SARS-CoV-2 Omicron Variants Show Attenuated Neurovirulence Compared with the Wild-Type Strain in Elderly Human Brain Spheroids.

Infection with severe acute respiratory syndrome coronavirus 2 Omicron variants still causes neurological complications in elderly individuals. However, whether and how aging brains are affected by Omicron variants in terms of neuroinvasiveness and neurovirulence are unknown. Here, we utilize resected paracarcinoma brain tissue from elderly individuals to generate primary brain spheroids (BSs) for investigating the replication capability of live wild-type (WT) strain and Omicron (BA.1/BA.2), as well as the mechanisms underlying their neurobiological effects. We find that both WT and Omicron BA.1/BA.2 are able to enter BSs but weakly replicate. There is no difference between Omicron BA.1/BA.2 and WT strains in neurotropism in aging BSs. However, Omicron BA.1/BA.2 exhibits ameliorating neurological damage. Transcriptional profiling indicates that Omicron BA.1/BA.2 induces a lower neuroinflammatory response than WT strain in elderly BSs, suggesting a mechanistic explanation for their attenuated neuropathogenicity. Moreover, we find that both Omicron BA.1/BA.2 and WT strain infections disrupt neural network activity associated with neurodegenerative disorders by causing neuron degeneration and amyloid-ß deposition in elderly BSs. These results uncover Omicron-specific mechanisms and cellular immune responses associated with severe acute respiratory syndrome coronavirus 2-induced neurological complications.

T-bet deficiency and Hic1 induction override TGF-ß-dependency in the formation of CD103+ intestine-resident memory CD8+ T cells.

Transforming growth factor ß (TGF-ß) represents a well-established signal required for tissue-resident memory T cell (TRM) formation at intestinal surfaces, regulating the expression of a large collection of genes coordinately promoting intestinal TRM differentiation. The functional contribution from each TGF-ß-controlled transcription factor is not entirely known. Here, we find that TGF-ß-induced T-bet downregulation and Hic1 induction represent two critical events during intestinal TRM differentiation. Importantly, T-bet deficiency significantly rescues intestinal TRM formation in the absence of the TGF-ß receptor. Hic1 induction further strengthens TRM maturation in the absence of TGF-ß and T-bet. Our results reveal that provision of certain TGF-ß-induced molecular events can partially replace TGF-ß signaling to promote the establishment of intestinal TRMs, which allows the functional dissection of TGF-ß-induced transcriptional targets and molecular mechanisms for TRM differentiation.

Distinct roles of TREM2 in central nervous system cancers and peripheral cancers.

Glioblastomas (GBM) are incurable central nervous system (CNS) cancers characterized by substantial myeloid cell infiltration. Whether myeloid cell-directed therapeutic targets identified in peripheral non-CNS cancers are applicable to GBM requires further study. Here, we identify that the critical immunosuppressive target in peripheral cancers, triggering receptor expressed on myeloid cells-2 (TREM2), is immunoprotective in GBM. Genetic or pharmacological TREM2 deficiency promotes GBM progression in vivo. Single-cell and spatial sequencing reveals downregulated TREM2 in GBM-infiltrated myeloid cells. TREM2 negatively correlates with immunosuppressive myeloid and T cell exhaustion signatures in GBM. We further demonstrate that during GBM progression, CNS-enriched sphingolipids bind TREM2 on myeloid cells and elicit antitumor responses. Clinically, high TREM2 expression in myeloid cells correlates with better survival in GBM. Adeno-associated virus-mediated TREM2 overexpression impedes GBM progression and synergizes with anti-PD-1 therapy. Our results reveal distinct functions of TREM2 in CNS cancers and support organ-specific myeloid cell remodeling in cancer immunotherapy.

IFI35 regulates non-canonical NF-κB signaling to maintain glioblastoma stem cells and recruit tumor-associated macrophages.

