In silico analysis of the transcriptome of human breast tumors, confirmed the association between RANK expression and stem cell and mesenchymal markers in ER+HER2- tumors. Importantly, exposure of ER+HER2- cells to continuous RANK pathway activation by exogenous RANKL, in vitro and in vivo, induced a negative feedback effect, independent of RANK levels, leading to the downregulation of HR and increased resistance to hormone therapy. These results suggest that ER+HER2- RANK-positive cells may constitute an important reservoir of slow cycling, therapy-resistance cancer cells; and that RANK pathway activation is deleterious in all ER+HER2- breast cancer cells, independently of RANK levels.The role of RANKL-RANK pathway in progesterone-driven mammary carcinogenesis and triple negative breast cancer tumorigenesis has been well characterized. However, and despite evidences of the existence of RANK-positive hormone receptor (HR)-positive breast tumors, the implication of RANK expression in HR-positive breast cancers has not been addressed before. Here, we report that RANK pathway affects the expression of cell cycle regulators and decreases sensitivity to fulvestrant of estrogen receptor (ER)-positive (ER+)/HER2- breast cancer cells, MCF-7 and T47D. Moreover, RANK overexpressing cells had a staminal and mesenchymal phenotype, with decreased proliferation rate and decreased susceptibility to chemotherapy, but were more invasive  Breast cancer is responsible for over 600,000 deaths per year worldwide , the vasThe receptor activator of nuclear factor-kB ligand (RANKL)-RANK pathway was first identified as mediator of T and dendritic cells interaction , but it The RANKL-RANK pathway also emerged as a major mediator of hormone-driven breast carcinogenesis. RANKL is expressed downstream of activated progesterone receptor (PR) in HR-positive mammary epithelial cells (MECs), and drives the proliferation of HR-negative RANK-expressing mammary stem cells (MaSCs) in a paracrine manner –12. OverIn clinical samples, RANK was found to be expressed by different solid tumors , and RANDespite evidences of heterogeneous RANK expression amongst breast tumors, the implication of RANK expression in HR-positive breast cancers have remained elusive. Here, we report that ER+HER2- RANK-overexpressing breast cancer cells have a staminal and mesenchymal phenotype, with decreased proliferation rate and decreased susceptibility to chemotherapy and fulvestrant. Moreover, continuous RANK pathway activation in ER+HER2- cells induces a negative feedback effect, independent of RANK levels, leading to the downregulation of HR and increased resistance to hormone therapy (HT). Therefore, RANKL-RANK pathway may affect the outcomes of ER+HER2- breast cancer.In this work we proposed to investigate the effect of RANK expression in ER+HER2- breast cancer. To address this, we overexpressed RANK in two different ER+HER2- breast cancer cell lines, MCF-7 and T47D, which express low endogenous levels of this gene (Supplementary Table 1). MDA-MB-231 TNBC cells, known to express functional RANK , 22, andExposure to exogenous RANKL had no effect on luminal cells’ proliferation; however, RANK OE cells were less proliferative upon release from serum starvation in comparison with parental counterparts . We therefore quantified each cell line’s doubling time, which was higher in RANK OE cells .Since proliferation rate was negatively affected, we questioned if RANK OE impacts the expression of ER, a major regulator of proliferation in ER+ cells. We analyzed ER levels by western blot, and found ER to be up-regulated in RANK OE cell lines, although to a higher extent in MCF-7 cells . HoweverBecause RANK OE cells were characterized by increased expression of ER but decreased sensitivity to estradiol, we questioned if this would affect the response to HT, standard of care for ER+ breast cancers in all settings. Drug sensitivity assays demonstrate that RANK OE cells had decreased sensitivity to fulvestrant but not to tamoxifen . TamoxifOE cells, but not in T47DOE. We hypothesize that this can be due to the fact that T47D cells exhibit a more mesenchymal phenotype in comparison with MCF-7, with basal increased levels of these proteins.Next, and based on the reported effects of RANK OE in TNBC, we analyzed the expression of known epithelial and mesenchymal cell markers and observed an increase in Snail in both RANK OE cell lines ; whereasper se, and stimuli with RANKL increased migration, although not significantly. Like in MDA-MB-231 cells, RANKL effect was abrogated by RANKL neutralization with an anti-RANKL specific antibody.Since increase in migration has been associated with RANK-pathway activation, we next assessed migration in the presence of 2.5 μg/ml RANKL, which was previously shown to induce cancer cells migration  and doxorubicin (Doxo), two chemotherapeutic agents commonly used to treat patients with breast cancer. RANK OE cells showed decreased sensitivity to both drugs, in comparison with parental cell lines . Chemore MCF-7OE , whereasn = 1015) (Supplementary Table 2). In this cohort, as expected, RANK expression was higher in ER-negative (ER-) tumors vs ER+HER2+/- tumors . Moreover, when considering all cases, RANKhigh was associated with ER, PR and HER2 negative status  (Supplementary Table 3). When considering ER+ or ER+HER2- cases, RANK expression was not significantly associated with any clinicopathologic characteristics.Next we questioned if increased expression of mesenchymal, stemness and chemoresistance-related markers is also associated with RANK expression in human ER+ tumors. To address this question, we analyzed the TCGA breast cancer cohort, including all female patients with known ER status . When considering all ER+ cases, independently of HER2 status, we observed the same associations, plus decreased E-cadherin in RANKhigh tumors .We interrogated this cohort for the expression of the genes altered in RANK OE cell lines. RANKnificant . We also ALDH1A3 , and incnd ABCG2 . CorrelaRANK expression (RANK+) by RT-qPCR. We analyzed the correlation between the expression of RANK and Vimentin, N-cadherin, Twist, Slug, Oct4 and Cyclin D1 . We observed a significant positive correlation between RANK and Vimentin  and Slug  . When considering only ER+RANK+ cases (25/57), we observed a significant positive correlation between RANK and Vimentin  and N-cadherin  .To further expand our results into the clinical setting, we analyzed a second cohort, in this case a cohort of 57 patients diagnosed with breast cancer at Hospital de Santa Maria (Supplementary Table 5). 33/57 (57.9%) samples were positive for in vitro, which is a limitation of our study, these findings reinforce the hypothesis that RANK is associated with mesenchymal ER+ breast cancers.Although we could not confirm all the associations we observed in vivo. First we used an orthotopic xenograft model in NSG mice supplemented with estradiol. MCF-7 RANK OE xenografts were significantly smaller than MCF-7 xenografts .We next aimed to characterize RANK OE cells nografts , concordnografts . RANK exOE2), this time by CRISPR-Cas9 gene activation . Again, RANK OE2 xenografts were significantly smaller than MCF-7 xenografts ; with lower Ki67  and higher desmoplasia . We confirmed the overexpression of RANK, N-cadherin, Vimentin, Snail, Slug and Twist in RANK OE2 xenografts . Notably, flow cytometry analysis of xenografts derived from the simultaneous inoculation of RANK OE2 and parental cells  revealed that MIX tumors were enriched in RANK OE2 cells (GFP+RFP+) in the end of the experiment . This may suggest that RANK OE cells have increased fitness in comparison with parental cells, and will be important to determine which factors promote the growth of RANK OE cells in these conditions, and which is the competition mechanism involved in this interaction.Difference in xenograft growth was so remarkable that we further confirmed our findings by deriving a second MCF-7 RANK OE cell line , which induced a strong ER downregulation .Since to RANKL , as wellto RANKL . Moreoveto RANKL , even inThe pharmacological inhibition of RANK pathway has gained particular attention in adjuvant control of bone recurrence from breast cancer or in the prevention of BRCA-related breast cancer, since its role in bone remodeling and breast carcinogenesis was clearly shown.n = 601) where 23 out of 160 RANK-positive cases were ER+  in breast cancer cell lines was derived from the Cancer Cell Line Encyclopedia (CCLE) database . Analyses were performed using GraphPad Prism8 software.Normalized gene expression [log2 FPKM (fragments per kilobase million)] and clinical data for 1015 breast tumors from The Cancer Genome Atlas (TCGA) breast cancer dataset  were provided by Dr. Sérgio Dias ; and T47D cell line was provided by Dr. Phyllippe Clézardin . Cells were cultured under standard conditions, used at low passage number, and tested for Mycoplasma contamination. For continuous RANKL exposure experiments, medium was supplemented with 1 μg/ml RANKL  every 48 h for 6 weeks. Doubling time was calculated in GraphPad Prism 8 software, using the exponential growth equation with least squares regression fitting model.Human breast carcinoma cell lines MCF-7TNFRSF11A) overexpression plasmid pReceiver-Lv121 ), and selected with 0.5 μg/mL (MCF-7 and MDA-MB-231) or 1.5 μg/ml (T47D) puromycin dihydrochloride . For CRISPR/Cas9 activation of RANK expression (MCF-7OE2), MCF-7 cells were transduced with huRANK lentiviral activation particles  or control lentiviral particles , and selected with 0.5 μg/mL puromycin dihydrochloride, 