Novel insights into paclitaxel's role on tumor-associated macrophages in enhancing PD-1 blockade in breast cancer treatment.

BACKGROUND: Triple-negative breast cancer (TNBC) poses unique challenges due to its complex nature and the need for more effective treatments. Recent studies showed encouraging outcomes from combining paclitaxel (PTX) with programmed cell death protein-1 (PD-1) blockade in treating TNBC, although the exact mechanisms behind the improved results are unclear. METHODS: We employed an integrated approach, analyzing spatial transcriptomics and single-cell RNA sequencing data from TNBC patients to understand why the combination of PTX and PD-1 blockade showed better response in TNBC patients. We focused on toll-like receptor 4 (TLR4), a receptor of PTX, and its role in modulating the cross-presentation signaling pathways in tumor-associated macrophages (TAMs) within the tumor microenvironment. Leveraging insights obtained from patient-derived data, we conducted in vitro experiments using immunosuppressive bone marrow-derived macrophages (iBMDMs) to validate if PTX could augment the cross-presentation and phagocytosis activities. Subsequently, we extended our study to an in vivo murine model of TNBC to ascertain the effects of PTX on the cross-presentation capabilities of TAMs and its downstream impact on CD8+ T cell-mediated immune responses. RESULTS: Data analysis from TNBC patients revealed that the activation of TLR4 and cross-presentation signaling pathways are crucial for the antitumor efficacy of PTX. In vitro studies showed that PTX treatment enhances the cross-presentation ability of iBMDMs. In vivo experiments demonstrated that PTX activates TLR4-dependent cross-presentation in TAMs, improving CD8+ T cell-mediated antitumor responses. The efficacy of PTX in promoting antitumor immunity was elicited when combined with PD-1 blockade, suggesting a complementary interaction. CONCLUSIONS: This study reveals how PTX boosts the effectiveness of PD-1 inhibitors in treating TNBC. We found that PTX activates TLR4 signaling in TAMs. This activation enhances their ability to present antigens, thereby boosting CD8+ T cell antitumor responses. These findings not only shed light on PTX's immunomodulatory role in TNBC but also underscore the potential of targeting TAMs' antigen presentation capabilities in immunotherapy approaches.

Identification of an Antagonist Targeting G Protein and ß-Arrestin Signaling Pathways of 5-HT7R.

In consideration of the limited number of FDA-approved drugs for autism spectrum disorder (ASD), significant efforts have been devoted to identifying novel drug candidates. Among these, 5-HT7R modulators have garnered considerable attention due to their potential in alleviating autism-like behaviors in ASD animal models. In this study, we designed and synthesized biphenyl-3-ylmethylpyrrolidines 3 and biphenyl-3-yl-dihydroimidazoles 4 as 5-HT7R modulators. Through extensive biological tests of 3 and 4 in G protein and ß-arrestin signaling pathways of 5-HT7R, it was determined that 2-(2'-methoxy-[1,1'-biphenyl]-3-yl)-4,5-dihydro-1H-imidazole 4h acted as a 5-HT7R antagonist in both signaling pathways. In in vivo study with Shank3-/- transgenic (TG) mice, the self-grooming behavior test was performed with 4h, resulting in a significant reduction in the duration of self-grooming. In addition, an immunohistochemical experiment with 4h restored reduced neurogenesis in Shank3-/- TG mice, which is confirmed by the quantification of doublecortin (DCX) positive neurons, suggesting the promising therapeutic potential of 4h.

A neural probe for concurrent real-time measurement of multiple neurochemicals with electrophysiology in multiple brain regions in vivo.

