A Self-Cascade Penetrating Brain Tumor Immunotherapy Mediated by Near-Infrared II Cell Membrane-Disrupting Nanoflakes via Detained Dendritic Cells.

Immunotherapy can potentially suppress the highly aggressive glioblastoma (GBM) by promoting T lymphocyte infiltration. Nevertheless, the immune privilege phenomenon, coupled with the generally low immunogenicity of vaccines, frequently hampers the presence of lymphocytes within brain tumors, particularly in brain tumors. In this study, the membrane-disrupted polymer-wrapped CuS nanoflakes that can penetrate delivery to deep brain tumors via releasing the cell-cell interactions, facilitating the near-infrared II (NIR II) photothermal therapy, and detaining dendritic cells for a self-cascading immunotherapy are developed. By convection-enhanced delivery, membrane-disrupted amphiphilic polymer micelles (poly(methoxypoly(ethylene glycol)-benzoic imine-octadecane, mPEG-b-C18) with CuS nanoflakes enhances tumor permeability and resides in deep brain tumors. Under low-power NIR II irradiation (0.8 W/cm2), the intense heat generated by well-distributed CuS nanoflakes actuates the thermolytic efficacy, facilitating cell apoptosis and the subsequent antigen release. Then, the positively charged polymer after hydrolysis of the benzoic-imine bond serves as an antigen depot, detaining autologous tumor-associated antigens and presenting them to dendritic cells, ensuring sustained immune stimulation. This self-cascading penetrative immunotherapy amplifies the immune response to postoperative brain tumors but also enhances survival outcomes through effective brain immunotherapy.

pH-Responsive ß-Glucans-Complexed mRNA in LNPs as an Oral Vaccine for Enhancing Cancer Immunotherapy.

mRNA vaccines for cancer immunotherapy are commonly delivered using lipid nanoparticles (LNPs), which, when administered intravenously, may accumulate in the liver, potentially limiting their therapeutic efficacy. To overcome this challenge, the study introduces an oral mRNA vaccine formulation tailored for efficient uptake by immune cells in the gastrointestinal (GI) tract, known for its high concentration of immune cells, including dendritic cells (DCs). This formulation comprises mRNA complexed with ß-glucans (ßGlus), a potential adjuvant for vaccines, encapsulated within LNPs (ßGlus/mRNA@LNPs). The ßGlus/mRNA complexes within the small compartments of LNPs demonstrate a distinctive ability to partially dissociate and reassociate, responding to pH changes, effectively shielding mRNA from degradation in the harsh GI environment. Upon oral administration to tumor-bearing mice, ßGlus/mRNA@LNPs are effectively taken up by intestinal DCs and local nonimmune cells, bypassing potential liver accumulation. This initiates antigen-specific immune responses through successful mRNA translation, followed by drainage into the mesenteric lymph nodes to stimulate T cells and trigger specific adaptive immune responses, ultimately enhancing antitumor effects. Importantly, the vaccine demonstrates safety, with no significant inflammatory reactions observed. In conclusion, the potential of oral ßGlus/mRNA@LNPs delivery presents a promising avenue in cancer immunotherapy, offering needle-free and user-friendly administration for widespread adoption and self-administration.

Revealing intact neuronal circuitry in centimeter-sized formalin-fixed paraffin-embedded brain.

Tissue-clearing and labeling techniques have revolutionized brain-wide imaging and analysis, yet their application to clinical formalin-fixed paraffin-embedded (FFPE) blocks remains challenging. We introduce HIF-Clear, a novel method for efficiently clearing and labeling centimeter-thick FFPE specimens using elevated temperature and concentrated detergents. HIF-Clear with multi-round immunolabeling reveals neuron circuitry regulating multiple neurotransmitter systems in a whole FFPE mouse brain and is able to be used as the evaluation of disease treatment efficiency. HIF-Clear also supports expansion microscopy and can be performed on a non-sectioned 15-year-old FFPE specimen, as well as a 3-month formalin-fixed mouse brain. Thus, HIF-Clear represents a feasible approach for researching archived FFPE specimens for future neuroscientific and 3D neuropathological analyses.

Asymmetric neurons are necessary for olfactory learning in the Drosophila brain.