5 μg/ml blasticidin S HCl  and 200 μg/mL hygromycin B . RANK overexpression was confirmed by RT-qPCR and flow cytometry. Next MCF-7OE2 cells were transduced with Cignal Lenti Positive Control (RFP) ready-to-transduce lentiviral particles , and selected with 0.5 μg/mL puromycin dihydrochloride, followed by RFP+ cell sorting in a FACS Aria III cell sorter (BD Biosciences).For RANK overexpression cells were transduced with RANK lentiviral overexpression particles , TNFSF11 , PGR , ESR1 , Twist , Slug , Vimentin , N-cadherin , Oct4  and GAPDH . Gene expression was normalized using the housekeeping gene GAPDH, and relative mRNA expression was calculated using the 2–ΔΔCt method.Cells or mouse tumors total RNA was extracted using the NZY Total RNA Isolation kit . RNA from human breast tumors was obtained from Biobanco-iMM. DNase I-treated RNA was reverse transcribed using the NZY M-MuLV First-Strand cDNA Synthesis kit  and Oligo (dT)20 primer; and cDNAs were amplified by real-time PCR using TaqMan Gene Expression Master Mix  or NZY qPCR Green, ROX . Specific primers included: For analysis of RANK pathway activation by RANKL, cells were serum-starved in low-serum medium (0.1% FBS) for 24 h, and stimulated with 1 μg/mL human RANKL  for the indicated time points. Specific primary antibodies included: mouse monoclonal anti β-Actin antibody , rabbit monoclonal anti α-Tubulin (11H10) , rabbit polyclonal anti-NFkB p65 , rabbit monoclonal anti-NF-kB p65 (D14E12) , rabbit monoclonal anti-Phospho-NF-kB p65 (Ser536) (93H1) , mouse monoclonal anti-IkBα (L35A5) , rabbit monoclonal anti-Phospho-IkBα (Ser32) (14D4) , rabbit polyclonal anti-Phospho-ERK1/2 (Thr-202/Tyr-204) , rabbit polyclonal anti-ERK1/2 (c-14) , rabbit polyclonal anti-Phospho-AKT1/2/3 (Ser-473) (D9E) , rabbit polyclonal anti-AKT1/2/3 (H-136) , rabbit monoclonal anti-Vimentin (D21H3) , rabbit monoclonal anti-N-Cadherin (D4R1H) , rabbit monoclonal anti-β-Catenin (D10A8) , rabbit monoclonal anti-Snail (C15D3) , rabbit monoclonal anti-Slug (C19G7) , rabbit monoclonal anti-E-Cadherin (24E10) , mouse monoclonal anti-Nanog (hNanog.2) , mouse monoclonal anti-Sox2 (245610) , mouse monoclonal anti-OCT4 (7F9.2) , rabbit monoclonal anti-Phospho-Rb (Ser807/811) (D20B12) , rabbit monoclonal anti-p21 Waf1/Cip1 (12D1) , rabbit monoclonal anti-Cyclin D1 (92G2) , mouse monoclonal anti-Cyclin E (HE12) , rabbit monoclonal anti-CDK2 (78B2) , rabbit monoclonal anti-CDK4 (D9G3E) , mouse monoclonal anti-CDK6 (DCS83) , rabbit monoclonal anti-p27 Kip1 (D69C12) , mouse monoclonal anti-p53 (Bp53-12) , mouse monoclonal anti-p18 INK4C (DCS118) , rabbit monoclonal anti-ERα (D6R2W) , mouse monoclonal anti-PR , rabbit monoclonal anti-Her2/ErbB2 , rabbit monoclonal anti-BCRP/ABCG2 , rabbit polyclonal anti-hsRANKL , goat polyclonal anti-OPG . Horseradish peroxidase (HRP)-conjugated specific secondary antibodies anti-mouse-HRP IgG , anti-rabbit-HRP IgG , and anti-goat-HRP IgG  were used. Band intensity was calculated using ImageJ software and normalized for β-Actin.For RANK expression analysis, trypsinized cells were incubated with mouse monoclonal antibody anti-RANK  and labelled with 1:100 Cy5 conjugated AffiniPure goat anti-mouse IgG . For ABACB1/MDR1 expression analysis, trypsinized cells were incubated with biotinylated mouse antibody anti-CD243  and labelled with anti-biotin-PE antibody . For cell cycle analysis cells were cultured under standard conditions for 24 h (asynchronous), synchronized by 24 h serum starvation, and released from serum starvation for another 24 h. Cell cycle analysis was performed using the Propidium iodide (PI) flow cytometry kit . Analysis was made using FlowJo V10 software.sRANKL in conditioned media was quantified by sandwich ELISA, using 1 μg/ml polyclonal rabbit anti-sRANKL antibody  as capture antibody, 0.5 μg/ml biotinylated rabbit anti-sRANKL antibody  as detection antibody, and ABTS  as substrate . RANKL  was used as standard; and protein concentration was normalized for media concentration factor and number of cells.TRAcP 5b was quantified in mouse serum using the MouseTRAP (TRAcP 5b) ELISA kit , according to the manufacturer instructions.Cells were seeded in 3D Tumorsphere Medium XF  in ultra-low attachment 6-well plates. After 7 days, average tumorsphere area was calculated by measuring all tumorspheres > 50 μm in diameter per well. Sphere Forming Capacity (SFC) (%) was determined as the number of mammospheres > 50 μm/number of cells seeded) × 100. 3D tumor spheroid invasion assay was performed as previously described [Cells were seeded in 96 well-plates, with or without paclitaxel , doxorubicin hydrochloride , tamoxifen , or fulvestrant . Medium was replaced every two days. After 72 h for paclitaxel and doxorubicin, and seven days for tamoxifen and fulvestrant, to assess the proliferative effect of estrogen, cells were cultured in 24-well plates, in phenol red-free DMEM:F12 medium  supplemented with 5% (v/v) charcoal stripped FBS , for five days, with or without 10 nM β-estradiol . Viability was assessed by Alamar blue assay .Migration of cancer cells was assessed using a 96-well chemotaxis chamber with polycarbonate filters (8 mm pore size) , as previously described . Briefly6 cells/ml. Tumor growth was monitored weekly by luminescence analysis. For circulating tumor cells (CTC) analysis, venous blood was collected by cardiac puncture before sacrifice, and analyzed for GFP and RFP expression in a BD LSRFortessa flow cytometer (BD Biosciences).All animal experiments were reviewed and approved by the Institutional Animal Welfare Body of the Institute of Molecular Medicine, and licensed by the national regulatory agency Direcção Geral de Alimentação e Veterinária (DGAV). Experiments were performed in four week old NOD scid gamma (NSG) mice (Charles River) supplemented with subcutaneous 17β-estradiol pellets , unless otherwise stated. For orthotopic xenografts, cells were resuspended in 50% phenol-free matrigel solution . For the experimental metastases model NSG mice were inoculated in the tail vein with 2.5 × 105 μm tissue sections from Formalin-Fixed Paraffin-Embedded (FFPE) samples were stained by immunohistochemistry (IHC) for the detection of Ki67, ER and VWF. Specific antibodies included rabbit anti-human Ki67 primary antibody , rabbit anti-human ERα  and polyclonal rabbit anti-human von Willebrand Factor (VWF) primary antibody , and EnVisionTM Detection System, rabbit/mouse . Ki67 was quantified as the percentage of DAB-stained nuclear area over total nuclear area (hematoxylin-stained nuclei regions), from 5 fields at 400× magnification, using the ImageJ software. Microvessel density (MVD) was determined using VWF IHC. A single microvessel was defined as previously described [t-test, one-way or two-way ANOVA were used as appropriate. Results are presented as mean ± SEM and p-value < 0.05 was considered statistically significant.Data was analyzed using GraphPad Prism8 software. The unpaired


Rationale: Stroke is a leading cause of adult disability worldwide, but no drug provides functional recovery during the repair phase. Accumulating evidence demonstrates that environmental enrichment (EE) promotes stroke recovery by enhancing network excitability. However, the complexities of utilizing EE in a clinical setting limit its translation.Methods: We used multifaceted approaches combining electrophysiology, chemogenetics, optogenetics, and floxed mice in a mouse photothrombotic stroke model to reveal the key target of EE-mediated stroke recovery.Results: EE reduced tonic gamma-aminobutyric acid (GABA) inhibition and facilitated phasic GABA inhibition in the peri-infarct cortex, thereby promoting network excitability and stroke recovery. These beneficial effects depended on GAT-1, a GABA transporter regulating both tonic and phasic GABA signaling, as EE positively regulated GAT-1 expression, trafficking, and function. Furthermore, GAT-1 was necessary for EE-induced network plasticity, including structural neuroplasticity, input synaptic strengthening in the peri-infarct cortex, output synaptic strengthening in the corticospinal tract, and sprouting of uninjured corticospinal axons across the midline into the territory of denervated spinal cord, and functional recovery from stroke. Moreover, restoration of GAT-1 function in the peri-infarct cortex by its overexpression showed similar beneficial effects on stroke recovery as EE exposure.Conclusion: GAT-1 is a key molecular substrate of the effects of EE on network excitability and consequent stroke recovery and can serve as a novel therapeutic target for stroke treatment during the repair phase. Stroke is a major cause of adult disability in much of the world Environmental enrichment (EE) is known to profoundly remodel the central nervous system (CNS) at molecular, anatomical, and functional levels Gat-1 is a hub gene Functional recovery from stroke is related to gamma-aminobutyric acid (GABA) transporters (GATs). Four subtypes of GATs have been identified: GAT-1, -2, -3 and betaine GABA transporter. Of these, GAT-1 is the most abundantly expressed transporter in neurons, and the predominantly glial GAT-3 is also expressed in neurons flox/floxSlc6a1mice (GAT-1flox/flox) with exon 6 flanked by loxP recombination sites were generated at Shanghai Model Organisms following the generation methods described in detail in the following section. Wild-type male adult (6-7 weeks) C57BL6/J mice  were purchased from the Model Animal Research Center of Nanjing University. Wild-type C57BL6/J mice  were used for primary neuron cultures. Wild-type neonatal C57BL6/J mice  were used for primary