Real-time monitoring of various neurochemicals with high spatial resolution in multiple brain regions in vivo can elucidate neural circuits related to various brain diseases. However, previous systems for monitoring neurochemicals have limitations in observing multiple neurochemicals without crosstalk in real time, and these methods cannot record electrical activity, which is essential for investigating neural circuits. Here, we present a real-time bimodal (RTBM) neural probe that uses monolithically integrated biosensors and multiple shanks to study the connectivity of neural circuits by measuring multiple neurochemicals and electrical neural activity in real time. Using the RTBM probe, we demonstrate concurrent measurements of four neurochemicals-glucose, lactate, choline, and glutamate without cross-talking each other-and electrical activity in real time in vivo. Additionally, we show the functional connectivity between the medial prefrontal cortex and mediodorsal thalamus through the simultaneous measurement of chemical and electrical signals. We expect that our device will contribute to not only elucidating the role of neurochemicals in neural circuits related to brain functions but also developing drugs for various brain diseases related to neurochemicals.

Neural probe system for behavioral neuropharmacology by bi-directional wireless drug delivery and electrophysiology in socially interacting mice.

Assessing the neurological and behavioral effects of drugs is important in developing pharmacological treatments, as well as understanding the mechanisms associated with neurological disorders. Herein, we present a miniaturized, wireless neural probe system with the capability of delivering drugs for the real-time investigation of the effects of the drugs on both behavioral and neural activities in socially interacting mice. We demonstrate wireless drug delivery and simultaneous monitoring of the resulting neural, behavioral changes, as well as the dose-dependent and repeatable responses to drugs. Furthermore, in pairs of mice, we use a food competition assay in which social interaction was modulated by the delivery of the drug, and the resulting changes in their neural activities are analyzed. During modulated food competition by drug injection, we observe changes in neural activity in mPFC region of a participating mouse over time. Our system may provide new opportunities for the development of studying the effects of drugs on behaviour and neural activity.

A Multimodal Multi-Shank Fluorescence Neural Probe for Cell-Type-Specific Electrophysiology in Multiple Regions across a Neural Circuit.

Cell-type-specific, activity-dependent electrophysiology can allow in-depth analysis of functional connectivity inside complex neural circuits composed of various cell types. To date, optics-based fluorescence recording devices enable monitoring cell-type-specific activities. However, the monitoring is typically limited to a single brain region, and the temporal resolution is significantly low. Herein, a multimodal multi-shank fluorescence neural probe that allows cell-type-specific electrophysiology from multiple deep-brain regions at a high spatiotemporal resolution is presented. A photodiode and an electrode-array pair are monolithically integrated on each tip of a minimal-form-factor silicon device. Both fluorescence and electrical signals are successfully measured simultaneously in GCaMP6f expressing mice, and the cell type from sorted neural spikes is identified. The probe's capability of combined electro-optical recordings for cell-type-specific electrophysiology at multiple brain regions within a neural circuit is demonstrated. The new experimental paradigm to enable the precise investigation of functional connectivity inside and across complex neural circuits composed of various cell types is expected.

Discovery of G Protein-Biased Antagonists against 5-HT7R.

5-HT7R belongs to a family of G protein-coupled receptors and is associated with a variety of physiological processes in the central nervous system via the activation of the neurotransmitter serotonin (5-HT). To develop selective and biased 5-HT7R ligands, we designed and synthesized a series of pyrazolyl-diazepanes 2 and pyrazolyl-piperazines 3, which were evaluated for binding affinities to 5-HTR subtypes and functional selectivity for G protein and ß-arrestin signaling pathways of 5-HT7R. Among them, 1-(3-(3-chlorophenyl)-1H-pyrazol-4-yl)-1,4-diazepane 2c showed the best binding affinity for 5-HT7R and selectivity over other 5-HTR subtypes. It was also revealed as a G protein-biased antagonist. The self-grooming behavior test was performed with 2c in vivo with Shank3-/- transgenic (TG) mice, wherein 2c significantly reduced self-grooming duration time to the level of wild-type mice. The results suggest that 5-HT7R could be a potential therapeutic target for treating autism spectrum disorder stereotypy.

Bimodal neural probe for highly co-localized chemical and electrical monitoring of neural activities in vivo.

