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Chimeric antigen receptor (CAR) T-cell immunotherapies for solid tumors face critical challenges such as heterogeneous antigen expression. We characterized stage-specific embryonic antigen-4 (SSEA-4) cell-surface glycolipid as a target for CAR T-cell therapy. SSEA-4 is mainly expressed during embryogenesis but is also found in several cancer types making it an attractive tumor-associated antigen. Anti-SSEA-4 CAR-T cells were generated and assessed preclinically in vitro and in vivo for antitumor response and safety. SSEA-4 CAR-T cells effectively eliminated SSEA-4-positive cells in all the tested cancer cell lines, whereas SSEA-4-negative cells lines were not targeted. In vivo efficacy and safety studies using NSG mice and the high-grade serous ovarian cancer cell line OVCAR4 demonstrated a remarkable and specific antitumor response at all the CAR T-cell doses used. At high T-cell doses, CAR T cell-treated mice showed signs of health deterioration after a follow-up period. However, the severity of toxicity was reduced with a delayed onset when lower CAR T-cell doses were used. Our data demonstrate the efficacy of anti-SSEA-4 CAR T-cell therapy; however, safety strategies, such as dose-limiting and/or equipping CAR-T cells with combinatorial antigen recognition should be implemented for its potential clinical translation., (©2023 American Association for Cancer Research.)

The immunoglobulin (Ig) superfamily members are involved in cell adhesion and migration, complex multistep processes that play critical roles in embryogenesis, wound healing, tissue formation, and many other processes, but their specific functions during embryonic development remain unclear. Here, we have studied the function of the immunoglobulin superfamily member 3 (IGSF3) by generating an Igsf3 knockout (KO) mouse model with CRISPR/Cas9-mediated genome engineering. By combining RNA and protein detection methodology, we show that during development, IGSF3 localizes to the neural crest and a subset of its derivatives, suggesting a role in normal embryonic and early postnatal development. Indeed, inactivation of Igsf3 impairs the ability of the vagal neural crest cells to migrate and normally innervate the intestine. The small intestine of Igsf3 KO mice shows reduced thickness of the muscularis externa and diminished number of enteric neurons. Also, misalignment of neurons and smooth muscle cells in the developing intestinal villi is detected. Taken together, our results suggest that IGSF3 functions contribute to the formation of the enteric nervous system. Given the essential role of the enteric nervous system in maintaining normal gastrointestinal function, our study adds to the pool of information required for further understanding the mechanisms of gut innervation and etiology behind bowel motility disorders., (© 2023. Springer Nature Limited.)

After seeing a dramatic increase in the development and use of immunotherapy and precision medicine over the past few decades, oncological care now embraces the start of the adoptive cell therapy (ACT) era. This impulse towards a new treatment paradigm has been led by chimeric antigen receptor (CAR) T cells, the only type of ACT medicinal product to be commercialized so far. Brought about by an ever-growing understanding of cellular engineering, CAR T cells are T lymphocytes genetically modified with an appropriate DNA construct, which endows them with expression of a CAR, a fusion protein between a ligand-specific recognition domain, often an antibody-like structure, and the activating signaling domain of the T cell receptor. Through this genetic enhancement, CAR T cells are engineered from a cancer patient's own lymphocytes to better target and kill their cancer cells, and the current amassed data on clinical outcomes point to a stream of bright developments in the near future. Herein, from concept design and present-day manufacturing techniques to pressing hurdles and bright discoveries around the corner, we review and thoroughly describe the state of the art in CAR T cell therapy.