Glioblastoma (GBM) is the most aggressive malignant primary brain tumor characterized by a highly heterogeneous and immunosuppressive tumor microenvironment (TME). The symbiotic interactions between glioblastoma stem cells (GSCs) and tumor-associated macrophages (TAM) in the TME are critical for tumor progression. Here, we identified that IFI35, a transcriptional regulatory factor, plays both cell-intrinsic and cell-extrinsic roles in maintaining GSCs and the immunosuppressive TME. IFI35 induced non-canonical NF-kB signaling through proteasomal processing of p105 to the DNA-binding transcription factor p50, which heterodimerizes with RELB (RELB/p50), and activated cell chemotaxis in a cell-autonomous manner. Further, IFI35 induced recruitment and maintenance of M2-like TAMs in TME in a paracrine manner. Targeting IFI35 effectively suppressed in vivo tumor growth and prolonged survival of orthotopic xenograft-bearing mice. Collectively, these findings reveal the tumor-promoting functions of IFI35 and suggest that targeting IFI35 or its downstream effectors may provide effective approaches to improve GBM treatment.

Efficacy and safety of tislelizumab plus lenvatinib as first-line treatment in patients with unresectable hepatocellular carcinoma: a multicenter, single-arm, phase 2 trial.

BACKGROUND: Lenvatinib is widely used in treatment of unresectable hepatocellular carcinoma (uHCC), but the benefit of its combination with immunotherapy needs to be verified. This study evaluated the efficacy and safety of tislelizumab plus lenvatinib in systemic treatment-naïve patients with uHCC. METHODS: In this multicenter, single-arm, phase 2 study, systemic treatment-naïve patients with uHCC received tislelizumab 200 mg every three weeks plus lenvatinib (bodyweight ≥ 60 kg: 12 mg; < 60 kg: 8 mg; once daily). Dose-limiting toxicities (DLTs) were evaluated in safety run-in phase to determine whether to enter the expansion phase. The primary endpoint was objective response rate (ORR) assessed by independent review committee (IRC) per Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST v1.1). Based on Simon's two-stage design, > 6 responders were needed in stage 1 (n = 30) to continue the study, and ≥ 18 responders were needed by the end of stage 2 (n = 60) to demonstrate statistical superiority to a historical control of lenvatinib monotherapy. RESULTS: Sixty-four patients were enrolled. No DLTs were reported. The study achieved statistical superiority (p = 0.0003) with 23 responders assessed by IRC per RECIST v1.1 in the first 60 patients of the efficacy evaluable analysis set (n = 62). After a median follow-up of 15.7 months, confirmed ORR and disease control rate were 38.7% (24/62, 95% confidence interval [CI], 26.6-51.9) and 90.3% (56/62, 95% CI, 80.1-96.4), respectively. Median progression-free survival was 8.2 months (95% CI, 6.8-not evaluable). Overall survival rate at 12 months was 88.6% (95% CI, 77.7-94.4). Grade ≥ 3 treatment-related adverse events occurred in 18 (28.1%) patients. CONCLUSIONS: Tislelizumab plus lenvatinib demonstrated promising antitumor activity with favourable tolerability as first-line therapy for patients with uHCC. TRIAL REGISTRATION: ClinicalTrials.gov (NCT04401800).

A comparative study of diffusion kurtosis imaging and diffusion tensor imaging in detecting corticospinal tract impairment in diffuse glioma patients.

PURPOSE: This study aimed to investigate the diagnostic performance of diffusion kurtosis imaging (DKI) and diffusion tensor imaging (DTI) in identifying aberrations in the corticospinal tract (CST), whilst elucidating the relationship between abnormalities of CST and patients' motor function. METHODS: Altogether 21 patients with WHO grade II or grade IV glioma were enrolled and divided into Group 1 and Group 2, according to the presence or absence of preoperative paralysis. DKI and DTI metrics were generated and projected onto the CST. Histograms of the CST along x, y, and z axes were developed based on DKI and DTI metrics, and compared subsequently to determine regions of aberrations on the fibers. The receiver operating characteristic curve was performed to investigate the diagnostic efficacy of DKI and DTI metrics. RESULTS: In Group 1, a significantly lower fractional anisotropy, radial kurtosis and mean kurtosis, and a higher mean diffusivity were found in the ipsilateral CST as compared to the contralateral CST. Significantly higher relative axial diffusivity, relative radial diffusivity, and relative mean diffusivity (rMD) were found in Group 1, as compared to Group 2. The relative volume of ipsilateral CST abnormalities higher than the maximum value of mean kurtosis combined with rMD exhibited the best diagnostic performance in distinguishing dysfunction of CST with an AUC of 0.93. CONCLUSION: DKI is sensitive in detecting subtle changes of CST distal from the tumor. The combination of DKI and DTI is feasible for evaluating the impairment of the CST.