Animals have complementary parallel memory systems that process signals from various sensory modalities. In the brain of the fruit fly Drosophila melanogaster, mushroom body (MB) circuitry is the primary associative neuropil, critical for all stages of olfactory memory. Here, our findings suggest that active signaling from specific asymmetric body (AB) neurons is also crucial for this process. These AB neurons respond to odors and electric shock separately and exhibit timing-sensitive neuronal activity in response to paired stimulation while leaving a decreased memory trace during retrieval. Our experiments also show that rutabaga-encoded adenylate cyclase, which mediates coincidence detection, is required for learning and short-term memory in both AB and MB. We observed additive effects when manipulating rutabaga co-expression in both structures. Together, these results implicate the AB in playing a critical role in associative olfactory learning and short-term memory.

Isotropic multi-scale neuronal reconstruction from high-ratio expansion microscopy with contrastive unsupervised deep generative models.

BACKGROUND AND OBJECTIVE: Current methods for imaging reconstruction from high-ratio expansion microscopy (ExM) data are limited by anisotropic optical resolution and the requirement for extensive manual annotation, creating a significant bottleneck in the analysis of complex neuronal structures. METHODS: We devised an innovative approach called the IsoGAN model, which utilizes a contrastive unsupervised generative adversarial network to sidestep these constraints. This model leverages multi-scale and isotropic neuron/protein/blood vessel morphology data to generate high-fidelity 3D representations of these structures, eliminating the need for rigorous manual annotation and supervision. The IsoGAN model introduces simplified structures with idealized morphologies as shape priors to ensure high consistency in the generated neuronal profiles across all points in space and scalability for arbitrarily large volumes. RESULTS: The efficacy of the IsoGAN model in accurately reconstructing complex neuronal structures was quantitatively assessed by examining the consistency between the axial and lateral views and identifying a reduction in erroneous imaging artifacts. The IsoGAN model accurately reconstructed complex neuronal structures, as evidenced by the consistency between the axial and lateral views and a reduction in erroneous imaging artifacts, and can be further applied to various biological samples. CONCLUSION: With its ability to generate detailed 3D neurons/proteins/blood vessel structures using significantly fewer axial view images, IsoGAN can streamline the process of imaging reconstruction while maintaining the necessary detail, offering a transformative solution to the existing limitations in high-throughput morphology analysis across different structures.

Ex Vivo Evaluation of Combination Immunotherapy Using Tumor-Microenvironment-on-Chip.

Combination immunotherapy has emerged as a promising strategy to address the challenges associated with immune checkpoint inhibitor (ICI) therapy in breast cancer. The efficacy of combination immunotherapy hinges upon the intricate and dynamic nature of the tumor microenvironment (TME), characterized by cellular heterogeneity and molecular gradients. However, current methodologies for drug screening often fail to accurately replicate these complex conditions, resulting in limited predictive capacity for treatment outcomes. Here, a tumor-microenvironment-on-chip (TMoC), integrating a circulation system and ex vivo tissue culture with physiological oxygen and nutrient gradients, is described. This platform enables spatial infiltration of cytotoxic CD8+ T cells and their targeted attack on the tumor, while preserving the high complexity and heterogeneity of the TME. The TMoC is employed to assess the synergistic effect of five targeted therapy drugs and five chemotherapy drugs in combination with immunotherapy, demonstrating strong concordance between chip and animal model responses. The TMoC holds significant potential for advancing drug development and guiding clinical decision-making, as it offers valuable insights into the complex dynamics of the TME.

In Situ Forming of Nitric Oxide and Electric Stimulus for Nerve Therapy by Wireless Chargeable Gold Yarn-Dynamos.