Investigation of the chemical and electrical signals of cells in vivo is critical for studying functional connectivity and brain diseases. Most previous studies have observed either the electrical signals or the chemical signals of cells because recording electrical signals and neurochemicals are done by fundamentally different methods. Herein, we present a bimodal MEMS neural probe that is monolithically integrated with an array of microelectrodes for recording electrical activity, microfluidic channels for sampling extracellular fluid, and a microfluidic interface chip for multiple drug delivery and sample isolation from the localized region at the cellular level. In this work, we successfully demonstrated the functionality of our probe by monitoring and modulating bimodal (electrical and chemical) neural activities through the delivery of chemicals in a co-localized brain region in vivo. We expect our bimodal probe to provide opportunities for a variety of in-depth studies of brain functions as well as for the investigation of neural circuits related to brain diseases.

Statin-mediated inhibition of RAS prenylation activates ER stress to enhance the immunogenicity of KRAS mutant cancer.

BACKGROUND: Statins preferentially promote tumor-specific apoptosis by depleting isoprenoid such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate. However, statins have not yet been approved for clinical cancer treatment due, in part, to poor understanding of molecular determinants on statin sensitivity. Here, we investigated the potential of statins to elicit enhanced immunogenicity of KRAS-mutant (KRASmut) tumors. METHODS: The immunogenicity of treated cancer cells was determined by western blot, flow cytometry and confocal microscopy. The immunotherapeutic efficacy of mono or combination therapy using statin was assessed in KRASmut tumor models, including syngeneic colorectal cancer and genetically engineered lung and pancreatic tumors. Using NanoString analysis, we analyzed how statin influenced the gene signatures associated with the antigen presentation of dendritic cells in vivo and evaluated whether statin could induce CD8+ T-cell immunity. Multiplex immunohistochemistry was performed to better understand the complicated tumor-immune microenvironment. RESULTS: Statin-mediated inhibition of KRAS prenylation provoked severe endoplasmic reticulum (ER) stress by attenuating the anti-ER stress effect of KRAS mutation, thereby resulting in the immunogenic cell death (ICD) of KRASmut cancer cells. Moreover, statin-mediated ICD enhanced the cross-priming ability of dendritic cells, thereby provoking CD8+ T-cell immune responses against KRASmut tumors. Combination therapy using statin and oxaliplatin, an ICD inducer, significantly enhanced the immunogenicity of KRASmut tumors and promoted tumor-specific immunity in syngeneic and genetically engineered KRASmut tumor models. Along with immune-checkpoint inhibitors, the abovementioned combination therapy overcame resistance to PD-1 blockade therapies, improving the survival rate of KRASmut tumor models. CONCLUSIONS: Our findings suggest that KRAS mutation could be a molecular target for statins to elicit potent tumor-specific immunity.

Dysfunction of X-linked inhibitor of apoptosis protein (XIAP) triggers neuropathological processes via altered p53 activity in Huntington's disease.

Mitochondrial dysfunction is associated with neuronal damage in Huntington's disease (HD), but the precise mechanism of mitochondria-dependent pathogenesis is not understood yet. Herein, we found that colocalization of XIAP and p53 was prominent in the cytosolic compartments of normal subjects but reduced in HD patients and HD transgenic animal models. Overexpression of mutant Huntingtin (mHTT) reduced XIAP levels and elevated mitochondrial localization of p53 in striatal cells in vitro and in vivo. Interestingly, XIAP interacted directly with the C-terminal domain of p53 and decreased its stability via autophagy. Overexpression of XIAP prevented mitochondrially targeted-p53 (Mito-p53)-induced mitochondrial oxidative stress and striatal cell death, whereas, knockdown of XIAP exacerbated Mito-p53-induced neuronal damage in vitro. In vivo transduction of AAV-shRNA XIAP in the dorsal striatum induced rapid onset of disease and reduced the lifespan of HD transgenic (N171-82Q) mice compared to WT littermate mice. XIAP dysfunction led to ultrastructural changes of the mitochondrial cristae and nucleus morphology in striatal cells. Knockdown of XIAP exacerbated neuropathology and motor dysfunctions in N171-82Q mice. In contrast, XIAP overexpression improved neuropathology and motor behaviors in both AAV-mHTT-transduced mice and N171-82Q mice. Our data provides a molecular and pathological mechanism that deregulation of XIAP triggers mitochondria dysfunction and other neuropathological processes via the neurotoxic effect of p53 in HD. Together, the XIAP-p53 pathway is a novel pathological marker and can be a therapeutic target for improving the symptoms in HD.