Threonine fuels glioblastoma through YRDC-mediated codon-biased translational reprogramming.

Cancers commonly reprogram translation and metabolism, but little is known about how these two features coordinate in cancer stem cells. Here we show that glioblastoma stem cells (GSCs) display elevated protein translation. To dissect underlying mechanisms, we performed a CRISPR screen and identified YRDC as the top essential transfer RNA (tRNA) modification enzyme in GSCs. YRDC catalyzes the formation of N6-threonylcarbamoyladenosine (t6A) on ANN-decoding tRNA species (A denotes adenosine, and N denotes any nucleotide). Targeting YRDC reduced t6A formation, suppressed global translation and inhibited tumor growth both in vitro and in vivo. Threonine is an essential substrate of YRDC. Threonine accumulated in GSCs, which facilitated t6A formation through YRDC and shifted the proteome to support mitosis-related genes with ANN codon bias. Dietary threonine restriction (TR) reduced tumor t6A formation, slowed xenograft growth and augmented anti-tumor efficacy of chemotherapy and anti-mitotic therapy, providing a molecular basis for a dietary intervention in cancer treatment.

Application of a Novel Miniaturized Histopathologic Microscope for Ex Vivo Identifying Cerebral Glioma Margins Rapidly During Surgery: A Parallel Control Study.

PURPOSE: The purpose of our study is to assess the clinical performance of the DiveScope, a novel handheld histopathologic microscope in rapidly differentiating glioma from normal brain tissue during neurosurgery. METHODS: Thirty-two ex vivo specimens from 18 patients were included in the present study. The excised suspicious tissue was sequentially stained with sodium fluorescein and methylene blue and scanned with DiveScope during surgery. The adjacent tissue was sent to the department of pathology for frozen section examination. They would eventually be sent to the pathology department later for hematoxylin and eosin staining for final confirmation. The positive likelihood ratio, negative likelihood ratio, sensitivity, specificity, and area under the curve of the device were calculated. In addition, the difference in time usage between DiveScope and frozen sections was compared for the initial judgment. RESULTS: The sensitivity and specificity of the DiveScope after analyzing hematoxylin and eosin -staining sections, were 88.29% and 100%, respectively. In contrast, the sensitivity and specificity of the frozen sections histopathology were 100% and 75%, respectively. The area under the curve of the DiveScope and the frozen sections histopathology was not significant ( P =0.578). Concerning time usage, DiveScope is significantly much faster than the frozen sections histopathology no matter the size of tissue. CONCLUSION: Compared with traditional pathological frozen sections, DiveScope was faster and displayed an equal accuracy for judging tumor margins intraoperatively.

Epithelial organoid supports resident memory CD8 T cell differentiation.

Resident Memory T cells (TRM) play a vital role in regional immune defense in barrier organs. Although laboratory rodents have been extensively used to study fundamental TRM biology, poor isolation efficiency, sampling bias and low cell survival rates have limited our ability to conduct TRM-focused high-throughput assays. Here, we engineered a murine vaginal epithelial organoid (VEO)-CD8 T cell co-culture system that supports CD8 TRM differentiation in vitro. The three-dimensional VEOs established from murine adult stem cells resembled stratified squamous vaginal epithelium and induced gradual differentiation of activated CD8 T cells into epithelial TRM. These in vitro generated TRM were phenotypically and transcriptionally similar to in vivo TRM, and key tissue residency features were reinforced with a second cognate-antigen exposure during co-culture. TRM differentiation was not affected even when VEOs and CD8 T cells were separated by a semipermeable barrier, indicating soluble factors' involvement. Pharmacological and genetic approaches showed that TGF-ß signaling played a crucial role in their differentiation. We found that the VEOs in our model remained susceptible to viral infections and the CD8 T cells were amenable to genetic manipulation; both of which will allow detailed interrogation of antiviral CD8 T cell biology in a reductionist setting. In summary, we established a robust model which captures bonafide TRM differentiation that is scalable, open to iterative sampling, and can be subjected to high throughput assays that will rapidly add to our understanding of TRM.