astrocyte cultures. Animals were maintained at 20 ± 2 °C and under a 12 h light/dark cycle with food and water provided ad libitum. Every effort was made to minimize the number of animals used and their suffering. All experimental procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Nanjing Medical University  and complied with the Animal Research: Reporting of In vivo Experiments (ARRIVE) guidelines.flox/floxSlc6a1(C57BL6/J background) mice were generated using the clustered regulatory inter spaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system. A Slc6a1 donor vector containing flox sites flanking exon 6 of the Slc6a1 gene was created. Two single-guide RNAs (sgRNAs) targeting intron 5 and intron 6 were transcribed in vitro. The sgRNA target site for intron 5 was 5'-CGT GTA AAG AGT CTA GAG CC-3'. The sgRNA site for intron 6 was 5'-TAA GGT GCA CGG CAG ATG TT-3'. The donor vector with the two sgRNAs and Cas9 mRNA was microinjected into C57BL6/J fertilized eggs. F0 generation mice positive for homologous recombination were identified by polymerase chain reaction (PCR) analysis using two pairs of primers. A 4.3 kb fragment of the 5' end was amplified from the recombination allele with the primers P5-forward: 5'-GGG ATT GGG CCG TGA AGT TTT-3' and P5-reverse: 5'-CGG AAG TTG GGT GTG ATG TAG AAG-3'. A 3.9 kb fragment of the 3' end was amplified from the recombination allele with the primers P3-forward: 5'-AAC AAC GTG CTC CAG CGA CAA TGA-3' and P3-reverse: 5'-TGG CCA GGC AAG GAG AAA GACT-3'. The PCR products were further confirmed by sequencing.Slc6a1 allele were then bred to obtain homozygous mice. The genotype of this mouse line was confirmed by the pair of primers flox/floxSlc6a1 forward: 5'-GGA GGT GGC CTG GAA CAG C-3' and reverse: 5'-CTC CTG CCT GAG ACC TTG TAG TG-3'.Chimeras were mated with C57BL6/J mice to generate germline transmission. Heterozygous mice having the floxed Focal cortical strokes were induced in adult male mice through photothrombosis using a well-established procedure In vivo MRI was performed using a small animal MRI scanner . Mice were anesthetized with 2% isoflurane. T2-TurboRARE imaging was conducted 1, 4, 12, 19, 26, and 33 days after stroke using a 2D fast-spin-echo sequence . Fourteen axial slices with a slice thickness of 1 mm, a matrix size of 256 × 256, and a field of view of 25 mm × 25 mm were positioned over the brain.3) were equipped with a variety of objects such as running wheels, tunnels, igloos, huts, retreats, and wooden toys. EE cages were fourteen times larger than standard housing (SH) cages (30×25×18 cm3). The objects in the EE cages were changed every other day to maintain a novel environment. Six mice were housed in each EE cage, while three mice were housed in each SH cage. Thus, the EE cage environment provided more opportunities for physical exercise, social interaction, and exposure to novel learning tasks compared with the SH cages. All mice received chow and water ad libitum.EE cages (90×70×30 cm12 virus particles/mL) and AAV-VGAT-hM4D (Gi)-mCherry (3×1012 virus particles/mL) were purchased from BrainVTA. AAV-CaMKIIα-hChR2 (E123A)-eYFP (5×1012 virus particles/mL), AAV-hSyn-GAT-1-3Flag (7×1012 virus particles/mL), AAV-hSyn-GAT-1-3Flag-T2A-EGFP (7×1012 virus particles/mL), AAV-hSyn-3Flag-T2A-EGFP (7×1012 virus particles/mL), AAV-CAG-GAT-3-3Flag-T2A-mCherry (7×1012 virus particles/mL), AAV-CAG-3Flag-T2A-mCherry (7×1012 virus particles/mL) were purchased from GeneChem Co., Ltd. AAV-CAG-EGFP (7×1012 virus particles/mL) and AAV-CAG-EGFP-T2A-Cre (7×1012 virus particles/mL) were purchased from Obio Technology. Viruses were stored at -80 °C freezer until the day of infusion.The adeno-associated virus (AAV) AAV-CaMKIIα-hM4D (Gi)-mCherry  were produced, as described previously 9 virus particles /mL) was used as control.shRNAs of histone deacetylase (HDAC) 1 and 2 were constructed and synthesized by GeneChem Co., Ltd. The target sequence used against mouse flox/flox mice were anesthetized with 2% isoflurane for stereotaxic viral injections. A small craniotomy was made targeting the motor cortex with stereotaxic coordinates . Glass capillaries with a tip size of 30 μm in outer diameter were loaded with virus. Then, 500 nL of virus-containing solution was injected into the motor cortex at an injection rate of 1 nL/s. After injection, injection needles were left in place for an additional 10 min to assure even distribution of the virus and were then slowly withdrawn.Six-week-old male C57BL6/J mice and GAT-1Immediately after photothrombotic stroke was induced, 26-gauge, 3.5 mm guide cannulae (RWD Life Sience) were implanted into the core of the infarction . The cannulae were fixed to the skull using adhesive luting cement and acrylic dental cement. Following surgery, stainless steel obturators were inserted into the guide cannulae to avoid obstruction until infusions were made. For drug infusions, obturators were removed and 33-gauge, 4.0 mm injectors (RWD Life Science) were placed into the guide cannulae. Injector tips extended 0.5 mm beyond the guide cannulae. During drug injection, 10 μM NO-711 (Tocris Bioscience), or 1 mg/mL TrkB-Fc (R&D system) were slowly infused at a flow rate of 0.2 μL/min to a total volume of 2 μL. Following infusion, the injectors were left in place for an additional 5 min to allow for diffusion of the drug. Following microinjection, the stainless steel obturators were subsequently reinserted into the guide cannulae.Mice were tested by grid-walking tasks and spontaneous forelimb (cylinder) tasks 7 days before surgery to establish baseline performance and subsequently 4, 12, 19, 26, and 33 days after stroke to evaluate functional deficits. The mice were identified by earmarks and numbered only after finishing the behavioral experiments.Grid-walking tasks were performed as previously described in detail with minor modifications th real time speed) by calculating the time (s) during each rear that the animal spent on either its right forelimb, left forelimb, or both forelimbs. Only rears in which both forelimbs could be clearly seen were included. The percentage of time spent on each limb was calculated and these data were used to derive an asymmetry index as follows:  - .Cylinder tasks were performed as previously described in detail with minor modifications th section throughout the infarct area was collected and processed for Nissl staining. The sections were immersed in Nissl staining solution for 10 min at 40 °C. Images were captured with a microscope .Infarct size was measured by Nissl staining according to the manufacturer's protocol (Beyotime Biotechnology). Briefly, coronal slices (40 μm) were cut using a vibrating blade microtome . Every 8Human cortical neurons were differentiated from human embryonic stem cells  according to the following protocol. hESCs were detached with 1 U/mL Dispase (Thermo Fisher Scientific) to form embryoid bodies in suspension in neural induction medium consisting of DMEM/F12 (Gibco), N2 supplement , and non-essential amino acids  for 7 days. Embryoid bodies were attached on day 7 and neural tube-like rosette structures were formed on days 10-16. Rosette colonies were detached by gently blowing with a pipette and suspended to form neural spheres the next day. For neuronal differentiation, the neural spheres were dissociated with TrypLE (Thermo Fisher Scientific) and attached onto a coverslip precoated with poly-L-ornithine (Sigma-Aldrich). The medium was refreshed every 5-6 days.4 cells/cm2 and infected with LV-GFP for morphological analysis.Primary neurons were isolated from E16 wild-type mice cortex and cultured on dishes coated with poly-L-ornithine in neurobasal medium (Gibco) containing 2% B27 supplement (Gibco), as reported previously Primary astrocytes were isolated from neonatal wild-type mice cortex and cultured on dishes coated with poly-L-ornithine in DMEM/F12 medium containing 10% fetal calf serum (Gibco), as reported previously in vivo conditions of ischemia, hESCs-differentiated cortical neurons , primary mouse cortical cultures (10-12 DIV), and primary mouse astrocyte cultures were rinsed twice with serum- and glucose-free medium and incubated with serum- and glucose-free medium in a hypoxia chamber . The chamber was flushed for 3 h with N2 and CO2 to maintain 2% O2 and 5% CO2. After oxygen glucose deprivation (OGD), the serum- and glucose-free medium was replaced with normal medium and the cultures were returned to a normoxic incubator. The cultures were treated with 0.5 μM trichostatin A  or a mixture of 0.5 μM TSA and 10 μM NO-711 (Tocris Bioscience) 24 h after OGD. An equal volume of vehicle was added as control. After 24 h of drug incubation, the cultures were fixed with 4% paraformaldehyde (PFA) and labeled for immunostaining. Astrocytes were harvested for Western blot analysis at 24, 48, and 72 h after TSA treatment. All cultures were maintained in an incubator  with a humidified atmosphere of 95% air and 5% CO2 at 37 °C, except during OGD.To mimic the g for 5 min to separate cytosol and membrane protein fractions. Phase partitioning resulted in the hydrophilic cytosol proteins layering at the top and the hydrophobic membrane proteins at the bottom. The isolated membrane and cytosol proteins were detected by immunoblotting. To test the purity of the isolated proteins, we measured β-actin (a cytosol protein) and E-cadherin (a membrane protein) by western blot.Membrane protein fractions were extracted using a kit (Merck) according to the manufacturer's protocol. Briefly, peri-infarct tissue or corresponding tissue of sham-operated mice was dissected and