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Neural stem cells comprise a small population of subependymal cells in the adult brain that divide asymmetrically under baseline conditions to maintain the stem cell pool and divide symmetrically in response to injury to increase their numbers. Using in vivo and in vitro models,we demonstrate that Wnt signaling plays a role in regulating the symmetric divisions of adult neural stem cells with no change in the proliferation kinetics of the progenitor population. Using BAT-gal transgenic reporter mice to identify cells with active Wnt signaling,we demonstrate that Wnt signaling is absent in stem cells in conditions where they are dividing asymmetrically and that it is upregulated when stem cells are dividing symmetrically,such as (a) during subependymal regeneration in vivo,(b) in response to stroke,and (c) during colony formation in vitro. Moreover,we demonstrate that blocking Wnt signaling in conditions where neural stem cells are dividing symmetrically inhibits neural stem cell expansion both in vivo and in vitro. Together,these findings reveal that the mechanism by which Wnt signaling modulates the size of the stem cell pool is by regulating the symmetry of stem cell division. View Publication

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the degeneration of motor neurons. Currently,there is no effective therapy for ALS. Stem cell transplantation is a potential therapeutic strategy for ALS,and the reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) represents a novel cell source. In this study,we isolated a specific neural stem cell (NSC) population from human iPSCs based on high aldehyde dehydrogenase activity,low side scatter and integrin VLA4 positivity. We assessed the therapeutic effects of these NSCs on the phenotype of ALS mice after intrathecal or intravenous injections. Transplanted NSCs migrated and engrafted into the central nervous system via both routes of injection. Compared with control ALS,treated ALS mice exhibited improved neuromuscular function and motor unit pathology and significantly increased life span,in particular with the systemic administration of NSCs (15%). These positive effects are linked to multiple mechanisms,including production of neurotrophic factors and reduction of micro- and macrogliosis. NSCs induced a decrease in astrocyte number through the activation of the vanilloid receptor TRPV1. We conclude that minimally invasive injections of iPSC-derived NSCs can exert a therapeutic effect in ALS. This study contributes to advancements in iPSC-mediated approaches for treating ALS and other neurodegenerative diseases. View Publication

We examined the unique contributions of the cytokines IL-21 and IL-4 on germinal center (GC) B cell initiation and subsequent maturation in a murine model system. Similar to other reports,we found T follicular helper cell expression of IL-21 begins prior to T follicular helper cell migration into the B cell follicle and precedes that of IL-4. Consistent with this timing,IL-21 signaling has a greater influence on the perifollicular pre-GC B cell transition to the intrafollicular stage. Notably,Bcl6hi B cells can form in the combined absence of IL-21R- and STAT6-derived signals; however,these nascent GC B cells cease to proliferate and are more prone to apoptosis. When B cells lack either IL-21R or STAT6,aberrant GCs form atypical centroblasts and centrocytes that differ in their phenotypic maturation and costimulatory molecule expression. Thus,IL-4 and IL-21 play nonredundant roles in the phased progression of GC B cell development that can initiate in the combined absence of these cytokine signals. View Publication