Choroid plexus mast cells drive tumor-associated hydrocephalus.

Tumor-associated hydrocephalus (TAH) is a common and lethal complication of brain metastases. Although other factors beyond mechanical obstructions have been suggested, the exact mechanisms are unknown. Using single-nucleus RNA sequencing and spatial transcriptomics, we find that a distinct population of mast cells locate in the choroid plexus and dramatically increase during TAH. Genetic fate tracing and intracranial mast-cell-specific tryptase knockout showed that choroid plexus mast cells (CPMCs) disrupt cilia of choroid plexus epithelia via the tryptase-PAR2-FoxJ1 pathway and consequently increase cerebrospinal fluid production. Mast cells are also found in the human choroid plexus. Levels of tryptase in cerebrospinal fluid are closely associated with clinical severity of TAH. BMS-262084, an inhibitor of tryptase, can cross the blood-brain barrier, inhibit TAH in vivo, and alleviate mast-cell-induced damage of epithelial cilia in a human pluripotent stem-cell-derived choroid plexus organoid model. Collectively, we uncover the function of CPMCs and provide an attractive therapy for TAH.

Fine scale hippocampus morphology variation cross 552 healthy subjects from age 20 to 80.

The cerebral cortex varies over the course of a person's life span: at birth, the surface is smooth, before becoming more bumpy (deeper sulci and thicker gyri) in middle age, and thinner in senior years. In this work, a similar phenomenon was observed on the hippocampus. It was previously believed the fine-scale morphology of the hippocampus could only be extracted only with high field scanners (7T, 9.4T); however, recent studies show that regular 3T MR scanners can be sufficient for this purpose. This finding opens the door for the study of fine hippocampal morphometry for a large amount of clinical data. In particular, a characteristic bumpy and subtle feature on the inferior aspect of the hippocampus, which we refer to as hippocampal dentation, presents a dramatic degree of variability between individuals from very smooth to highly dentated. In this report, we propose a combined method joining deep learning and sub-pixel level set evolution to efficiently obtain fine-scale hippocampal segmentation on 552 healthy subjects. Through non-linear dentation extraction and fitting, we reveal that the bumpiness of the inferior surface of the human hippocampus has a clear temporal trend. It is bumpiest between 40 and 50 years old. This observation should be aligned with neurodevelopmental and aging stages.

The spatiotemporal heterogeneity of the biophysical microenvironment during hematopoietic stem cell development: from embryo to adult.

Hematopoietic stem cells (HSCs) with the ability to self-renew and differentiate are responsible for maintaining the supply of all types of blood cells. The complex and delicate microenvironment surrounding HSCs is called the HSC niche and can provide physical, chemical, and biological stimuli to regulate the survival, maintenance, proliferation, and differentiation of HSCs. Currently, the exploration of the biophysical regulation of HSCs remains in its infancy. There is evidence that HSCs are susceptible to biophysical stimuli, suggesting that the construction of engineered niche biophysical microenvironments is a promising way to regulate the fate of HSCs in vitro and ultimately contribute to clinical applications. In this review, we introduced the spatiotemporal heterogeneous biophysical microenvironment during HSC development, homeostasis, and malignancy. Furthermore, we illustrated how these biophysical cues contribute to HSC behaviors, as well as the possible mechanotransduction mechanisms from the extracellular microenvironment into cells. Comprehending the important functions of these biophysical regulatory factors will provide novel approaches to resolve clinical problems.

FOXM1 maintains fatty acid homoeostasis through the SET7-H3K4me1-FASN axis.