homogenized until intact pieces were no longer visible. The homogenized protein mixture was incubated with gentle agitation then centrifugated at 1000 ×Western blot was performed as described in detail previously The detailed methods for immunofluorescence of brain sections and cultured cells were described in detail previously 3, and crosslinking was reversed by incubating the samples overnight at 65 °C. Purified DNA samples were normalized and subjected to real-time PCR for 45 cycles, using primer pairs specific to 150-250 bp segments corresponding to mouse gene promoter regions .ChIP assays were performed using a kit (EMD Millipore) according to the manufacturer's protocol. Briefly, peri-infarct tissue or corresponding tissue of sham-operated mice was homogenized, chemically cross-linked in 1% formaldehyde for 10 min, and then quenched with 125 mM glycine for 5 min. Chromatin was sheared to 200-1000 bp using a sonifier  set to 45% maximum amplitude for twenty-five 20 s pulses with 50 s pauses. The samples were immersed in an ice bath during shearing. After centrifugation, cell debris was removed and the supernatants were diluted with ChIP dilution buffer. A fraction of the diluted supernatant was collected as immunoprecipitation input control. The remainder was incubated with Protein A-Sepharose beads for 30 min at 4 °C to reduce nonspecific background. The beads were removed by centrifugation, and the immunoprecipitating antibody against acetylated histone H4  was added to the supernatant and incubated overnight. Then, immune complexes were enriched by incubation with Protein A-Sepharose beads for 1 h at 4 °C. The beads were collected and subjected to a series of sequential washes. Chromatin was eluted from the beads by agitation in elution buffer containing 1% sodium dodecyl sulfate and 0.1 M NaHCOT method. All reactions were performed in triplicate.Real-time PCR was performed with TB-Green reagents (TaKaRa) using a real-time PCR detection system . The relative quantities of immunoprecipitated DNA fragments were calculated by the ΔΔCGat-1 forward: CAT GGG TGG ACC TAT GCC TC, Gat-1 reverse: AGG GAC ACA ATG GTA GAC GC; Gat-3 forward: CTG CAT GGT GCA TGG TCA TTG, Gat-3 reverse: GCT GAG TAT CCT GCT TCA CCT; Bdnf-p1 forward: TGA TCA TCA CTC ACG ACC ACG, Bdnf-p1 reverse: CAG CCT CTC TGA GCC AGT TAC G; Bdnf-p4 forward: GCG CGG AAT TCT GAT TCT GGT AAT, Bdnf-p4 reverse, GAG AGG GCT CCA CGC TGC CTT GAC G.The primer sequences used for PCR were 2, 7 mM MgCl2, 1.3 mM NaH2PO4, 25 mM NaHCO3, 1.3 mM sodium ascorbate, and 0.6 mM sodium pyruvate (pH 7.4). The slices were transferred to an interface-style chamber and incubated in artificial cerebrospinal fluid (ACSF) containing 10 mM glucose, 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1.3 mM MgCl2, 1.3 mM NaH2PO4, 25 mM NaHCO3, 1.3 mM sodium ascorbate, and 0.6 mM sodium pyruvate. The slices were recovered at 34 °C for at least 1 h before recording. All external solutions were saturated with 95% O 2 / 5% CO2 gas.Under deep ethyl ether anesthesia, mice were decapitated and their brains and spinal cords were quickly removed. Slices  were cut in ice-cold cutting solution containing 110 mM choline chloride, 20 mM glucose, 2.5 mM KCl, 0.5 mM CaClSlices or hESCs-differentiated neurons (43-45 DIV) were transferred to a recording chamber that was continuously perfused with oxygenated ACSF . The slices were visualized using an upright microscope  with a 5× 0.1 NA or 60× 1.0 NA objective and infrared and differential interference contrast optics. The fluorescent neurons were visualized under an Olympus X51W microscope equipped with a 60× water-immersion lens and illuminated with a mercury lamp. Whole-cell recordings were made in pyramidal neurons from peri-infarct tissues or tissues from corresponding locations in sham-operated mice using patch-clamp electrodes . All recordings were low-pass filtered at 2 kHz and sampled at 10 kHz using a digitizer . Access resistance was 5-25 MΩ and was monitored throughout the experiment (neurons with access resistance changes > 20% were excluded). Data were collected with pClamp 10.3 software and analysed using Clamfit 10.3 (Molecular Devices). All experiments were performed by a researcher blinded to group allocation.To reconstruct the morphology of the putative pyramidal neurons, 0.2% biocytin (Sigma-Aldrich) was added to the internal solution. After recording, the micropipette was gently retracted from the cell to achieve an outside-out patch for better morphological reconstruction of biocytin-filled neurons. Slices were fixed with 4% PFA and processed for post hoc immunohistochemistry.Itonic) and mean phasic current recordings, patch-clamp electrodes were filled with an intracellular solution containing 120 mM CsMeSO4, 10 mM CsCl, 5 mM TEA-Cl, 1.5 mM MgCl2, 10 mM HEPES, 0.1 mM EGTA, 2 mM Na-ATP, 0.5 mM Na-GTP, and 5 mM QX-314 with the pH adjusted to 7.25-7.30 by CsOH (275-285 mOsmol). To replenish the extracellular GABA concentration reduced by the high-flow perfusion of the slices, the recording ACSF was supplemented with 5 μM GABA. Itonic and mean phasic current were recorded by voltage clamping at +10 mV using an amplifier . Itonic was recorded as the baseline shift after administration of 100 μM bicuculline methiodide . Tonic current density was calculated as follows: current amplitude/membrane capacitance. The amplitude and frequency of spontaneous inhibitory post-synaptic current (sIPSC) before BMI administration were analyzed using MiniAnalysis Program 6.0 (Synaptosoft Inc.).For tonic inhibitory current .For action potential (AP) recordings, glass pipettes were loaded with an intracellular solution containing 70 mM potassium gluconate, 70 mM KCl, 2 mM NaCl, 2 mM MgCl2, 2 mM MgCl2, 10 mM HEPES, and 4 mM K2ATP4 (pH 7.25-7.30).For spontaneous postsynaptic current (sPSC) recordings in hESCs-differentiated neurons, the membrane potential was held at -70 mV with the following intracellular solution: 140 mM CsF, 10 mM BAPTA, 1.0 mM CaCl2, 0.5 mM EGTA, 10 mM HEPES, 4 mM ATP, 5 mM QX-314 with the pH adjusted to 7.25-7.30 by CsOH (275-285 mOsmol). To study mEPSC activity in isolation, 0.5 μM tetrodotoxin and 20 μM BMI were added to block action potentials and GABAA receptor-mediated currents, respectively. The recordings were longer than 5 min. Data were analyzed using MiniAnalysis Program 6.0 (Synaptosoft Inc.). Up to 100 events from each neuron were selected at a fixed sampling interval to generate cumulative probabilities.For miniature excitatory postsynaptic current (mEPSC) recordings, microelectrodes were filled with internal pipette solution, containing 132.5 mM cesium gluconate, 17.5 mM CsCl, 2 mM MgCl2, 0.5 mM EGTA, 10 mM HEPES, 4 mM ATP, and 5 mM QX-314 with the pH adjusted to 7.25-7.30 by CsOH (275-285 mOsmol). We added 10 μM NBQX and 20 μM BMI to block AMPA receptor-mediated currents and GABAA receptor-mediated currents, respectively. Optogenetic stimulation during recordings was performed using a 470 nm light-emitting diode (LED) source coupled to a fiber guide and a collimator lens to focus the light spot around the recorded neuron.For channelrhodopsin-2 (ChR2)-mediated current recordings, microelectrodes were filled with internal pipette solution containing 132.5 mM cesium gluconate, 17.5 mM CsCl, 2 mM MgClFor LFP recordings, microelectrodes (1-2 MΩ) were filled with ACSF. A collimated 470 nm LED was used to photostimulate ChR2-expressing peri-infarct motor cortex fibers in the C5-C7 cervical spinal cord segment. The slices were stimulated by a single 20 ms light pulse repeated every 4 s for 20 repetitions.+ fibers (from intact to lesioned side) was manually counted by an investigator blinded to group allocation. Three sections (80 µm apart) at the level of the medullary pyramids from each mouse were assessed.Tract tracing was used to assess post-stroke axonal sprouting, as described previously All mice were numbered by earmarks and were randomly divided into groups using a table of random numbers generated using Microsoft Excel. Modified neurological severity score, which is positively correlated with infarct volume, was tested 3 days after stroke to assess neurological outcome t-test (Stata 9.0 software). The threshold level of significance was set at p < 0.05. The sample size was obtained with power analysis and sample size software using a significance level of α = 0.05 with 90% power to detect statistical differences. For animal studies, the sample size was predetermined by our prior experiments. All experiments and data analyses were conducted blinded.Data are presented as mean ± standard error of the mean (SEM). Comparisons among multiple groups in the behavioral assessments were made with two-way repeated-measures ANOVA followed by post hoc Bonferroni test (SPSS Statistics 22 software), and other comparisons among multiple groupswere made with one-way ANOVA followed by post hoc Scheffe test (Stata 9.0 software). Comparisons between two groups were made with two-tailed student's Figure ). Peri-infarct pyramidal neurons were recorded in whole-cell mode and identified using biocytin . Whole-cell voltage-clamp recordings in acute brain slices showed that EE blocked the stroke-induced increase in GABAAR-mediated Itonic in layer 5 pyramidal neurons . Additionally, EE significantly increased sIPSC frequency but not amplitude compared to SH , suggesting an enhanced phasic GABA inhibition through presynaptic mechanisms. Tonic inhibition in layer 5 neurons in the neocortex plays a critical role in the control of network excitability Figure ), indicating an EE-induced excitability enhancement. The