Endogenous signals, namely nitric oxide (NO) and electrons, play a crucial role in regulating cell fate as well as the vascular and neuronal systems. Unfortunately, utilizing NO and electrical stimulation in clinical settings can be challenging due to NO's short half-life and the invasive electrodes required for electrical stimulation. Additionally, there is a lack of tools to spatiotemporally control gas release and electrical stimulation. To address these issues, an "electromagnetic messenger" approach that employs on-demand high-frequency magnetic field (HFMF) to trigger NO release and electrical stimulation for restoring brain function in cases of traumatic brain injury is introduced. The system comprises a NO donor (poly(S-nitrosoglutathione), pGSNO)-conjugated on a gold yarn-dynamos (GY) and embedded in an implantable silk in a microneedle. When subjected to HFMF, conductive GY induces eddy currents that stimulate the release of NO from pGSNO. This process significantly enhances neural stem cell (NSC) synapses' differentiation and growth. The combined strategy of using NO and electrical stimulation to inhibit inflammation, angiogenesis, and neuronal interrogation in traumatic brain injury is demonstrated in vivo.

Single-Cell Transcriptomics Reveals Cellular Heterogeneity and Complex Cell-Cell Communication Networks in the Mouse Cornea.

Purpose: To generate a single-cell RNA-sequencing (scRNA-seq) map and construct cell-cell communication networks of mouse corneas. Methods: C57BL/6 mouse corneas were dissociated to single cells and subjected to scRNA-seq. Cell populations were clustered and annotated for bioinformatic analysis using the R package "Seurat." Differential expression patterns were validated and spatially mapped with whole-mount immunofluorescence staining. Global intercellular signaling networks were constructed using CellChat. Results: Unbiased clustering of scRNA-seq transcriptomes of 14,732 cells from 40 corneas revealed 17 cell clusters of six major cell types: nine epithelial cell, three keratocyte, two corneal endothelial cell, and one each of immune cell, vascular endothelial cell, and fibroblast clusters. The nine epithelial cell subtypes included quiescent limbal stem cells, transit-amplifying cells, and differentiated cells from corneas and two minor conjunctival epithelial clusters. CellChat analysis provided an atlas of the complex intercellular signaling communications among all cell types. Conclusions: We constructed a complete single-cell transcriptomic map and the complex signaling cross-talk among all cell types of the cornea, which can be used as a foundation atlas for further research on the cornea. This study also deepens the understanding of the cellular heterogeneity and heterotypic cell-cell interaction within corneas.

Electronic Preresonance Stimulated Raman Scattering Spectromicroscopy Using Multiple-Plate Continuum.

Stimulated Raman scattering (SRS) spectromicroscopy is a powerful technique that enables label-free detection of chemical bonds with high specificity. However, the low Raman cross section due to typical far-electronic resonance excitation seriously restricts the sensitivity and undermines its application to bio-imaging. To address this bottleneck, the electronic preresonance (EPR) SRS technique has been developed to enhance the Raman signals by shifting the excitation frequency toward the molecular absorption. A fundamental weakness of the previous demonstration is the lack of dual-wavelength tunability, making EPR-SRS only applicable to a limited number of species in the proof-of-concept experiment. Here, we demonstrate the EPR-SRS spectromicroscopy using a multiple-plate continuum (MPC) light source able to examine a single vibration mode with independently adjustable pump and Stokes wavelengths. In our experiments, the C═C vibration mode of Alexa 635 is interrogated by continuously scanning the pump-to-absorption frequency detuning throughout the entire EPR region enabled by MPC. The results exhibit 150-fold SRS signal enhancement and good agreement with the Albrecht A-term preresonance model. Signal enhancement is also observed in EPR-SRS images of the whole Drosophila brain stained with Alexa 635. With the improved sensitivity and potential to implement hyperspectral measurement, we envision that MPC-EPR-SRS spectromicroscopy can bring the Raman techniques closer to a routine in bio-imaging.

Macrophage-Hitchhiked Orally Administered ß-Glucans-Functionalized Nanoparticles as "Precision-Guided Stealth Missiles" for Targeted Pancreatic Cancer Therapy.