Reprogramming of metabolic genes and subsequent alterations in metabolic phenotypes occur widely in malignant tumours, including glioblastoma (GBM). FOXM1 is a potent transcription factor that plays an oncogenic role by regulating the expression of many genes. As a SET domain containing protein, SET7 is a protein lysine methyltransferase which monomethylates histone proteins and other proteins. The epigenetic modification of histones regulates gene expressions by epigenetically modifying promoters of DNAs and inter vening in tumor development. Activation of FASN increased de novo fatty acid (FA) synthesis, a hallmark of cancer cells. Here, we report that FOXM1 may directly promote the transcription of SET7 and activate SET7-H3K4me1-FASN axis, which results in the maintenance of de novo FA synthesis.

Circular RNA encoded MET variant promotes glioblastoma tumorigenesis.

Activated by its single ligand, hepatocyte growth factor (HGF), the receptor tyrosine kinase MET is pivotal in promoting glioblastoma (GBM) stem cell self-renewal, invasiveness and tumorigenicity. Nevertheless, HGF/MET-targeted therapy has shown limited clinical benefits in GBM patients, suggesting hidden mechanisms of MET signalling in GBM. Here, we show that circular MET RNA (circMET) encodes a 404-amino-acid MET variant (MET404) facilitated by the N6-methyladenosine (m6A) reader YTHDF2. Genetic ablation of circMET inhibits MET404 expression in mice and attenuates MET signalling. Conversely, MET404 knock-in (KI) plus P53 knock-out (KO) in mouse astrocytes initiates GBM tumorigenesis and shortens the overall survival. MET404 directly interacts with the MET ß subunit and forms a constitutively activated MET receptor whose activity does not require HGF stimulation. High MET404 expression predicts poor prognosis in GBM patients, indicating its clinical relevance. Targeting MET404 through a neutralizing antibody or genetic ablation reduces GBM tumorigenicity in vitro and in vivo, and combinatorial benefits are obtained with the addition of a traditional MET inhibitor. Overall, we identify a MET variant that promotes GBM tumorigenicity, offering a potential therapeutic strategy for GBM patients, especially those with MET hyperactivation.

Dual Role of CXCL8 in Maintaining the Mesenchymal State of Glioblastoma Stem Cells and M2-Like Tumor-Associated Macrophages.

PURPOSE: The dynamic interplay between glioblastoma stem cells (GSC) and tumor-associated macrophages (TAM) sculpts the tumor immune microenvironment (TIME) and promotes malignant progression of glioblastoma (GBM). However, the mechanisms underlying this interaction are still incompletely understood. Here, we investigate the role of CXCL8 in the maintenance of the mesenchymal state of GSC populations and reprogramming the TIME to an immunosuppressive state. EXPERIMENTAL DESIGN: We performed an integrative multi-omics analyses of RNA sequencing, GBM mRNA expression datasets, immune signatures, and epigenetic profiling to define the specific genes expressed in the mesenchymal GSC subsets. We then used patient-derived GSCs and a xenograft murine model to investigate the mechanisms of tumor-intrinsic and extrinsic factor to maintain the mesenchymal state of GSCs and induce TAM polarization. RESULTS: We identified that CXCL8 was preferentially expressed and secreted by mesenchymal GSCs and activated PI3K/AKT and NF-κB signaling to maintain GSC proliferation, survival, and self-renewal through a cell-intrinsic mechanism. CXCL8 induced signaling through a CXCR2-JAK2/STAT3 axis in TAMs, which supported an M2-like TAM phenotype through a paracrine, cell-extrinsic pathway. Genetic- and small molecule-based inhibition of these dual complementary signaling cascades in GSCs and TAMs suppressed GBM tumor growth and prolonged survival of orthotopic xenograft-bearing mice. CONCLUSIONS: CXCL8 plays critical roles in maintaining the mesenchymal state of GSCs and M2-like TAM polarization in GBM, highlighting an interplay between cell-autonomous and cell-extrinsic mechanisms. Targeting CXCL8 and its downstream effectors may effectively improve GBM treatment.

Upstream open reading frame-encoded MP31 disrupts the mitochondrial quality control process and inhibits tumorigenesis in glioblastoma.