EE-induced excitability enhancement of principle neurons in the peri-infarct cortex is necessary for stroke recovery . Moreover, we investigated whether the excitability of inhibitory interneurons is implicated in EE-induced stroke recovery and found that silencing the activity of peri-infarct interneurons also cancelled the beneficial effects of EE on stroke recovery , suggesting the importance of phasic GABA signaling in EE-mediated stroke recovery. Additionally, EE exposure during the repair phase of stroke did not affect infarct volume .To determine whether tonic and phasic GABA inhibition are implicated in EE-induced neuronal excitability in the peri-infarct zone during the repair phase of stoke, we induced photothrombotic stroke in the forelimb motor cortex of mice, in which the ischemic infarct core and peri-infarct zone were well defined Gat-1 is a hub gene ) and the expression and function of GAT-1 are critically regulated by BDNF Gat-1, Gat-3, and Bdnf. EE effectively reversed the stroke-induced downregulation of promoter region acetylation of Gat-1, Bdnf-p1, and Bdnf-p4 but not Gat-3 in the peri-infarct cortex . More importantly, consistent with the ChIP assays, stroke-induced downregulation of GAT-1 expression in the peri-infarct cortex was rescued by EE exposure . Furthermore, immunofluorescence of GAT-1 in the peri-infarct cortex also showed that EE relieved the decrease in GAT-1 caused by stroke . To test whether EE inhibits GAT-1 internalization as BDNF does (17), membrane and cytosol protein fractions were extracted from the peri-infarct cortex and the corresponding cortex area of sham-operated mice. β-actin was detected in the cytosol fractions but not in the membrane protein fractions, while E-cadherin was detected in the membrane protein fractions but not in the cytosol protein fractions , confirming the purity of the isolated membrane and cytosol proteins. Immunoblotting analysis showed that EE reversed the stroke-induced decrease in GAT-1 in the plasma membrane fraction and increase in the cytosol fraction , suggesting that EE may modify the trafficking of GAT-1. To further explore the role of BDNF in GAT-1 trafficking, TrkB-Fc, a mature BDNF scavenger ), suggesting that BDNF deficiency leads to trafficking of GAT-1 from membrane to cytosol. Given that the beneficial effects of EE have been associated with chromatin remodeling in vitro to mimic EE conditioning in vivo. Immunofluorescence of GAT-1 in hESCs-derived neurons that underwent this mimetic ischemia confirmed that TSA reversed the GAT-1 internalization caused by stroke . Consistent with the reported literature ). However, immunoblotting analysis showed that ischemia and TSA treatment had no effect on the expression of GAT-1 in astrocytes , suggesting that EE exposure mainly upregulated the expression of GAT-1 in neurons. HDAC2 has been reported to be a critical factor in EE-mediated stroke ). Thus, GAT-1 may be downstream of BDNF, although HDAC2 negatively regulates the expression of BDNF and GAT-1 at the same time. To understand the relationship between EE and GAT-1 function in regulating phasic and tonic GABA inhibition, we exposed mice to EE or SH on days 5-11 and performed electrophysiology on day 12 after stroke. We incubated acute brain slices from stroke mice with NO-711, a GAT-1-selective antagonist, and recorded tonic GABA currents and sIPSCs in the peri-infarct pyramidal neurons. NO-711-induced increases in Itonic in sham and EE-treated stroke mice were significantly higher than those in SH-treated stroke mice , suggesting that stroke caused GAT-1 dysfunction and EE restored GAT-1 function. Furthermore, NO-711 diminished sIPSC frequency in all groups , suggesting that GAT-1 mediated phasic GABA inhibition through presynaptic mechanisms and NO-711 abolished the effect of EE on sIPSC frequency , indicating that EE modifies phasic GABA inhibition through GAT-1. Together, these data demonstrate that EE positively regulates the expression and function of GAT-1 in the peri-infarct cortex during the repair phase of stroke.GAT-1 preferentially expressed in neurons DNF does , membranDNF does , membran). The ratio between mushroom-like and thin spines (M/T) is an indicator of spine maturation ). Thus, GAT-1 function is implicated in the epigenetic regulation of structural neuroplasticity.Chronically raised tonic inhibition antagonizes neuronal plasticity Figure ), mEPSC frequency was significantly decreased in SH-treated mice subjected to stroke compared to sham-operated mice, exposure to EE reversed the stroke-induced decrease in mEPSC frequency, and NO-711 abolished the promotion effect of EE on mEPSCs, indicating that GAT-1 is critical for EE-induced input synaptic strengthening in the peri-infarct cortex.Dendritic spines are the postsynaptic sites of excitatory synapses and receive excitatory synaptic inputs Figure ). Three weeks later, ChR2-eYFP, identified as green fluorescence, was abundantly expressed in surviving neurons in the peri-infarct cortex . Furthermore, ChR2-expressing peri-infarct motor cortex fibers abundantly projected to the C5-C7 cervical spinal cord segment . To validate the functionality of expressed ChR2, we illuminated the peri-infarct cortex in the acute slices with blue light (470 nm) . The illumination resulted in inward currents in transduced pyramidal neurons under voltage-clamp recording conditions at a holding potential of -70 mV that followed the light power density, confirming the functionality of expressed ChR2 . Next, we recorded LFPs in the C5-C7 cervical spinal cord segment by photostimulating the fibers projected from the peri-infarct cortex . Stroke caused a substantial decrease in synaptic responses in the CST, which was ameliorated by EE exposure. Interestingly, NO-711 abolished the EE-dependent enhancement in LFPs . Thus, GAT-1 is required for EE-induced output synaptic strengthening in the CST.Corticospinal tract (CST) lesion is a biomarker for stroke motor outcomes flox/floxmouse line . We infused AAV-CAG-EGFP-Cre into the motor cortex of GAT-1flox/floxmice to specifically delete local GAT-1. Stroke was induced 3 days after virus microinjection. Then, mice were exposed to EE or SH on days 5-11 after stroke. The tract tracer BDA was microinjected into the contralesional cortex on day 34 after stroke . Fourteen days later, we performed fluorescence staining of BDA in the contralesional motor cortex and spinal cord. BDA fluorescence in the contralesional motor cortex was comparable between groups, suggesting that BDA injection was consistent . EE exposure significantly increased the number of BDA+ fibers crossing the midline into the denervated spinal cord at the C7 level, and the EE-induced sprouting of uninjured corticospinal axons was abolished by deleting GAT-1 in the peri-infarct cortex . Together, the function of GAT-1 in the peri-infarct cortex is crucial for EE-induced neuronal plasticity in the cortex and CST after stroke.Axonal sprouting is an attractive mechanism underlying the restoration of neuronal networks and functions after stroke Figure S9). To determine whether GAT-1 can serve as the target molecule, mice were exposed to EE or SH and NO-711 or vehicle was infused daily into the peri-infarct cortex on days 5-11 after stroke. Motor function was measured 7 days before stroke and 4, 12, 19, 26, and 33 days after stroke . Mice subjected to photothrombotic stroke in the forelimb motor cortex showed a marked impairment in motor function compared to sham-operated mice. The mice exposed to EE displayed significantly better motor function than mice exposed to SH on days 12-33 after stroke, and treatment with NO-711 abolished the EE-mediated functional recovery from stroke . To further confirm whether GAT-1 is a key mediator for EE-dependent functional recovery, AAV-CAG-EGFP-Cre was microinjected into the motor cortex of GAT-1flox/floxmice 3 days before stroke to specifically delete peri-infarct GAT-1, and motor function was measured 7 days before and 4, 12, 19, 26, and 33 days after stroke . GAT-1 was almost completely deleted in the AAV-Cre-infected cells in the peri-infarct cortex . In wild-type animals, EE exposure significantly ameliorated motor function, while it had no effect in mice with GAT-1 conditional knockout in the peri-infarct cortex , suggesting that GAT-1 is essential for EE-induced functional recovery after stroke.Owing to the difficulties in translating EE from the bench to the bedside in stroke Figure ). AAV-GAT-1-3Flag effectively infected the peri-infarct cortex  and significantly upregulated GAT-1 in the peri-infarct cortex . Furthermore, as an important control of GAT-1 overexpression, we generated an AAV vector expressing GAT-3 (AAV-CAG-GAT-3-3Flag-T2A-mCherry) and its control AAV-CAG-3Flag-T2A-mCherry. We infused these vectors into the motor cortex of mice 3 days before stroke, and performed immunoblots on day 5 after stroke. The immunoblots confirmed that AAV-CAG-GAT-3-3Flag-T2A-mCherry significantly upregulated GAT-3 in the peri-infarct area . To visualize peri-infarct pyramidal apical dendritic spines, LV-Ubi-GFP was microinjected into the motor cortex of mice 10 days before stroke. AAV-hSyn-GAT-1-3Flag or AAV-CAG-GAT-3-3Flag-T2A-mCherry was microinjected 7 days later. Immunofluorescence staining was performed 5 days after stroke. GAT-1 but not GAT-3 overexpression attenuated the stroke-induced decrease in spine density in the peri-infarct area , suggesting that GAT-1 is critical for structural plasticity after stroke. The NO-711-induced increase in Itonic in AAV-control-treated sham mice was significantly higher than