The prognosis in cases of pancreatic ductal adenocarcinoma (PDAC) with current treatment modalities is poor owing to the highly desmoplastic tumor microenvironment (TME). Herein, a ß-glucans-functionalized zinc-doxorubicin nanoparticle system (ßGlus-ZnD NPs) that can be orally administered, is developed for targeted PDAC therapy. Following oral administration in PDAC-bearing mice, ßGlus-ZnD NPs actively target/transpass microfold cells, overcome the intestinal epithelial barrier, and then undergo subsequent phagocytosis by endogenous macrophages (ßGlus-ZnD@Mϕ). As hitchhiking cellular vehicles, ßGlus-ZnD@Mϕ transits through the intestinal lymphatic system and enters systemic circulation, ultimately accumulating in the tumor tissue as a result of the tumor-homing and "stealth" properties that are conferred by endogenous Mϕ. Meanwhile, the Mϕ that hitchhikes ßGlus-ZnD NPs is activated to produce matrix metalloproteinases, destroying the desmoplastic stromal barrier, and differentiates toward the M1 -like phenotype, modulating the TME and recruiting effector T cells, ultimately inducing apoptosis of the tumor cells. The combination of ßGlus-ZnD@Mϕ and immune checkpoint blockade effectively inhibits the growth of the primary tumor and suppresses the development of metastasis. It thus represents an appealing approach to targeted PDAC therapy.

Towards stimulated Raman scattering spectro-microscopy across the entire Raman active region using a multiple-plate continuum.

Stimulated Raman scattering (SRS) has attracted increasing attention in bio-imaging because of the ability toward background-free molecular-specific acquisitions without fluorescence labeling. Nevertheless, the corresponding sensitivity and specificity remain far behind those of fluorescence techniques. Here, we demonstrate SRS spectro-microscopy driven by a multiple-plate continuum (MPC), whose octave-spanning bandwidth (600-1300 nm) and high spectral energy density (∼1 nJ/cm-1) enable spectroscopic interrogation across the entire Raman active region (0-4000 cm-1), SRS imaging of a Drosophila brain, and electronic pre-resonance (EPR) detection of a fluorescent dye. We envision that utilizing MPC light source will substantially enhance the sensitivity and specificity of SRS by implementing EPR mode and spectral multiplexing via accessing three or more coherent wavelengths.

Wireless charging-mediated angiogenesis and nerve repair by adaptable microporous hydrogels from conductive building blocks.

Traumatic brain injury causes inflammation and glial scarring that impede brain tissue repair, so stimulating angiogenesis and recovery of brain function remain challenging. Here we present an adaptable conductive microporous hydrogel consisting of gold nanoyarn balls-coated injectable building blocks possessing interconnected pores to improve angiogenesis and recovery of brain function in traumatic brain injury. We show that following minimally invasive implantation, the adaptable hydrogel is able to fill defects with complex shapes and regulate the traumatic brain injury environment in a mouse model. We find that placement of this injectable hydrogel at peri-trauma regions enhances mature brain-derived neurotrophic factor by 180% and improves angiogenesis by 250% in vivo within 2 weeks after electromagnetized stimulation, and that these effects facilitate neuron survival and motor function recovery by 50%. We use blood oxygenation level-dependent functional neuroimaging to reveal the successful restoration of functional brain connectivity in the corticostriatal and corticolimbic circuits.

Rabies Virus Glycoprotein-Mediated Transportation and T Cell Infiltration to Brain Tumor by Magnetoelectric Gold Yarnballs.

T lymphocyte infiltration with immunotherapy potentially suppresses most devastating brain tumors. However, local immune privilege and tumor heterogeneity usually limit the penetration of immune cells and therapeutic agents into brain tumors, leading to tumor recurrence after treatment. Here, a rabies virus glycoprotein (RVG)-camouflaged gold yarnball (RVG@GY) that can boost the targeting efficiency at a brain tumor via dual hierarchy- and RVG-mediated spinal cord transportation, facilitating the decrease of tumor heterogeneity for T cell infiltration, is developed. Upon magnetoelectric irradiation, the electron current generated on the GYs activates the electrolytic penetration of palbociclib-loaded dendrimer (Den[Pb]) deep into tumors. In addition, the high-density GYs at brain tumors also induces the disruption of cell-cell interactions and T cell infiltration. The integration of the electrolytic effects and T cell infiltration promoted by drug-loaded RVG@GYs deep in the brain tumor elicits sufficient T cell numbers and effectively prolongs the survival rate of mice with orthotopic brain tumors.

Mild Chronic Colitis Triggers Parkinsonism in LRRK2 Mutant Mice Through Activating TNF-α Pathway.

BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) is a common risk gene for Parkinson's disease (PD) and inflammatory bowel disorders. However, the penetrance of the most prevalent LRRK2 mutation, G2019S, is <50%. Factors other than genetic mutations are needed in PD process. OBJECTIVES: To examine whether and how gut inflammation may act as an environmental trigger to neurodegeneration in PD. METHODS: A mild and chronic dextran sodium sulfate (DSS)-induced colitis mice model harboring LRRK2 G2019S mutation was established. The colitis severity, immune responses, locomotor function, dopaminergic neuron, and microglia integrity were compared between littermate controls, transgenic LRRK2 wild type (WT), and LRRK2 G2019S mice. RESULTS: The LRRK2 G2019S mice are more vulnerable to DSS-induced colitis than littermate controls or LRRK2 WT animals with increased intestinal expressions of pattern-recognition receptors, including toll-like receptors (TLRs), nuclear factor (NF)-κB activation, and pro-inflammatory cytokines secretion, especially tumor necrosis factor (TNF)-α. Notably, the colonic expression of α-synuclein was significantly increased in LRRK2 G2019S colitis mice. We subsequently observed more aggravated locomotor defect, microglia activation, and dopaminergic neuron loss in LRRK2 G2019S colitis mice than control animals. Treatment with anti-TNF-α monoclonal antibody, adalimumab, abrogated both gut and neuroinflammation, mitigated neurodegeneration, and improved locomotor function in LRRK2 G2019S colitis mice. Finally, we validated increased colonic expressions of LRRK2, TLRs, and NF-κB pathway proteins and elevated plasma TNF-α level in PD patients compared to controls, especially in those with LRRK2 risk variants. CONCLUSIONS: Our findings demonstrate that chronic colitis promotes parkinsonism in genetically susceptible mice and TNF-α plays a detrimental role in the gut-brain axis of PD. © 2021 International Parkinson and Movement Disorder Society.

Dihydroceramide desaturase promotes the formation of intraluminal vesicles and inhibits autophagy to increase exosome production.

Exosomes are important for cell-cell communication. Deficiencies in the human dihydroceramide desaturase gene, DEGS1, increase the dihydroceramide-to-ceramide ratio and cause hypomyelinating leukodystrophy. However, the disease mechanism remains unknown. Here, we developed an in vivo assay with spatially controlled expression of exosome markers in Drosophila eye imaginal discs and showed that the level and activity of the DEGS1 ortholog, Ifc, correlated with exosome production. Knocking out ifc decreased the density of the exosome precursor intraluminal vesicles (ILVs) in the multivesicular endosomes (MVEs) and reduced the number of exosomes released. While ifc overexpression and autophagy inhibition both enhanced exosome production, combining the two had no additive effect. Moreover, DEGS1 activity was sufficient to drive ILV formation in vitro. Together, DEGS1/Ifc controls the dihydroceramide-to-ceramide ratio and enhances exosome secretion by promoting ILV formation and preventing the autophagic degradation of MVEs. These findings provide a potential cause for the neuropathy associated with DEGS1-deficient mutations.

Impact of a Desmoplastic Tumor Microenvironment for Colon Cancer Drug Sensitivity: A Study with 3D Chimeric Tumor Spheroids.