Discovery of G Protein-Biased Ligands against 5-HT7R.

There has been significant attention concerning the biased agonism of G protein-coupled receptors (GPCRs), and it has resulted in various pharmacological benefits. 5-HT7R belongs to a GPCR, and it is a promising pharmaceutical target for the treatment of neurodevelopmental and neuropsychiatric disorders. Based on our previous research, we synthesized a series of 6-chloro-2'-methoxy biphenyl derivatives 1, 2, and 3 with a variety of amine scaffolds. These compounds were evaluated for their binding affinities to 5-HTR subtypes and their functional selectivity toward the Gs protein and the ß-arrestin signaling pathways of 5-HT7R. Among them, 2-(6-chloro-2'-methoxy-[1,1'-biphenyl]-3-yl)-N-ethylethan-1-amine, 2b, was found to be a G-protein-biased ligand of 5-HT7R. In an in vivo study with Shank3 transgenic mice, the self-grooming behavior test was performed with 2b, which increased the duration of self-grooming. The experiments further suggested that 5-HT7R is associated with autism spectrum disorders (ASDs) and could be a therapeutic target for the treatment of stereotypy in ASDs.

Xenogenization of tumor cells by fusogenic exosomes in tumor microenvironment ignites and propagates antitumor immunity.

Many cancer patients not responding to current immunotherapies fail to produce tumor-specific T cells for various reasons, such as a lack of recognition of cancer cells as foreign. Here, we suggest a previously unidentified method for xenogenizing (turning self to non-self) tumors by using fusogenic exosomes to introduce fusogenic viral antigens (VSV-G) onto the tumor cell surface. We found that xenogenized tumor cells were readily recognized and engulfed by dendritic cells; thereby, tumor antigens were efficiently presented to T lymphocytes. Moreover, exosome-VSV-G itself acts as a TLR4 agonist and stimulates the maturation of dendritic cells, leading to CD8+ T cell cross-priming. The administration of these exosomes in multiple tumor mouse models xenogenized tumor cells, resulting in tumor growth inhibition. The combinatorial treatment with anti-PD-L1 exhibited complete tumor regression (30%) and better long-term overall survival. These results suggest that tumor xenogenization by fusogenic exosomes provides a previously unidentified novel strategy for cancer immunotherapy.

Rhizolutin, a Novel 7/10/6-Tricyclic Dilactone, Dissociates Misfolded Protein Aggregates and Reduces Apoptosis/Inflammation Associated with Alzheimer's Disease.

Rhizolutin (1) was discovered as a natural product of ginseng-rhizospheric Streptomyces sp. WON17. Its structure features an unprecedented 7/10/6-tricyclic dilactone carbon skeleton composed of dimethylcyclodecatriene flanked by a 7-membered and a 6-membered lactone ring based on spectroscopic analysis. During an unbiased screening of natural product libraries, this novel compound was found to dissociate amyloid-ß (Aß) plaques and tau tangles, which are key pathological hallmarks of Alzheimer's disease (AD). Rhizolutin treatment of APP/PS1 double transgenic mice with AD significantly dissociated hippocampal plaques. In vitro, rhizolutin substantially decreased Aß-induced apoptosis and inflammation in neuronal and glial cells. Our findings introduce a unique chemical entity that targets Aß and tau concurrently by mimicking misfolded protein clearance mechanisms of immunotherapy, which is prominently investigated in clinical trials.

Multifunctional multi-shank neural probe for investigating and modulating long-range neural circuits in vivo.