that in AAV-control-treated stroke mice , again confirming GAT-1 dysfunction in peri-infarct neurons. A similar trend was observed in mice treated with AAV-GAT-1-3Flag , suggesting restoration of GAT-1 function. Moreover, peri-infarct pyramidal neurons from AAV-GAT-1-3Flag-treated mice showed significantly increased sIPSC frequency but not amplitude compared to AAV-control-treated sham and stroke mice . To test whether GAT-3 overexpression can rescue electrophysiological defects in GAT-1 conditional knockout neurons, AAV-CAG-EGFP-Cre was microinjected into the motor cortex of GAT-1flox/floxmice 10 days before stroke to specifically delete peri-infarct GAT-1. AAV-CAG-GAT-3-3Flag-T2A-mCherry was microinjected into the motor cortex of GAT-1flox/floxmice 7 days later to overexpress GAT-3. Tonic inhibitory currents were recorded 5 days after stroke. Importantly, GAT-3 overexpression did not rescue the GAT-1 knockdown-induced increase in Itonic in peri-infarct layer 5 pyramidal neurons . Collectively, overexpression of GAT-1 restored the roles of GAT-1 in regulating phasic and tonic inhibition in the peri-infarct cortex. Importantly, overexpression of GAT-1 but not GAT-3 significantly ameliorated stroke-induced functional impairment 12 to 33 days after stroke compared with AAV-control-treated stroke mice . Thus, GAT-1 function is crucial for functional recovery from stroke.To directly evaluate the role of GAT-1 in functional recovery from stroke, we generated an AAV vector expressing GAT-1 (AAV-hSyn-GAT-1-3Flag-T2A-EGFP) and its control AAV-hSyn-3Flag-T2A-EGFP. We infused these vectors into the motor cortex of mice 3 days before stroke, performed immunoblots, immunofluorescence staining, and electrophysiological recordings on day 5 after stroke, and measured motor function 7 days before and 4, 12, 19, 26, and 33 days after stroke . Therefore, GAT-1 but not other GATs, is a good target for treating stroke during the repair phase.Extracellular concentration of GABA is effectively controlled by GATs, which critically determines tonic conductance, neuronal excitability threshold, and LTP induction Figure ). Although tonic GABAergic inhibition controls network excitability AR antagonist that reverses tonic inhibition in peri-infarct tissue, did not improve clinical outcome in patients after stroke AR exhibits both synaptic phasic and extrasynaptic tonic inhibition AR antagonist enhances network excitability by reducing tonic inhibition, blocking α5 GABAAR may also impair GABAergic synaptic transmission, a process that contributes to the homeostasis of circuit excitability Figure ). Additionally, elimination of phasic GABA signaling by silencing the activity of GABAergic inhibitory neurons abolished the effect of EE on functional recovery from stroke . Therefore, reducing tonic and enhancing phasic GABA inhibition at the same time may modify network excitability in a regular and controlled manner, thereby improving stroke recovery. Indeed, regular plasticity is of critical importance for stroke recovery Structural and functional plasticity is fundamental for functional recovery from stroke Bdnf in the peri-infarct cortex and promotes stroke recovery Bdnf . Simultaneously, the expression and membrane localization of GAT-1 are positively regulated by BDNF  and associated with the beneficial effects of EE Figure 6). Thus, targeting GAT-1 may have advantages over other EE-related target molecules, such as BDNF and HDACs.BDNF is crucial for EE-induced neuroplasticity and stroke recovery Figure 8). From these results, we identify a novel therapeutic target for functional recovery from stroke. Drugs targeting GAT-1 may mimic the impact of EE and offer a novel therapeutic strategy for stroke recovery within a feasible time window. Given that EE-induced network plasticity benefits various neuropsychiatric disorders, targeting GAT-1 may bring hope to the treatment of other diseases and is not limited to stroke.In summary, our findings indicate that EE-induced network plasticity enhancement and consequent stroke recovery during the repair phase depend on GAT-1-mediated amplification of phasic GABA signaling and reduction of tonic GABA signaling (Supplementary figures.Click here for additional data file.


Scientific Reports 10.1038/s41598-019-44541-4, published online 30 May 2019Correction to: In Figure 7, the sample labels for panels A and B are incorrectly ordered. The correct Figure 7 appears below as Figure


In this study, we hypothesized that significantly altering the suppressive tumor immune landscape in NSCLC could potentially improve ICB efficacy. Using sub-therapeutic doses of our Salmonella typhimurium-based therapy targeting the suppressive molecule indoleamine 2,3-dioxygenase (shIDO-ST) in tumor-bearing mice, we observed dramatic changes in immune subset phenotypes that included increases in antigen presentation markers, decreased regulatory T cell frequency and overall reduced checkpoint protein expression. Combination shIDO-ST treatment with anti-PD-1/CTLA-4 antibodies enhanced tumor growth control, compared to either treatment alone, which was associated with significant intratumoral infiltration by CD8+ and CD4+ T cells. Ultimately, we show that increases in antigen presentation markers and infiltration by T cells is correlated with significantly increased survival in NSCLC patients. These results suggest that the success of ICB therapy may be more accurately predicted by taking into account multiple factors such as potential for antigen presentation and immune subset repertoire in addition to markers already being considered. Alternatively, combination treatment with agents such as shIDO-ST could be used to create a more conducive tumor microenvironment for improving responses to ICB.Therapeutic options for non-small cell lung cancer (NSCLC) treatment have changed dramatically in recent years with the advent of novel immunotherapeutic approaches. Among these, immune checkpoint blockade (ICB) using monoclonal antibodies has shown tremendous promise in approximately 20% of patients. In order to better predict patients that will respond to ICB treatment, biomarkers such as tumor-associated CD8 Howevs (TCRs) ,4. An ims (TCRs) . Antibods (TCRs) ,9,10. Ses (TCRs) ,12.The presence of another cell surface molecule, cytotoxic T lymphocyte-associated protein 4 (CTLA-4), suppresses T cell responses by acting as a homolog for CD28 that competes for binding to activating MHC co-receptors present on antigen presenting cells (APCs) . Treatme+ T cells (Tregs) and myeloid derived suppressor cells (MDSCs) [Two major cell types have been connected to checkpoint blockade failure: regulatory CD4 (MDSCs) ,19,20. T (MDSCs) . MDSCs s (MDSCs) ,23,24 wh (MDSCs) . MDSCs c (MDSCs) , inhibit (MDSCs) ,28. Fina (MDSCs) .Recently, a novel subset of anti-tumor neutrophils have been identified in the early stages of NSCLC that are capable of inducing anti-tumor immunity through attainment of antigen presentation capabilities ,31,32,33Salmonella typhimurium-based platform targeting IDO (shIDO-ST) is engulfed by tumor-infiltrating neutrophils, leading to hyperactivation of their cytotoxic activity and subsequent tumor regression [Utilizing various cancer models, we have confirmed that our gression ,35. In tgression ,37. Altegression ,39.LSL-G12D/+; Trp53R270H/+Kras (KP) mice were provided by Dr. Thomas Ludwig  [® (CRL-1642). Cells were maintained in RPMI media containing 10% FBS, 2mM l-glutamine and pen/strep. Prior to subcutaneous implantation into C57BL/6 mice, cells were passaged ≤5 times and maintained at ≤80% confluency.Experiments involving animals were performed according to the standards of the Institution Animal Care and Use Committee (IACUC) at the City of Hope ). Male C57BL/6 mice, 8–10 weeks old, were obtained from breeding colonies housed at the City of Hope Animal Research Center. OH, USA) . The Lew® (202165™). YS1646 was cultured in modified LB media containing MgSO4 and CaCl2 in place of NaCl. The pAKlux2 plasmid was a kind gift from Attila Karsi . ShScr and shIDO plasmids  [YS1646 was obtained from ATCCCA, USA)  were ele6 cfu of shScr, shIDO-ST or HBSS volume equivalent followed by 2 × 106 cfu on the second and 1 × 106 cfu on the third consecutive day intravenously via the retro-orbital vein. Blood and spleens were collected after euthanization 48 or 72 h after the third ST administration. For peritoneal neutrophil isolation, 4 × 107 cfu of shScr, shIDO-ST or HBSS volume equivalent was injected into the intraperitoneal cavity of mice followed by 2 × 107 cfu on the second day. Three hours after the second administration, mice were sedated and 5 mL of HBSS was injected into the intraperitoneal cavity; the abdomen was massaged to dislodge cells into the fluid. Then, the HBSS with cells was removed using a large gauge needle. The solution was then overlaid on top of a 0/52/62.5/78 percent Percoll gradient and centrifuged at 700× g for 30 min without acceleration or brake. Individual layers were removed by transfer pipette to individual tubes for washing. Wright’s staining  was performed on 10–25 µL of washed layers that were smeared and allowed to dry on slides before fixation in 100% methanol.For blood and spleen neutrophil isolations, on the first day mice were administered 5 × 10Tumor growth in the lungs of KP mice was initiated through inhalation of adenovirus expressing Cre recombinase , Lot#Ad4067), according to the intranasal method described in DuPage, et al. 2009 . IntranaIHC was performed on Ventana Discovery Ultra IHC autostainer  according to manufacturer’s protocols. Briefly, tissue samples