they mostly operate as a rigid type due to the need for prior calibration of a fibre probe. Furthermore, most implementations work in fluorescence mode rather than label-free imaging mode, making them unsuitable for general medical diagnosis. Herein, we report a fully flexible ultrathin fibre endoscope taking 3D holographic images of unstained tissues with 0.85-µm spatial resolution. Using a bare fibre bundle as thin as 200-µm diameter, we design a lensless Fourier holographic imaging configuration to selectively detect weak reflections from biological tissues, a critical step for label-free endoscopic reflectance imaging. A unique algorithm is developed for calibration-free holographic image reconstruction, allowing us to image through a narrow and curved passage regardless of fibre bending. We demonstrate endoscopic reflectance imaging of unstained rat intestine tissues that are completely invisible to conventional endoscopes. The proposed endoscope will expedite a more accurate and earlier diagnosis than before with minimal complications.

Through-skull brain imaging in vivo at visible wavelengths via dimensionality reduction adaptive-optical microscopy.

Compensation of sample-induced optical aberrations is crucial for visualizing microscopic structures deep within biological tissues. However, strong multiple scattering poses a fundamental limitation for identifying and correcting the tissue-induced aberrations. Here, we introduce a label-free deep-tissue imaging technique termed dimensionality reduction adaptive-optical microscopy (DReAM) to selectively attenuate multiple scattering. We established a theoretical framework in which dimensionality reduction of a time-gated reflection matrix can attenuate uncorrelated multiple scattering while retaining a single-scattering signal with a strong wave correlation, irrespective of sample-induced aberrations. We performed mouse brain imaging in vivo through the intact skull with the probe beam at visible wavelengths. Despite the strong scattering and aberrations, DReAM offered a 17-fold enhancement of single scattering-to-multiple scattering ratio and provided high-contrast images of neural fibers in the brain cortex with the diffraction-limited spatial resolution of 412 nanometers and a 33-fold enhanced Strehl ratio.

High-throughput volumetric adaptive optical imaging using compressed time-reversal matrix.

Deep-tissue optical imaging suffers from the reduction of resolving power due to tissue-induced optical aberrations and multiple scattering noise. Reflection matrix approaches recording the maps of backscattered waves for all the possible orthogonal input channels have provided formidable solutions for removing severe aberrations and recovering the ideal diffraction-limited spatial resolution without relying on fluorescence labeling and guide stars. However, measuring the full input-output response of the tissue specimen is time-consuming, making the real-time image acquisition difficult. Here, we present the use of a time-reversal matrix, instead of the reflection matrix, for fast high-resolution volumetric imaging of a mouse brain. The time-reversal matrix reduces two-way problem to one-way problem, which effectively relieves the requirement for the coverage of input channels. Using a newly developed aberration correction algorithm designed for the time-reversal matrix, we demonstrated the correction of complex aberrations using as small as 2% of the complete basis while maintaining the image reconstruction fidelity comparable to the fully sampled reflection matrix. Due to nearly 100-fold reduction in the matrix recording time, we could achieve real-time aberration-correction imaging for a field of view of 40 × 40 µm2 (176 × 176 pixels) at a frame rate of 80 Hz. Furthermore, we demonstrated high-throughput volumetric adaptive optical imaging of a mouse brain by recording a volume of 128 × 128 × 125 µm3 (568 × 568 × 125 voxels) in 3.58 s, correcting tissue aberrations at each and every 1 µm depth section, and visualizing myelinated axons with a lateral resolution of 0.45 µm and an axial resolution of 2 µm.

Time-resolved detection of early-arriving ballistic waves in a quasi-diffusive regime.

Ballistic waves directly carry image information in imaging through a scattering medium, but they are often obscured by much intense multiple-scattered waves. Detecting early arriving photons has been an effective method to extract ballistic waves in the transmission-mode imaging. However, it has been difficult to identify the temporal distribution of ballistic waves relative to the multiple scattering waves in the quasi-diffusive regime. Here, we present a method to separately quantify ballistic and multiple-scattered waves at their corresponding flight times even when multiple scattering is much stronger than the ballistic waves. This is realized by measuring the transmission matrix of an object embedded within scattering medium and comparing the coherent accumulation of ballistic waves with their incoherent addition. To further elucidate the temporal behavior of ballistic waves in quasi-diffusive regime, we analyze the flight time difference between ballistic and multiple-scattered waves and the effect of coherence gating on their relative intensities for the scattering medium of different thicknesses. The presented method to distinctively detect the temporal behavior of ballistic and multiple-scattered waves will lay a foundation to exploit multiple-scattered waves for deep-tissue imaging.

Single-shot multiple-depth macroscopic imaging by spatial frequency multiplexing.

We present a low-coherence interferometric imaging system designed for 3-dimensional (3-D) imaging of a macroscopic object through a narrow passage. Our system is equipped with a probe-type port composed of a bundle fiber for imaging and a separate multimode optical fiber for illumination. To eliminate the need for mechanical depth scanning, we employ a spatial frequency multiplexing method by installing a 2-D diffraction grating and an echelon in the reference arm. This configuration generates multiple reference beams, all having different path lengths and propagation directions, which facilitates the encoding of different depth information in a single interferogram. We demonstrate the acquisition of 9 depth images at the interval of 250 µm for a custom-made cone and a plaster teeth model. The proposed system minimizes the need for mechanical scanning and achieves a wide range of depth coverage, significantly increasing the speed of 3-D imaging for macroscopic objects.

FABP7 Facilitates Uptake of Docosahexaenoic Acid in Glioblastoma Neural Stem-like Cells.

