Angiopoietin 1 and integrin beta 1b are vital for zebrafish brain development.

Introduction: Angiopoietin 1 (angpt1) is essential for angiogenesis. However, its role in neurogenesis is largely undiscovered. This study aimed to identify the role of angpt1 in brain development, the mode of action of angpt1, and its prime targets in the zebrafish brain. Methods: We investigated the effects of embryonic brain angiogenesis and neural development using qPCR, in situ hybridization, microangiography, retrograde labeling, and immunostaining in the angpt1sa14264, itgb1bmi371, tekhu1667 mutant fish and transgenic overexpression of angpt1 in the zebrafish larval brains. Results: We showed the co-localization of angpt1 with notch, delta, and nestin in the proliferation zone in the larval brain. Additionally, lack of angpt1 was associated with downregulation of TEK tyrosine kinase, endothelial (tek), and several neurogenic factors despite upregulation of integrin beta 1b (itgb1b), angpt2a, vascular endothelial growth factor aa (vegfaa), and glial markers. We further demonstrated that the targeted angpt1sa14264 and itgb1bmi371 mutant fish showed severely irregular cerebrovascular development, aberrant hindbrain patterning, expansion of the radial glial progenitors, downregulation of cell proliferation, deficiencies of dopaminergic, histaminergic, and GABAergic populations in the caudal hypothalamus. In contrast to angpt1sa14264 and itgb1bmi371 mutants, the tekhu1667 mutant fish regularly grew with no apparent phenotypes. Notably, the neural-specific angpt1 overexpression driven by the elavl3 (HuC) promoter significantly increased cell proliferation and neuronal progenitor cells but decreased GABAergic neurons, and this neurogenic activity was independent of its typical receptor tek. Discussion: Our results prove that angpt1 and itgb1b, besides regulating vascular development, act as a neurogenic factor via notch and wnt signaling pathways in the neural proliferation zone in the developing brain, indicating a novel role of dual regulation of angpt1 in embryonic neurogenesis that supports the concept of angiopoietin-based therapeutics in neurological disorders.

Development of a microfluidic electroosmosis pump on a chip for steady and continuous fluid delivery.

Infusion therapy is the most common form of therapy used in health care. However, the existing infusion devices show higher flow discrepancies as flow rates decrease to a few nL min-1. As a result, dosing errors can contribute to the morbidity and mortality of patients. In the scope of project 18HLT08 MeDD II - Metrology for drug delivery, this investigation aims at the development of a silicon microchip flow pump capable of steadily and continuously dispense very low flow rates of a few nL min-1. The fabrication methodologies explored here use a combination of typical cleanroom micro/nanofabrication techniques and off-the-shelf equipment. Preliminary tests show flow rates as low as 45 nL min-1 can be obtained in this microfluidic electroosmotic pump. The experimental flow rates are in good agreement with results predicted by multiphysics simulation, with less than 8% deviation ratio. This cost effective electroosmotic micropump has the potential to act as a steady and continuous drug delivery system to neonatal patients as well as to organs on chip (OoC), determining the stability of the shear stress imposed on the cells or the right cell culture medium conditions.

Single-cell characterization of human GBM reveals regional differences in tumor-infiltrating leukocyte activation.

Glioblastoma (GBM) harbors a highly immunosuppressive tumor microenvironment (TME) which influences glioma growth. Major efforts have been undertaken to describe the TME on a single-cell level. However, human data on regional differences within the TME remain scarce. Here, we performed high-depth single-cell RNA sequencing (scRNAseq) on paired biopsies from the tumor center, peripheral infiltration zone and blood of five primary GBM patients. Through analysis of >45,000 cells, we revealed a regionally distinct transcription profile of microglia (MG) and monocyte-derived macrophages (MdMs) and an impaired activation signature in the tumor-peripheral cytotoxic-cell compartment. Comparing tumor-infiltrating CD8+ T cells with circulating cells identified CX3CR1high and CX3CR1int CD8+ T cells with effector and memory phenotype, respectively, enriched in blood but absent in the TME. Tumor CD8+ T cells displayed a tissue-resident memory phenotype with dysfunctional features. Our analysis provides a regionally resolved mapping of transcriptional states in GBM-associated leukocytes, serving as an additional asset in the effort towards novel therapeutic strategies to combat this fatal disease.

Targeting the Siglec-sialic acid axis promotes antitumor immune responses in preclinical models of glioblastoma.

Glioblastoma (GBM) is the most aggressive form of primary brain tumor, for which effective therapies are urgently needed. Cancer cells are capable of evading clearance by phagocytes such as microglia- and monocyte-derived cells through engaging tolerogenic programs. Here, we found that high expression of sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) correlates with reduced survival in patients with GBM. Using microglia- and monocyte-derived cell-specific knockouts of Siglec-E, the murine functional homolog of Siglec-9, together with single-cell RNA sequencing, we demonstrated that Siglec-E inhibits phagocytosis by these cells, thereby promoting immune evasion. Loss of Siglec-E on monocyte-derived cells further enhanced antigen cross-presentation and production of pro-inflammatory cytokines, which resulted in more efficient T cell priming. This bridging of innate and adaptive responses delayed tumor growth and resulted in prolonged survival in murine models of GBM. Furthermore, we showed the combinatorial activity of Siglec-E blockade and other immunotherapies demonstrating the potential for targeting Siglec-9 as a treatment for patients with GBM.