were sectioned at a thickness of 5 μm and put on positively charged glass slides. Deparaffinization, rehydration, endogenous peroxidase activity inhibition and antigen retrieval were all performed on the automated stainer. Slides were then incubated with primary antibodies, followed by DISCOVERY HQ and DISCOVERY HQ-HRP system, visualized with ChromoMap DAB detection Kit . The slides were then counterstained with haematoxylin  and coverslipped. Antibodies used: CD4, CD8a, and CD11c at a dilution of 1:100 . DAB staining out of total nuclei per field was done using ImageJ . DAB and hematoxylin channels were separated using color deconvolution (H-DAB preset), and thresholds were set to cover positive staining area for DAB and positive staining nuclei for hematoxylin. DAB-positive nuclei were quantified by dividing DAB threshold area by DAB threshold area plus hematoxylin threshold area.5) were implanted subcutaneously in HBSS. Tumors were allowed to grow to an average of ~200 mm3 and then injected retro-orbitally with 1 × 106 ST electroporated with the pAKlux2 plasmid. Mice were imaged for luminescence in the Small Animal Imaging Core at City of Hope using the Lago biphotonic imaging system .The right-side midsections of mice were shaved and LLC1 cells (2 × 105 per mouse) in the right-side midsection of the mice. After 6 days of growth, treatment with shScr-ST or shIDO-ST began with three consecutive daily doses of 1 × 106 cfu per mouse. On the first day of ST treatment, mice were concurrently given 200 µg of anti-PD-1 (clone J43) antibody or IgG equivalent  and 75 µg of anti-CTLA-4 (9H10) antibody or IgG equivalent . Treatments continued every three days until the end of the experiment (maximum tumor growth of 15 mm diameter for control groups). Tumor volumes were measured thrice weekly using manual calipers. Lower doses from previously published regimens of anti-PD-1 [LLC1 cells were injected subcutaneously  for 30 min at 4 °C. Cells were washed in flow wash buffer (PBS with 0.05% sodium azide and 1% FBS) and stained with surface antibodies for 40 min at 4 °C. Cells were washed in flow buffer and fixed in flow buffer plus 1% PFA before filtering through 40 μm mesh strainer/tube . Flow cytometry was performed on the BD FACSCelesta cytometer and data was analyzed using FlowJo Version 10 . For flow cytometry from spleens, spleens were removed and mashed in complete RPMI medium (10% FBS with glutamine and pen/strep) on a 70 µm mesh strainer with a syringe plunger. Cells were washed in PBS and stained using the above protocol. For flow cytometry on tumor cells, tumors were excised and digested mechanically by mincing with a sterile scalpel before digestion in 1 mg/mL collagenase I  plus 1% FBS for 1.5 h shaking at 200 rpm in a 37 °C incubator. Dissociated tumor cells were spun at 450 g for 10 min and filtered through a 70 μm strainer. After washing, these cells were stained for flow cytometry following the above protocol. Flow cytometry antibodies used from BD biosciences : CD45-PerCPCy5.5 (550994), CD8-APC-R700 (564983), CD4-APC-H7 (560246), Ly6G-BV605 (563005), Ly6C-FITC (561085), CD11b-APC (561690), PD-1-BV421 (562584), CTLA-4-PE (553720), CD45-APC-R700 (565478), CD3-PerCPCy5.5 (551071), NK1.1-BV650 (564143), CD11c-BV605 (563057), CD45-BV650 (563410), CD4-BV786 (563331), CD25-PerCPcy5.5 (551071), MHCII-PerCPCy5.5 (562363), CD11c-APC-R700 (565871), IFN-gamma-APC (554413), CD27-BV450 (561245), andCD62L-BV605 (562720). Flow cytometry antibodies used from eBiosciences : F4/80-Super Bright 780 (78-4801-82), PD-L1-Super Bright 645 (64-5982-82), PD-L1-Super Bright 780 (78-5982-80), CD86-Super Bright 645 (64-0862-80), FoxP3-APC (17-5773-82), and Granzyme B-PE (12-8898-80). For intracellular staining against CTLA-4, cells were permeabilized using the Cytofix/Cytoperm Plus kit . For intracellular staining against FoxP3, cells were permeabilized using the FoxP3/transcription factor staining buffer set from eBiosciences .n = 513) were selected from the pan-cancer RNA-seq dataset for analysis of overall survival comparing high and low expression of CD11c (ITGAX) and MHCII . Three restriction scenarios were used: (1) no restriction of subtypes, no restriction of cellular content, (2) no restriction of subtypes, restriction to samples with enriched CD4 and CD8 content and decreased Treg content, and (3) no restriction of subtypes, restriction to samples with decreased CD4 and CD8 content and enriched Treg content.KMplot.com  was usedAll statistical analyses were done using the Prism software by GraphPad (V8). Unless otherwise noted in figure legends, statistics were obtained by performing a two-way ANOVA followed by Tukey’s multiple comparisons test.p < 0.022) in shIDO-ST-treated mice. The percentage of Ly6G+ neutrophils was also significantly elevated (p < 0.0007) in the spleens of mice administered shIDO-ST, compared to shScr-ST control treatment . Purified neutrophil populations were then fixed and incubated with the ovalbumin synthetic peptide SIINFEKL, followed by incubation with splenocytes from OT-I mice transgenic for the SIINFEKL-specific T cell receptor [+IFNγ+ T cells. In shIDO-ST-treated mice, the percentage of CD8+IFNγ+ T cells was significantly higher compared to shScr-ST-treated mice (p < 0.05), indicating an enhanced ability of shIDO-ST-induced neutrophils to augment SIINFEKL-specific T cell responses. As expected, these results were only observed in the high-density (activated) neutrophil fraction. Low-density neutrophils from shIDO-ST- or shScr-ST-treated mice did not significantly induce CD8+IFNγ+ responses  ,52 in oru LUX-ST  resultedshIDO-ST ,35,54,55r growth B and didr growth C,D. ThesLSL-G12D/+Kras; R270H/+Trp53(KP) mouse model of NSCLC, where Kras and p53 mutations can be activated in the lung by intranasal administration of adenovirus encoding Cre recombinase (Ad-cre) [6 cfu) [+ cells (p < 0.0001) in transformed lung tissue (as determined by IHC), while CD4+ and CD8+ cells remained unchanged and were largely confined to blood vessels with little infiltration into tumor tissue (We next examined the effects of low-dose shIDO-ST treatment on immune cell infiltration in a (Ad-cre) . Followi [6 cfu) ,35 and ir tissue E. As the+ and CD8+ T cells and myeloid-derived immune subsets 48 h post-shIDO-ST treatment were comparable to HBSS (no-ST) and shScr-ST control groups   D. These + cells are significantly increased in shIDO-ST-treated mice compared to shScr-ST-treated mice (p < 0.0001)  , possibl 0.0001) . Splenic 0.0001) . Howeverted mice A. In addNK cells B. FurtheNK cells C. Decrea+ (+ T cells (p = 0.0007), shIDO-ST + IgG (p = 0.0020) and shScr-ST + ICB (p = 0.0020). This reduced tumor growth remained significant through the end of the study, while tumor growth in control groups did not differ significantly throughout the study   B. Total n groups C. Viabiln groups .+ and CD8+ T cells compared to all other treatment groups (p < 0.0001), as well as significantly higher numbers of CD11c+ cells  [+ and CD8+ cells in tumors, most markers remained unchanged between shScr-ST + ICB and shIDO-ST + ICB groups except for NK1.1, which was significantly decreased in shIDO-ST + ICB treated tumors compared to shScr-ST + ICB treated tumors   , CD62L (priming) , interfepriming)  and NK1.priming) . Out of  0.0027) C. Howeveincrease D. Tregs increase E and, siincrease F.+ and CD8+ T cell content as well as decreased Treg content, similar to results observed using shIDO-ST+ICB. Furthermore, MHCII expression in tumors with decreased CD4+ and CD8+ T cells and increased Tregs did not correlate with changes in survival. Increased CD11c+ expression significantly correlated with increased overall survival in tumors with enriched CD4+ and CD8+ T cells with decreased Tregs as well as in tumors with decreased CD4+ and CD8+ T cells with increased Tregs  similar to kynurenine (the product of tryptophan conversion by IDO). In addition, to the deleterious effects of kynurenine on T cells ,79, agontype DCs . These dThe ability of shIDO-ST to decrease the suppressive immune phenotype of the TME in the LLC1 NSCLC model, represents a promising mechanism to increase the action of ICB treatments in NSCLC patients. Recently, the CheckMate 9LA phase 3 clinical trial determined that the first line combination of nivolumab (anti-PD-1) and ipilimumab (anti-CTLA-4) with chemotherapy significantly increased overall survival in stage IV and recurrent NSCLC compared to chemotherapy alone . It is a