Three-dimensional (3D) spheroid culture provides opportunities to model tumor growth closer to its natural context. The collagen network in the extracellular matrix supports autonomic tumor cell proliferation, but its presence and role in tumor spheroids remain unclear. In this research, we developed an in vitro 3D co-culture model in a microwell 3D (µ-well 3D) cell-culture array platform to mimic the tumor microenvironment (TME). The modular setup is used to characterize the paracrine signaling molecules and the role of the intraspheroidal collagen network in cancer drug resistance. The µ-well 3D platform is made up of poly(dimethylsiloxane) that contains 630 round wells for individual spheroid growth. Inside each well, the growth surface measured 500 µm in diameter and was functionalized with the amphiphilic copolymer. HCT-8 colon cancer cells and/or NIH3T3 fibroblasts were seeded in each well and incubated for up to 9 days for TME studies. It was observed that NIH3T3 cells promoted the kinetics of tumor organoid formation. The two types of cells self-organized into core-shell chimeric tumor spheroids (CTSs) with fibroblasts confined to the shell and cancer cells localized to the core. Confocal microscopy analysis indicated that a type-I collagen network developed inside the CTS along with increased TGF-ß1 and α-SMA proteins. The results were correlated with a significantly increased stiffness in 3D co-cultured CTS up to 52 kPa as compared to two-dimensional (2D) co-culture. CTS was more resistant to 5-FU (IC50 = 14.0 ± 3.9 µM) and Regorafenib (IC50 = 49.8 ± 9.9 µM) compared to cells grown under the 2D condition 5-FU (IC50 = 12.2 ± 3.7 µM) and Regorafenib (IC50 = 5.9 ± 1.9 µM). Targeted collagen homeostasis with Sclerotiorin led to damaged collagen structure and disrupted the type-I collagen network within CTS. Such a treatment significantly sensitized collagen-supported CTS to 5-FU (IC50 = 4.4 ± 1.3 µM) and to Regorafenib (IC50 = 0.5 ± 0.2 µM). In summary, the efficient formation of colon cancer CTSs in a µ-well 3D culture platform allows exploration of the desmoplastic TME. The novel role of intratumor collagen quality as a drug sensitization target warrants further investigation.

Light field microscopy based on structured light illumination.

In this Letter, we present the modeling, design, and characterization of a light sheet-based structured light illumination (SLI) light field microscopy (LFM) system for fast 3D imaging, where a digital micromirror device is employed to rapidly generate designed sinusoidal patterns in the imaging field. Specifically, we sequentially obtain uniformly illuminated and structured light field images, followed by post-processing with a new, to the best of our knowledge, algorithm that combines the deconvolution and HiLo algorithms. This enables fast volumetric imaging with improved optical cross-sectioning capability at a speed of 50 volumes per second over an imaging field of 250×250×80µm3 in the x, y, and z axis, respectively. Mathematical models have been derived to explain the performance enhancement due to suppressed background noises. To verify the results, imaging experiments on fluorescence beads, fern spore, and Drosophila brain samples, have been performed. The results indicate that the light sheet-based SLI-LFM presents a fast 3D imaging solution with substantially improved optical cross-sectioning capability in comparison with a standard light sheet-based LFM. The new light field imaging method may find important applications in the field of biophotonics.

VCP maintains nuclear size by regulating the DNA damage-associated MDC1-p53-autophagy axis in Drosophila.

The maintenance of constant karyoplasmic ratios suggests that nuclear size has physiological significance. Nuclear size anomalies have been linked to malignant transformation, although the mechanism remains unclear. By expressing dominant-negative TER94 mutants in Drosophila photoreceptors, here we show disruption of VCP (valosin-containing protein, human TER94 ortholog), a ubiquitin-dependent segregase, causes progressive nuclear size increase. Loss of VCP function leads to accumulations of MDC1 (mediator of DNA damage checkpoint protein 1), connecting DNA damage or associated responses to enlarged nuclei. TER94 can interact with MDC1 and decreases MDC1 levels, suggesting that MDC1 is a VCP substrate. Our evidence indicates that MDC1 accumulation stabilizes p53A, leading to TER94K2A-associated nuclear size increase. Together with a previous report that p53A disrupts autophagic flux, we propose that the stabilization of p53A in TER94K2A-expressing cells likely hinders the removal of nuclear content, resulting in aberrant nuclear size increase.

5D superresolution imaging for a live cell nucleus.

With a spatial resolution breaking the diffraction limit of light, superresolution imaging allows the visualization of detailed structures of organelles such as mitochondria, cytoskeleton, nucleus, and so on. With multi-dimensional imaging (x, y, z, t, λ), namely, multi-color 3D live imaging enables us fully understand the function of the cell. It is necessary to analyze structural changes or molecular interactions across a large volume in 3D with different labelled targets. To achieve this goal, scientists recently have expanded the original 2D superresolution microscopic tools into 3D imaging techniques. In this review, we will discuss recent development in superresolution microscopy for live imaging with minimal phototoxicity. We will focus our discussion on the cell nucleus where the genetic materials are stored and processed. Machine learning algorism will be introduced to improve the axial resolution of superresolution imaging.