Immunotherapy of glioblastoma explants induces interferon-γ responses and spatial immune cell rearrangements in tumor center, but not periphery.

A patient-tailored, ex vivo drug response platform for glioblastoma (GBM) would facilitate therapy planning, provide insights into treatment-induced mechanisms in the immune tumor microenvironment (iTME), and enable the discovery of biomarkers of response. We cultured regionally annotated GBM explants in perfusion bioreactors to assess iTME responses to immunotherapy. Explants were treated with anti-CD47, anti-PD-1, or their combination, and analyzed by multiplexed microscopy [CO-Detection by indEXing (CODEX)], enabling the spatially resolved identification of >850,000 single cells, accompanied by explant secretome interrogation. Center and periphery explants differed in their cell type and soluble factor composition, and responses to immunotherapy. A subset of explants displayed increased interferon-γ levels, which correlated with shifts in immune cell composition within specified tissue compartments. Our study demonstrates that ex vivo immunotherapy of GBM explants enables an active antitumoral immune response within the tumor center and provides a framework for multidimensional personalized assessment of tumor response to immunotherapy.

Severe Neuro-COVID is associated with peripheral immune signatures, autoimmunity and neurodegeneration: a prospective cross-sectional study.

Growing evidence links COVID-19 with acute and long-term neurological dysfunction. However, the pathophysiological mechanisms resulting in central nervous system involvement remain unclear, posing both diagnostic and therapeutic challenges. Here we show outcomes of a cross-sectional clinical study (NCT04472013) including clinical and imaging data and corresponding multidimensional characterization of immune mediators in the cerebrospinal fluid (CSF) and plasma of patients belonging to different Neuro-COVID severity classes. The most prominent signs of severe Neuro-COVID are blood-brain barrier (BBB) impairment, elevated microglia activation markers and a polyclonal B cell response targeting self-antigens and non-self-antigens. COVID-19 patients show decreased regional brain volumes associating with specific CSF parameters, however, COVID-19 patients characterized by plasma cytokine storm are presenting with a non-inflammatory CSF profile. Post-acute COVID-19 syndrome strongly associates with a distinctive set of CSF and plasma mediators. Collectively, we identify several potentially actionable targets to prevent or intervene with the neurological consequences of SARS-CoV-2 infection.

Co-evolution of matrisome and adaptive adhesion dynamics drives ovarian cancer chemoresistance.

Due to its dynamic nature, the evolution of cancer cell-extracellular matrix (ECM) crosstalk, critically affecting metastasis and treatment resistance, remains elusive. Our results show that platinum-chemotherapy itself enhances resistance by progressively changing the cancer cell-intrinsic adhesion signaling and cell-surrounding ECM. Examining ovarian high-grade serous carcinoma (HGSC) transcriptome and histology, we describe the fibrotic ECM heterogeneity at primary tumors and distinct metastatic sites, prior and after chemotherapy. Using cell models from systematic ECM screen to collagen-based 2D and 3D cultures, we demonstrate that both specific ECM substrates and stiffness increase resistance to platinum-mediated, apoptosis-inducing DNA damage via FAK and ß1 integrin-pMLC-YAP signaling. Among such substrates around metastatic HGSCs, COL6 was upregulated by chemotherapy and enhanced the resistance of relapse, but not treatment-naïve, HGSC organoids. These results identify matrix adhesion as an adaptive response, driving HGSC aggressiveness via co-evolving ECM composition and sensing, suggesting stromal and tumor strategies for ECM pathway targeting.

Microglia-Centered Combinatorial Strategies Against Glioblastoma.

Tumor-associated microglia (MG) and macrophages (MΦ) are important components of the glioblastoma (GBM) immune tumor microenvironment (iTME). From the recent advances in understanding how MG and GBM cells evolve and interact during tumorigenesis, we emphasize the cooperation of MG with other immune cell types of the GBM-iTME, mainly MΦ and T cells. We provide a comprehensive overview of current immunotherapeutic clinical trials and approaches for the treatment of GBM, which in general, underestimate the counteracting contribution of immunosuppressive MG as a main factor for treatment failure. Furthermore, we summarize new developments and strategies in MG reprogramming/re-education in the GBM context, with a focus on ways to boost MG-mediated tumor cell phagocytosis and associated experimental models and methods. This ultimately converges in our proposal of novel combinatorial regimens that locally modulate MG as a central paradigm, and therefore may lead to additional, long-lasting, and effective tumoricidal responses.