Investigation and modulation of neural circuits in vivo at the cellular level are very important for studying functional connectivity in a brain. Recently, neural probes with stimulation capabilities have been introduced, and they provided an opportunity for studying neural activities at a specific region in the brain using various stimuli. However, previous methods have a limitation in dissecting long-range neural circuits due to inherent limitations on their designs. Moreover, the large size of the previously reported probes induces more significant tissue damage. Herein, we present a multifunctional multi-shank MEMS neural probe that is monolithically integrated with an optical waveguide for optical stimulation, microfluidic channels for drug delivery, and microelectrode arrays for recording neural signals from different regions at the cellular level. In this work, we successfully demonstrated the functionality of our probe by confirming and modulating the functional connectivity between the hippocampal CA3 and CA1 regions in vivo.

Combined Rho-kinase inhibition and immunogenic cell death triggers and propagates immunity against cancer.

Activation of T cell immune response is critical for the therapeutic efficacy of cancer immunotherapy. Current immunotherapies have shown remarkable clinical success against several cancers; however, significant responses remain restricted to a minority of patients. Here, we show a therapeutic strategy that combines enhancing the phagocytic activity of antigen-presenting cells with immunogenic cell death to trigger efficient antitumour immunity. Rho-kinase (ROCK) blockade increases cancer cell phagocytosis and induces antitumour immunity through enhancement of T cell priming by dendritic cells (DCs), leading to suppression of tumour growth in syngeneic tumour models. Combining ROCK blockade with immunogenic chemotherapy leads to increased DC maturation and synergistic CD8+ cytotoxic T cell priming and infiltration into tumours. This therapeutic strategy effectively suppresses tumour growth and improves overall survival in a genetic mouse mammary tumour virus/Neu tumour model. Collectively, these results suggest that boosting intrinsic cancer immunity using immunogenic killing and enhanced phagocytosis is a promising therapeutic strategy for cancer immunotherapy.

Four-week individual caging of male ICR mice alters body composition without change in body mass.

Understanding the physiological implications of caging conditions for mice is crucial in improving the replicability and reliability of animal research. Individual caging of mice is known to alter mouse psychology, such as triggering depression-like symptoms in mice, suggesting that caging conditions could have negative effects on mice. Therefore, we hypothesized that individual caging could affect the physical composition of outbred mice. To investigate this, dual X-ray absorptiometry (DXA) was used to compare the mass, bone mineral content (BMC), bone mineral density (BMD), lean tissue percentage and fat tissue percentage between group and individual caged mice. We also conducted open field test to compare mouse activities in different caging conditions. Our results showed significantly reduced BMD and lean tissue percentage and significantly increased fat tissue percentage in individually-caged male mice. Furthermore, there were no differences in body mass and activity between the grouped and individual mice, suggesting that these physical alterations were not induced by group-related activity. In this study, we conclude that individual caging could alter the body composition of mice without affecting external morphology.

Age-dependent inverse correlations in CSF and plasma amyloid-ß(1-42) concentrations prior to amyloid plaque deposition in the brain of 3xTg-AD mice.

Amyloid-ß (Aß) plays a critical role as a biomarker in Alzheimer's disease (AD) diagnosis. In addition to its diagnostic potential in the brain, recent studies have suggested that changes of Aß level in the plasma can possibly indicate AD onset. In this study, we found that plasma Aß(1-42) concentration increases with age, while the concentration of Aß(1-42) in the cerebrospinal fluid (CSF) decreases in APPswe, PS1M146V and TauP301L transgenic (3xTg-AD) mice, if measurements were made before formation of ThS-positive plaques in the brain. Our data suggests that there is an inverse correlations between the plasma and CSF Aß(1-42) levels until plaques form in transgenic mice's brains and that the plasma Aß concentration possesses the diagnostic potential as a biomarker for diagnosis of early AD stages.

Neural probes with multi-drug delivery capability.