Cellular interactions with the extracellular environment are modulated by cell surface polysialic acid (PSA) carried by the neural cell adhesion molecule (NCAM). PSA-NCAM is involved in cellular processes such as differentiation, plasticity, and migration, and is elevated in Alzheimer's disease as well as in metastatic tumour cells. Our previous work demonstrated that insulin enhances the abundance of cell surface PSA by inhibiting PSA-NCAM endocytosis. In the present study we have identified a mechanism for insulin-dependent inhibition of PSA-NCAM turnover affecting cell migration. Insulin enhanced the phosphorylation of the focal adhesion kinase leading to dissociation of αv-integrin/PSA-NCAM clusters, and promoted cell migration. Our results show that αv-integrin plays a key role in the PSA-NCAM turnover process. αv-integrin knockdown stopped PSA-NCAM from being endocytosed, and αv-integrin/PSA-NCAM clusters co-labelled intracellularly with Rab5, altogether indicating a role for αv-integrin as a carrier for PSA-NCAM during internalisation. Furthermore, inhibition of p-FAK caused dissociation of αv-integrin/PSA-NCAM clusters and counteracted the insulin-induced accumulation of PSA at the cell surface and cell migration was impaired. Our data reveal a functional association between the insulin/p-FAK-dependent regulation of PSA-NCAM turnover and cell migration through the extracellular matrix. Most importantly, they identify a novel mechanism for insulin-stimulated cell migration., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The human brain is a highly vascular organ in which the blood-brain barrier (BBB) tightly regulates molecules entering the brain. Pericytes are an integral cell type of the BBB, regulating vascular integrity, neuroinflammation, angiogenesis and wound repair. Despite their importance, identifying pericytes amongst other perivascular cell types and deciphering their specific role in the neurovasculature remains a challenge. Using primary adult human brain cultures and fluorescent-activated cell sorting, we identified two CD73(+)CD45(-) mesenchymal populations that showed either high or low CD90 expression. CD90 is known to be present on neurons in the brain and peripheral blood vessels. We found in the human brain, that CD90 immunostaining localised to the neurovasculature and often associated with pericytes. In vitro, CD90(+) cells exhibited higher basal proliferation, lower expression of markers αSMA and CD140b, produced less extracellular matrix (ECM) proteins, and exhibited lesser pro-inflammatory responses when compared to the CD90(-) population. Thus, CD90 distinguishes two interrelated, yet functionally distinct pericyte populations in the adult human brain that may have discrete roles in neurovascular function, immune response and scar formation.

It is now well established that the human brain continuously produces new stem cells until well into old age. One of these stem-cell rich areas in the human brain is the sub-ventricular zone (SVZ). The human SVZ is organized in four distinctive layers containing type A, B and C cells. To date, no studies have investigated the distribution of inhibitory neurotransmitters such as γ-aminobutyric acid (GABA) and their respective receptors on the different cell types in the human SVZ. GABA(A) receptors (GABA(A)R) are ubiquitously expressed, inhibitory heteropentameric chloride ion channels comprised of a variety of subunits that are targeted by many prescribed drugs. In this study we present detailed immunohistochemical data on the regional and cellular localization of α₁, α₂, α3, β₂,₃ and γ₂ subunits of GABA(A)R in the human SVZ. The results from our double and triple labeling studies demonstrate that the cell types and subunit composition throughout the SVZ is heterogeneous; the thickness of the SVZ and GABA(A)R α₂ and γ₂ expression is increased especially in the vicinity of large SVZ blood vessels. GABA(A)R γ₂ is the most specific to the SVZ and present on various cells that express, either glial fibrillary acidic protein (GFAPδ) or polysialic acid-neural cell adhesion molecule (PSA-NCAM) separately, or together in a respective ratio of 7:6:2. Proliferating (type C) cells in the SVZ express GAD65/67, GFAPδ and GABA(A)R β₂,₃ receptor subunits. Within the SVZ the majority of cells have an unexpected nuclear GABA(A)R β₂,₃ expression that is inversely proportional to that of PCNA (proliferating cell nuclear antigen marker), which is a very different pattern of expression compared with underlying caudate nucleus cells. Taken together our results provide a detailed description of the chemo-architecture of the adult human SVZ demonstrating the importance of GABA and GABA(A) receptors on the various cell types in the SVZ., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cellular interactions mediated by the neural cell adhesion molecule (NCAM) are critical in cell migration, differentiation and plasticity. Switching of the NCAM-interaction mode, from adhesion to signalling, is determined by NCAM carrying a particular post-translational modification, polysialic acid (PSA). Regulation of cell-surface PSA-NCAM is traditionally viewed as a direct consequence of polysialyltransferase activity. Taking advantage of the polysialyltransferase Ca²⁺-dependent activity, we demonstrate in TE671 cells that downregulation of PSA-NCAM synthesis constitutes a necessary but not sufficient condition to reduce cell-surface PSA-NCAM; instead, PSA-NCAM turnover required internalization of the molecule into the cytosol. PSA-NCAM internalization was specifically triggered by collagen in the extracellular matrix (ECM) and prevented by insulin-like growth factor (IGF1) and insulin. Our results pose a novel role for IGF1 and insulin in controlling cell migration through modulation of PSA-NCAM turnover at the cell surface. Neural cell adhesion molecules (NCAMs) are critically involved in cell differentiation and migration. Polysialylation (PSA)/desialylation of NCAMs switches their functional interaction mode and, in turn, migration and differentiation. We have found that the desialylation process of PSA-NCAM occurs via endocytosis, induced by collagen-IV and blocked by insulin-like growth factor (IGF1) and insulin, suggesting a novel association between PSA-NCAM, IGF1/insulin and brain/tumour plasticity., (© 2013 International Society for Neurochemistry.)