BACKGROUND: Mitochondrial hyperpolarization achieved by the elevation of mitochondrial quality control (MQC) activity is a hallmark of glioblastoma (GBM). Therefore, targeting the MQC process to disrupt mitochondrial homeostasis should be a promising approach for GBM therapy. METHODS: We used 2-photon fluorescence microscopy, Fluorescence-Activated Cell Sorting, and confocal microscopy with specific fluorescent dyes to detect the mitochondrial membrane potential (MMP) and mitochondrial structures. Mitophagic flux was measured with mKeima. RESULTS: MP31, a phosphatase and tensin homolog (PTEN) uORF-translated and mitochondria-localized micropeptide, disrupted the MQC process and inhibited GBM tumorigenesis. Re-expression of MP31 in patient-derived GBM cells induced MMP loss to trigger mitochondrial fission but blocked mitophagic flux, leading to the accumulation of damaged mitochondria in cells, followed by reactive oxygen species production and DNA damage. Mechanistically, MP31 inhibited lysosome function and blocked lysosome fusion with mitophagosomes by competing with V-ATPase A1 for lactate dehydrogenase B (LDHB) binding to induce lysosomal alkalinization. Furthermore, MP31 enhanced the sensitivity of GBM cells to TMZ by suppressing protective mitophay in vitro and in vivo, but showed no side effects on normal human astrocytes or microglia cells (MG). CONCLUSIONS: MP31 disrupts cancerous mitochondrial homeostasis and sensitizes GBM cells to current chemotherapy, without inducing toxicity in normal human astrocytes and MG. MP31 is a promising candidate for GBM treatment.

Induction of Transmucosal Protection by Oral Vaccination with an Attenuated Chlamydia.

Chlamydia muridarum has been used to study chlamydial pathogenesis because it induces mice to develop hydrosalpinx, a pathology observed in C. trachomatis-infected women. We identified a C. muridarum mutant that is no longer able to induce hydrosalpinx. In the current study, we evaluated the mutant as an attenuated vaccine. Following an intravaginal immunization with the mutant, mice were protected from hydrosalpinx induced by wild-type C. muridarum. However, the mutant itself productively colonized the mouse genital tract and produced infectious organisms in vaginal swabs. Nevertheless, the mutant failed to produce infectious shedding in the rectal swabs following an oral inoculation. Importantly, mice orally inoculated with the mutant mounted transmucosal immunity against challenge infection of wild-type C. muridarum in the genital tract. The protection was detected as early as day 3 following the genital challenge infection and the orally immunized mice were protected from any significant pathology in the upper genital tract. However, the same orally immunized mice failed to prevent the colonization of wild-type C. muridarum in the gastrointestinal tract. The transmucosal immunity induced by the oral mutant was further validated in the airway. The orally vaccinated mice were protected from both lung infection and systemic toxicity caused by intranasally inoculated wild-type C. muridarum although the same mice still permitted the gastrointestinal colonization by the wild-type C. muridarum. These observations suggest that the mutant C. muridarum may be developed into an intracellular oral vaccine vector (or IntrOv) for selectively inducing transmucosal immunity in extra-gut tissues.

Depleting CD103+ resident memory T cells in vivo reveals immunostimulatory functions in oral mucosa.

The oral mucosa is a frontline for microbial exposure and juxtaposes several unique tissues and mechanical structures. Based on parabiotic surgery of mice receiving systemic viral infections or co-housing with microbially diverse pet shop mice, we report that the oral mucosa harbors CD8+ CD103+ resident memory T cells (TRM), which locally survey tissues without recirculating. Oral antigen re-encounter during the effector phase of immune responses potentiated TRM establishment within tongue, gums, palate, and cheek. Upon reactivation, oral TRM triggered changes in somatosensory and innate immune gene expression. We developed in vivo methods for depleting CD103+ TRM while sparing CD103neg TRM and recirculating cells. This revealed that CD103+ TRM were responsible for inducing local gene expression changes. Oral TRM putatively protected against local viral infection. This study provides methods for generating, assessing, and in vivo depleting oral TRM, documents their distribution throughout the oral mucosa, and provides evidence that TRM confer protection and trigger responses in oral physiology and innate immunity.