N6-methyladenosine (m6A) modification is involved in diverse immunoregulation,while the relationship between m6A modification and immune tolerance post kidney transplantation remains unclear. Expression of Wilms tumor 1-associating protein (WTAP),an m6A writer,was firstly detected in tolerant kidney transplant recipients (TOL). Then the role of WTAP on regulatory T (Treg) cell differentiation and function in CD4+ T cells from kidney transplant recipients with immune rejection (IR) was investigated. The potential target of WTAP and effect of WTAP on immune tolerance in vivo were subsequently verified. WTAP was upregulated in CD4+ T cells of TOL and positively correlated with Treg cell proportion. In vitro,WTAP overexpression promoted Treg cell differentiation and enhanced Treg cell-mediated suppression toward na?ve T cells. Forkhead box other 1 (Foxo1) was identified as a target of WTAP. WTAP enhanced m6A modification of Foxo1 mRNA in coding sequence (CDS) region,leading to up-regulation of Foxo1. Overexpression of m6A demethylase removed the effect of WTAP overexpression,while Foxo1 overexpression reversed these effects. WTAP overexpression alleviated allograft rejection in model mice,as evidenced by reduced inflammatory response and increased Treg population. Our study suggests that WTAP plays a positive role in induction of immune tolerance post kidney transplant by promoting Treg cell differentiation and function. leading to up-regulation of Foxo1. Overexpression of m6A demethylase removed the effect of WTAP overexpression while Foxo1 overexpression reversed these effects. WTAP overexpression alleviated allograft rejection in model mice as evidenced by reduced inflammatory response and increased Treg population. Our study suggests that WTAP plays a positive role in induction of immune tolerance post kidney transplant by promoting Treg cell differentiation and function." View Publication

Central nervous system (CNS) cancers are responsible for high rates of morbidity and mortality worldwide. Malignant CNS tumors such as adult Glioblastoma (GBM) and pediatric embryonal CNS tumors such as medulloblastoma (MED) and atypical teratoid rhabdoid tumors (ATRT) present relevant therapeutic challenges due to the lack of response to classic treatment regimens with radio and chemotherapy. Recent findings on the Zika virus’ (ZIKV) ability to infect and kill CNS neoplastic cells draw attention to the virus’ oncolytic potential. Studies demonstrating the safety of using ZIKV for treating malignant CNS tumors,enabling the translation of this approach to clinical trials,are scarce in the literature. Here we developed a co-culture model of mature human cerebral organoids assembled with GBM,MED or ATRT tumor cells and used these assembloids to test ZIKV oncolytic effect,replication potential and preferential targeting between normal and cancer cells. Our hybrid co-culture models allowed the tracking of tumor cell growth and invasion in cerebral organoids. ZIKV replication and ensuing accumulation in the culture medium was higher in organoids co-cultured with tumor cells than in isolated control organoids without tumor cells. ZIKV infection led to a significant reduction in tumor cell proportion in organoids with GBM and MED cells,but not with ATRT. Tumoroids (3D cultures of tumor cells alone) were efficiently infected by ZIKV. Interestingly,ZIKV rapidly replicated in GBM,MED,and ATRT tumoroids reaching significantly higher viral RNA accumulation levels than co-cultures. Moreover,ZIKV infection reduced viable cells number in MED and ATRT tumoroids but not in GBM tumoroids. Altogether,our findings indicate that ZIKV has greater replication rates in aggressive CNS tumor cells than in normal human cells comprising cerebral organoids. However,such higher ZIKV replication in tumor cells does not necessarily parallels oncolytic effects,suggesting cellular intrinsic and extrinsic factors mediating tumor cell death by ZIKV. View Publication

SummaryBackgroundPresence of nerves in tumours,by axonogenesis and neurogenesis,is gaining increased attention for its impact on cancer initiation and development,and the new field of cancer neuroscience is emerging. A recent study in prostate cancer suggested that the tumour microenvironment may influence cancer progression by recruitment of Doublecortin (DCX)-expressing neural progenitor cells (NPCs). However,the presence of such cells in human breast tumours has not been comprehensively explored.MethodsHere,we investigate the presence of DCX-expressing cells in breast cancer stromal tissue from patients using Imaging Mass Cytometry. Single-cell analysis of 372,468 cells across histopathological images of 107 breast cancers enabled spatial resolution of neural elements in the stromal compartment in correlation with clinicopathological features of these tumours. In parallel,we established a 3D in vitro model mimicking breast cancer neural progenitor-innervation and examined the two cell types as they co-evolved in co-culture by using mass spectrometry-based global proteomics.FindingsStromal presence of DCX + cells is associated with tumours of higher histological grade,a basal-like phenotype,and shorter patient survival in tumour tissue from patients with breast cancer. Global proteomics analysis revealed significant changes in the proteomic landscape of both breast cancer cells and neural progenitors in co-culture.InterpretationThese results support that neural involvement plays an active role in breast cancer and warrants further studies on the relevance of nerve elements for tumour progression.FundingThis work was supported by the 10.13039/501100005416Research Council of Norway through its Centre of Excellence funding scheme,project number 223250 (to L.A.A),the 10.13039/100008730Norwegian Cancer Society (to L.A.A. and H.V.),the Regional Health Trust Western Norway (Helse Vest) (to L.A.A.),the 10.13039/501100008728Meltzer Research Fund (to H.V.) and the 10.13039/100000002National Institutes of Health (NIH)/10.13039/100000057NIGMS grant R01 GM132129 (to J.A.P.). View Publication