P19 embryonal carcinoma (EC) cells are an invaluable tool for approximating the mechanisms that govern neuronal differentiation but with an enormous degree of simplification and have primarily been used to model the early stages of neurogenesis. However, they are often cultured under conditions that promote unrestricted non-neuronal growth that compromises neuronal viability. In this study we report an improved method to differentiate P19 EC cells that gives rise to high yields of functionally and morphologically mature neurons while significantly reducing the over-growth of non-neuronal cells in the cultures. In this protocol, P19 EC cells are induced in Minimum Essential Medium alpha supplemented with all-trans retinoic acid (RA) and 2.5% serum, and cultured as a monolayer. After RA-induction, cells are cultured on Matrigel coated-plates using defined media comprised of Neurobasal-A medium temporally supplemented with N2 and then B-27 for the remaining culture period. By treating the culture with Cytosine β-d-arabinofuranoside and 2'-Deoxycytidine for five days, the cultures are reliably promoted toward the neuronal differentiation vs non-neuronal differentiation, this accounting for a progressive neuronal enrichment of the cultures reaching 56% after 20 days of culture. P19-derived neural progenitor cells progressively expressed neuronal markers such as NeuN, Calretinin, Calbindin and Synapsin I in close resemblance to that occurring in vivo in the central nervous system (CNS). Furthermore, RA-induced P19 EC cells progressively acquired functional neuronal traits and after approximately 3 weeks in culture revealed mature neurophysiological properties, characteristics of CNS neurons. This protocol allows for a more specific assessment of the neuronal differentiation processes in vitro., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In the mammalian brain, olfaction is an important sense that is used to detect odors of different kinds that can warn of off food, to produce a mothering instinct in a flock or group of animals, and to warn of danger such as fire or poison. The olfactory system is made up of a long-distance rostral migratory stream that arises from the subventricular zone in the wall of the lateral ventricle, mainly comprises neuroblasts, and stretches all the way through the basal forebrain to terminate in the olfactory bulb. The olfactory bulb receives a constant supply of new neurons that allow ongoing integration of new and different smells, and these are integrated into either the granule cell layer or the periglomerular layer. The continuous turnover of neurons in the olfactory bulb allows us to study the proliferation, migration, and differentiation of neurons and their application in therapies for neurodegenerative diseases. In this chapter, we will examine the notion that the olfactory system might be the route of entry for factors that cause or contribute to neurodegeneration in the central nervous system. We will also discuss the enzymes that may be involved in the addition of polysialic acid to neural cell adhesion molecule, which is vital for allowing the neuroblasts to move through the rostral migratory stream. Finally, we will discuss a possible role of endosialidases for removing polysialic acid from neural cell adhesion molecules, which causes neuroblasts to stop migrating and terminally differentiate into olfactory bulb interneurons.