Glioblastoma (GBM) is an aggressive tumor with a dismal prognosis. Neural stem-like cells contribute to GBM's poor prognosis by driving drug resistance and maintaining cellular heterogeneity. GBM neural stem-like cells express high levels of brain fatty acid-binding protein (FABP7), which binds to polyunsaturated fatty acids (PUFAs) ω-6 arachidonic acid (AA) and ω-3 docosahexaenoic acid (DHA). Similar to brain, GBM tissue is enriched in AA and DHA. However, DHA levels are considerably lower in GBM tissue compared to adult brain. Therefore, it is possible that increasing DHA content in GBM, particularly in neural stem-like cells, might have therapeutic value. Here, we examine the fatty acid composition of patient-derived GBM neural stem-like cells grown as neurosphere cultures. We also investigate the effect of AA and DHA treatment on the fatty acid profiles of GBM neural stem-like cells with or without FABP7 knockdown. We show that DHA treatment increases DHA levels and the DHA:AA ratio in GBM neural stem-like cells, with FABP7 facilitating the DHA uptake. We also found that an increased uptake of DHA inhibits the migration of GBM neural stem-like cells. Our results suggest that increasing DHA content in the GBM microenvironment may reduce the migration/infiltration of FABP7-expressing neural stem-like cancer cells.

Trastuzumab Mechanism of Action; 20 Years of Research to Unravel a Dilemma.

Trastuzumab as a first HER2-targeted therapy for the treatment of HER2-positive breast cancer patients was introduced in 1998. Although trastuzumab has opened a new avenue to treat patients with HER2-positive breast cancer and other types of cancer, some patients are not responsive or become resistant to this treatment. So far, several mechanisms have been suggested for the mode of action of trastuzumab; however, the findings regarding these mechanisms are controversial. In this review, we aimed to provide a detailed insight into the various mechanisms of action of trastuzumab.

Super resolution microscopy reveals DHA-dependent alterations in glioblastoma membrane remodelling and cell migration.

Brain fatty acid binding protein (FABP7; B-FABP) promotes glioblastoma (GBM) cell migration and is associated with tumor infiltration, properties associated with a poor prognosis in GBM patients. FABP7-expressing neural stem-like cells are known to drive tumor migration/infiltration and resistance to treatment. We have previously shown that FABP7's effects on cell migration can be reversed when GBM cells are cultured in medium supplemented with the omega-3 fatty acid, docosahexaenoic acid (DHA). Here, we use super-resolution imaging on patient-derived GBM stem-like cells to examine the importance of FABP7 and its fatty acid ligands in mitigating GBM cell migration. As FABPs are involved in fatty acid transport from membrane to cytosol, we focus on the effect of FABP7 and its ligand DHA on GBM membrane remodeling, as well as FABP7 nanoscale domain formation on GBM membrane. Using quantitative plasma membrane lipid order imaging, we show that FABP7 expression in GBM cells correlates with increased membrane lipid order, with DHA dramatically decreasing lipid order. Using super-resolution stimulated emission depletion (STED) microscopy, we observe non-uniform distribution of FABP7 on the surface of GBM cells, with FABP7 forming punctate nanoscale domains of ∼100 nm in diameter. These nanodomains are particularly enriched at the migrating front of GBM cells. Interestingly, FABP7 nanodomains are disrupted when GBM cells are cultured in DHA-supplemented medium. We demonstrate a tight link between cell migration, a higher membrane lipid order and increased FABP7 nanoscale domains. We propose that DHA-mediated disruption of membrane lipid order and FABP7 nanodomains forms the basis of FABP7/DHA-mediated inhibition of cell migration in GBM.

Single-shot multi-depth full-field optical coherence tomography using spatial frequency division multiplexing.

Fast 3D volumetric imaging has been essential for biology, medicine and industrial inspections, and various optical coherence tomography (OCT) methods have been developed to meet such needs. Point-scanning based approaches, such as swept-source OCT and spectral domain OCT, can obtain a depth information at once, but they require lateral scan for full 3D imaging. On the contrary, full-field OCT needs the scanning of imaging depth while it records a full lateral information at once. Here, we present a full-field OCT system that can obtain multi-depth information at once by a single-shot recording. We combine a 2D diffraction grating and a custom-made echelon to prepare multiple reference beams having different pathlengths and propagating angles. By recording a single interference image between the reflected wave from a sample and these multiple reference beams, we reconstruct full-field images at multiple depths associated with the pathlengths of the individual reference beams. We demonstrated the single-shot recording of 7 different depth images at 10 µm for biological tissues. Our method can potentially be useful for applications where high-speed recording of multiple en-face images is crucial.

Optical transfer function of time-gated coherent imaging in the presence of a scattering medium.

Optical imaging of objects embedded within scattering media such as biological tissues suffers from the loss of resolving power. In our previous work, we proposed an approach called collective accumulation of single scattering (CASS) microscopy that attenuates this detrimental effect of multiple light scattering by combining the time-gated detection and spatial input-output correlation. In the present work, we perform a rigorous theoretical analysis on the effect of multiple light scattering to the optical transfer function of CASS microscopy. In particular, the spatial frequency-dependent signal to noise ratio (SNR) is derived depending on the intensity ratio of the single- and multiple-scattered waves. This allows us to determine the depth-dependent resolving power. We conducted experiments using a Siemens star-like target having various spatial frequency components and supported the theoretical derived SNR spectra. Our study provides a theoretical framework for understanding the effect of multiple light scattering in high-resolution and deep-tissue optical imaging.