Chemotherapy remains the mainstay treatment for triple-negative breast cancer (TNBC) due to the lack of specific targets. Given a modest response of immune checkpoint inhibitors in TNBC patients,improving immunotherapy is an urgent and crucial task in this field. CD73 has emerged as a novel immunotherapeutic target,given its elevated expression on tumor,stromal,and specific immune cells,and its established role in inhibiting anti-cancer immunity. CD73-generated adenosine suppresses immunity by attenuating tumor-infiltrating T- and NK-cell activation,while amplifying regulatory T cell activation. Chemotherapy often leads to increased CD73 expression and activity,further suppressing anti-tumor immunity. While debulking the tumor mass,chemotherapy also enriches heterogenous cancer stem cells (CSC),potentially leading to tumor relapse. Therefore,drugs targeting both CD73,and CSCs hold promise for enhancing chemotherapy efficacy,overcoming treatment resistance,and improving clinical outcomes. However,safe and effective inhibitors of CD73 have not been developed as of now. We used in silico docking to screen compounds that may be repurposed for inhibiting CD73. The efficacy of these compounds was investigated through flow cytometry,RT-qPCR,CD73 activity,cell viability,tumorsphere formation,and other in vitro functional assays. For assessment of clinical translatability,TNBC patient-derived xenograft organotypic cultures were utilized. We also employed the ovalbumin-expressing AT3 TNBC mouse model to evaluate tumor-specific lymphocyte responses. We identified quercetin and luteolin,currently used as over-the-counter supplements,to have high in silico complementarity with CD73. When quercetin and luteolin were combined with the chemotherapeutic paclitaxel in a triple-drug regimen,we found an effective downregulation in paclitaxel-enhanced CD73 and CSC-promoting pathways YAP and Wnt. We found that CD73 expression was required for the maintenance of CD44 high CD24 low CSCs,and co-targeting CD73,YAP,and Wnt effectively suppressed the growth of human TNBC cell lines and patient-derived xenograft organotypic cultures. Furthermore,triple-drug combination inhibited paclitaxel-enriched CSCs and simultaneously improved lymphocyte infiltration in syngeneic TNBC mouse tumors. Conclusively,our findings elucidate the significance of CSCs in impairing anti-tumor immunity. The high efficacy of our triple-drug regimen in clinically relevant platforms not only underscores the importance for further mechanistic investigations but also paves the way for potential development of new,safe,and cost-effective therapeutic strategies for TNBC. View Publication

This study investigates the impact of minimal residual disease (MRD) on relapse in patients with acute myeloid leukemia (AML),focusing on its interaction with immune cells function. A total of 49 AML patients were enrolled in this prospective study and categorized into four groups: MRD − positive with relapse,MRD − positive without relapse,MRD − negative with relapse,and MRD − negative without relapse. Peripheral blood T lymphocyte subpopulations were analyzed using ten-color flow cytometry. CD4 + T cells were co-cultured with leukemia cell lines to assess the impact of CD4 + T cells on leukemia cell proliferation,apoptosis,and cytokine release. In MRD − positive patients,relapsed individuals exhibited significantly higher levels of CD4 + T cells,regulatory T (Treg) cells,and CD4 + CD45RA + naïve T cells compared to non-relapsed patients ( P < 0.0001,P = 0.0016,and P = 0.0066,respectively). Conversely,in MRD − negative patients,relapsed individuals showed a significantly lower percentage of Treg cells ( P = 0.0068). Furthermore,we observed that CD4 + T cells were associated with enhanced leukemia cell proliferation and reduced apoptosis,along with markedly increased IL-10 expression. The available data raise the possibility that CD4 + T cell-derived IL-10 participates in immune microenvironment regulation,a process that may have implications for MRD maintenance and disease recurrence in AML. View Publication