Multi-functional neural probes are promising platforms to conduct efficient and effective in-depth studies of brain by recording neural signals as well as modulating the signals with various stimuli. Here we present a neural probe with an embedded microfluidic channel (chemtrode) with multi-drug delivery capability suitable for small animal experiments. We integrated a staggered herringbone mixer (SHM) in a 3-inlet microfluidic chip directly into our chemtrode. This chip, which also serves as a compact interface for the chemtrode, allows for efficient delivery of small volumes of multiple or concentration-modulated drugs via chaotic mixing. We demonstrated the successful infusion of combinatorial inputs of three chemicals with a low flow rate (170 nl min(-1)). By sequentially delivering red, green, and blue inks from each inlet and conducting visual inspections at the tip of the chemtrode, we measured a short residence time of 14 s which corresponds to a small swept volume of 66 nl. Finally, we demonstrated the potential of our proposed chemtrode as an enabling tool through extensive in vivo mice experiments. Through simultaneous infusions of a chemical (pilocarpine or tetrodotoxin (TTX) at inlet 1), a buffer solution (saline at inlet 2), and 4',6-diamidino-2-phenylindole (DAPI at inlet 3) locally into a mouse brain, we not only modulated the neural activities by varying the concentration of the chemical but also locally stained the cells at our target region (CA1 in hippocampus). More specifically, infusion of pilocarpine with a higher concentration resulted in an increase in neural activities while infusion of TTX with a higher concentration resulted in a distinctive reduction. For each chemical, we acquired multiple sets of data using only one mouse through a single implantation of the chemtrode. Our proposed chemtrode offers 1) multiplexed delivery of three drugs through a compact packaging with a small swept volume and 2) simultaneous recording to monitor near real-time effects on neural signals, which allows for more versatile in vivo experiments with a minimum number of animals to be sacrificed.

Taurine in drinking water recovers learning and memory in the adult APP/PS1 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a lethal progressive neurological disorder affecting the memory. Recently, US Food and Drug Administration mitigated the standard for drug approval, allowing symptomatic drugs that only improve cognitive deficits to be allowed to accelerate on to clinical trials. Our study focuses on taurine, an endogenous amino acid found in high concentrations in humans. It has demonstrated neuroprotective properties against many forms of dementia. In this study, we assessed cognitively enhancing property of taurine in transgenic mouse model of AD. We orally administered taurine via drinking water to adult APP/PS1 transgenic mouse model for 6 weeks. Taurine treatment rescued cognitive deficits in APP/PS1 mice up to the age-matching wild-type mice in Y-maze and passive avoidance tests without modifying the behaviours of cognitively normal mice. In the cortex of APP/PS1 mice, taurine slightly decreased insoluble fraction of Aß. While the exact mechanism of taurine in AD has not yet been ascertained, our results suggest that taurine can aid cognitive impairment and may inhibit Aß-related damages.

Upregulation of TrkB by forskolin facilitated survival of MSC and functional recovery of memory deficient model rats.

Mesenchymal stem cells (MSCs) are effective vectors in delivering a gene of interest into degenerating brain. In ex vivo gene therapy, viability of transplanted MSCs is correlated with the extent of functional recovery. It has been reported that BDNF facilitates survival of MSCs but dividing MSCs do not express the BDNF receptor, TrkB. In this study, we found that the expression of TrkB is upregulated in human MSCs by the addition of forskolin (Fsk), an activator of adenylyl cyclase. To increase survival rate of MSCs and their secretion of tropic factors that enhance regeneration of endogenous cells, we pre-exposed hMSCs with Fsk and transduced with BDNF-adenovirus before transplantation into the brain of memory deficient rats, a degenerating brain disease model induced by ibotenic acid injection. Viability of MSCs and expression of a GABA synthesizing enzyme were increased. The pre-treatment improved learning and memory, as detected by the behavioral tests including Y-maze task and passive avoidance test. These results suggest that TrkB expression of hMSCs elevates the neuronal regeneration and efficiency of BDNF delivery for treating degenerative neurological diseases accompanying memory loss.