Spontaneous control of human immunodeficiency virus (HIV) is generally associated with an enhanced capacity of CD8+ T cells to eliminate infected CD4+ T cells,but the molecular characteristics of these highly functional CD8+ T cells are largely unknown. In the present study,using single-cell analysis,it was shown that HIV-specific,central memory CD8+ T cells from spontaneous HIV controllers (HICs) and antiretrovirally treated non-controllers have opposing transcriptomic profiles. Genes linked to effector functions and survival are upregulated in cells from HICs. In contrast,genes associated with activation,exhaustion and glycolysis are upregulated in cells from non-controllers. It was shown that HIV-specific CD8+ T cells from non-controllers are largely glucose dependent,whereas those from HICs have more diverse metabolic resources that enhance both their survival potential and their capacity to develop anti-HIV effector functions. The functional efficiency of the HIV-specific CD8+ T cell response in HICs is thus engraved in their memory population and related to their metabolic programme. Metabolic reprogramming in vitro through interleukin-15 treatment abrogated the glucose dependency and enhanced the antiviral potency of HIV-specific CD8+ T cells from non-controllers. View Publication

Proteomics of dystrophic muscle samples is limited by the amount of protein that can be extracted from patient biopsies. Cells and tissues derived from patient-derived induced pluripotent stem cells (iPSCs) can be an expandable alternative source. We have patterned iPSCs from three Duchenne muscular dystrophy (DMD) patient lines into skeletal muscle cells using a two-dimensional as well as our three-dimensional organoid differentiation system. Probes with sufficient protein amounts could be extracted and prepared for mass spectrometry. In total,3007 proteins in 2D and 2709 proteins in 3D were detected in DMD patient probes. A total of 83 proteins in 2D and 338 proteins in 3D can be described as differentially expressed between DMD and control patient probes in a post hoc test. We have identified and we propose Myosin-9,Collagen 18A,Tropomyosin 1,BASP1,RUVBL1,and NCAM1 as proteins specifically altered in their expression in DMD for further investigation. Proteomics of skeletal muscle organoids resulted in greater consistency of results between cell lines in comparison to the two-dimensional myogenic differentiation protocol. View Publication

A number of human malignancies exhibit sustained stimulation,mutation,or gene amplification of the receptor tyrosine kinase human mesenchymal-epithelial transition factor (c-Met). ARQ 197 is a clinically advanced,selective,orally bioavailable,and well tolerated c-Met inhibitor,currently in Phase 3 clinical testing in non-small cell lung cancer patients. Herein,we describe the molecular and structural basis by which ARQ 197 selectively targets c-Met. Through our analysis we reveal a previously undisclosed,novel inhibitory mechanism that utilizes distinct regulatory elements of the c-Met kinase. The structure of ARQ 197 in complex with the c-Met kinase domain shows that the inhibitor binds a conformation that is distinct from published kinase structures. ARQ 197 inhibits c-Met autophosphorylation and is highly selective for the inactive or unphosphorylated form of c-Met. Through our analysis of the interplay between the regulatory and catalytic residues of c-Met,and by comparison between the autoinhibited canonical conformation of c-Met bound by ARQ 197 to previously described kinase domains of type III receptor tyrosine kinases,we believe this to be the basis of a powerful new in silico approach for the design of similar inhibitors for other protein kinases of therapeutic interest. View Publication