St6gal1-deficient mice. However, the blood-borne pool of ST6GAL1, upregulated during systemic inflammation, can also extrinsically modify leukocyte cell surfaces. We show that B cell independent, extracellular ST6GAL1 enhances B cell IgG production and increases blood IgG titers. B cells of mice lacking the hepatocyte specific St6gal1 promoter have reduced sialylation of cell surface CD22 and CD45 and produce less IgG upon stimulation. Sialylation of B cells by extracellular ST6GAL1 boosts expression of IgM, IgD, and CD86, proliferation, and IgG production in vitro. In vivo, elevation of blood ST6GAL1 enhances B cell development and systemic IgG in a CD22-dependent manner. Our data point to a function of an extracellular glycosyltransferase in promoting humoral immunity. Manipulation of systemic ST6GAL1 may represent an effective therapeutic approach for humoral insufficiency.Humoral immunity is an effective but metabolically expensive defense mechanism. It is unclear whether systemic cues exist to communicate the dynamic need for antigen presentation and immunoglobulin production. Here, we report a novel role for the liver-produced, acute phase reactant ST6GAL1 in IgG production. B cell expression of ST6GAL1, a sialyltransferase mediating the attachment of α2,6-linked sialic acids on N-glycans, is classically implicated in the dysregulated B cell development and immunoglobulin levels of  We report a novel role for the liver-produced, acute phase protein ST6GAL1 in enhancing B cell development and in IgG production in a CD22-dependent manner Acute phase reactants are widely used as biomarkers of rheumatic and inflammatory diseases , 2. Althcis potentiates antigen-induced activation, marginal zone B cell development, as well as T-dependent and T-independent immune responses . Blood was collected from the retro-orbital venous sinus at indicated time points post-BMT for serum analysis and cell sorting. Unless otherwise stated, mice between 6 and 12 weeks of age were used for experiments. Roswell Park Institutional Animal Care and Use Committee approved maintenance of animals and all procedures used under protocol 1071M.C57BL/6J (WT) and B6.μMT mice were purchased from Jackson Laboratory. erations , 31. μMTeviously . CD22-KOFor flow cytometry, anti-CD45.1-PerCP/Cy5.5 (A20), anti-CD45.2-FITC (104), anti-B220-PE/Cy7 (RA3-6B2), anti-CD19-BV510 (GD5), anti-IgD-PE (11-26c.2a), anti-CD23-APC/Cy7 (B3B4), anti-IgM-APC (RMM-1), anti-CD21-BV421 (7E9), anti-CD24-PE (30-F1), anti-CD138-BV421 (281-2), anti-BP-1-PerCP/Cy5.5 (6C3), anti-CD93-PerCP/Cy5.5 (AA4.1), anti-CD86-APC/Cy7 (GL-1), anti-IgG1-FITC (RMG1-1), anti-IgG2a-APC (RMG2a-62), anti-IgG2b-PE (RMG2b-1), anti-IgG3-biotin (RMG3-1) were purchased from BioLegend. SNA-FITC was purchased from Vector Laboratories. Anti-pZap-70 (Y319)/pSyk (Y352)-AlexaFluor488 (65E4) was purchased from Cell Signaling Technology. For magnetic cell separation, biotinylated anti-IgM (RMM-1), anti-B220 (RA3-6B2), and anti-Gr1 (RB6-8C5), were purchased from BD Pharmingen, and anti-IgD-PE (11-26c.2a) from Biolegend. Streptavidin-conjugated microbeads or anti-PE microbeads (Miltenyi Biotechnology) were used to pull down labeled cells. For microscopy, anti-IgM-Cy3 (EMD Millipore), anti-IgD-FITC , anti-B220 , and PNA-Fluorescein lectin (Vector Biolabs) were used. For western blotting, anti-β-actin , anti-ST6GAL1 (R&D Biosystems), anti-CD22 , anti-B220/CD45-biotin , anti-IgM-biotin , anti-rat-HRP , streptavidin-HRP , anti-goat-HRP , and goat anti-mouse-HRP (Bio-Rad).in vitro conditioned supernatant by ELISA (Bethyl Laboratories). For total IgG, serum samples were diluted between 1:5000 and 1:50,000, and conditioned supernatant diluted 1:3. Standards of mouse serum were included in duplicate for quantification of unknowns. For antigen-specific IgG, plates were coated overnight with 10 μg/ml NP-Ova before blocking. IgG titers were quantified by comparison with standards in anti-IgG coated wells in parallel. Absorbance (650 nm) was quantified using a Synergy HTX Reader (Biotek). For analysis, standard values were modeled by best-fit equations (linear or logarithmic), which were used to infer concentrations of unknowns.IgG was quantified in serum and Bone marrow cells were collected by flushing femurs, splenocytes by dissociating and filtering spleens, and peripheral blood collected in citrate-based anticoagulant. All tissues were subjected to ACK lysis to remove anucleated cells. Cells were stained in flow cytometry buffer  with indicated antibodies at 1:100-1:200 dilution. For intracellular p-Syk staining, B cells were stimulated for indicated times, then fixed in 5% formalin for 10 min, washed, and resuspended in BD Cytoperm buffer (BD Biosciences) for 20 min. Cells were then incubated in BD Cytoperm buffer with anti-pSyk antibody (1:100) for 30 min, washed, and analyzed by flow cytometry. All flow cytometry data was collected on BD LSRII cytometer and analyzed with FlowJo software.St6gal1-KO mice, and serial blood samples collected between 1 and 9 days after administration. IgG concentrations, quantitated by ELISA, were plotted and fitted to exponential equations, which were used to determine the half-life of IgG.Hundred μg of Chrompure mouse polyclonal IgG (Jackson ImmunoResearch) was injected intraperitoneally into μMT or μMT/dP1 mice were isolated by magnetic separation . Enriched cells were washed and enumerated, then activated by functional grade mouse anti-IgM (eBioscience), anti-CD40  and either IL-4  or LPS (25 μg/ml) for 3 or 6 days in complete culture medium at 37°C. Cells were then centrifuged at 1000 rpm and cell-free supernatant collected and stored at −80°C until further analysis.Mature B cells (IgD+/IgM-low) from peripheral blood of BMT chimeras at 4–6 weeks post-transplant were sorted by fluorescence-activated cell sorting using BD FacsAria cytometer. Purity was routinely 95%+. In other experiments, bone marrow IgD+ mature B cells from WT or Ex vivo cultured cells were pelleted, then resuspended in TRI reagent (MRC Inc.) and frozen at −80°C. RNA was extracted under RNAse-free conditions according to manufacturer guidelines, then quantified and all samples normalized prior to cDNA synthesis of 750–2000 ng of RNA by iScript cDNA Synthesis kit (Bio-Rad). cDNA was amplified by previously described primers using iQ SYBR Green kit (Bio-Rad) (Bio-Rad) . All traEquivalent numbers of IgD+ bone marrow mature B cells were subjected to membrane protein extraction using Mem-PER Plus kit (Thermo Fisher Scientific). Protein concentration was quantitated by BCA protein assay test (Thermo Fisher Scientific) and normalized prior to overnight incubation with 100 μl of pre-blocked SNA-agarose or agarose beads (Vector Laboratories). Unbound supernatant was saved, and beads were extensively washed before bound protein was boiled off in 50 μl Laemmli buffer with 2-mercaptoethanol. Samples were resolved in a 4–15% gradient gel (Bio-Rad), then transferred to methanol-activated PVDF membranes, blocked for 1 h in 3% BSA solution, and incubated with primary antibodies overnight. Membranes were washed, incubated in secondary antibody for 1 h, then developed with Pierce ECL WB Substrate (Thermo Fisher Scientific) and immediately imaged using ChemiDoc Touch (Bio-Rad).Developing B cells were isolated from bone marrow as described previously . Briefly–7 moles CMP-Neu5Ac (Sigma Aldrich), and either 30 μg ST6GAL1 or equivalent volume of mock solution by tail vein injection. Animals were sacrificed 4 days later for analysis of bone marrow and splenic B cells by flow cytometry and microscopy.Wild-type mice were treated with 60 ng BAFF, 10peanut agglutinin (PNA)-FITC (Vector Biolabs). Slides were washed and incubated with secondary antibody for 1 h where necessary. Stained slides were mounted in 10% glycerol with glass cover slips, then visualized immediately using a Nikon Eclipse E600 microscope with EXFO X-cite 120 light source. Spot RT3 camera and Spot Software were used to capture images.Snap-frozen spleens were sectioned onto charged glass slides, fixed in acetone at −20°C, then rehydrated twice in PBS. Slides were blocked for 1 h in 5% BSA, then stained overnight with anti-IgD-FITC and anti-IgM-Cy3 or anti-B220-biotin and E. coli. Successful insertion was confirmed by two restriction digests. Mice were intravenously administered 20 μg of empty vector or rST6-containing plasmids in a volume of 2 ml saline. Blood collections were made prior to transfection and at indicated times. Tissue analysis by flow cytometry was performed upon sacrifice. Quantification of sialyltransferase activity in serum was performed using an artificial O-benzyl-linked disaccharide acceptor, as described previously (pLive plasmids (Mirus Bio) containing AFP enhancer and a downstream minimal albumin promoter were purchased. Rat secretory ST6GAL1 DNA sequence (∼600 bp) was inserted between introns 1 and 2 and expressed in transformed eviously .St6gal1 leads to humoral immunodeficiency, with severely reduced antigen-induced proliferation, antibody production, and marginal zone B cells  and were natively deficient in IgM+ immature and mature B cells, ensuring that all mature B cells were donor-derived and St6gal1-competent  and co-stimulatory CD86 . Fo. FoSt6ga1 status . At 8 we1 status . To unde1 status . Mature ory CD86 . We also/− hosts . To test10 weeks . St6gal1antation . Importaantation .St6gal1 gene in mice and humans  and germline transcripts for IgG1, IgG2a, IgG2b, and IgG3 remained unchanged, suggesting isotype switching was unaltered by circulating liver-derived ST6GAL1 . Interestingly, we observed a reduction in α2,6-sialylated CD45 and CD22 in dP1 B cells, despite equivalent cytosolic levels of ST6GAL1 protein within B cells of both genotypes  or mock control. Ex vivo maturation of immature B cells into functionally responsive mature B cells by BAFF treatment has been previously reported  on B cell development. All mice also received intravenous CMP-Sia sugar donor. In order to enhance B cell development, mice were administered concurrent recombinant murine BAFF, as has been reported elsewhere . After 4cle size . Flow cycle size  42, 43), 43−/− mcle size . Within cle size . Treatme in vivo .in vivo IgG production, we developed a model in which systemic ST6GAL1 levels are artificially elevated by hepatic overexpression. A plasmid encoding the secretory form of rat St6gal1 under the albumin promoter was constructed, ensuring hepatocyte-restricted expression of the transgene. The expressed enzyme lacks the transmembrane domain and is thus uniformly secreted  plasmid exhibited a significant and prolonged increase in blood sialyltransferase activity up to at least 8 weeks post-transfection (p < 0.001). This effect was abrogated in CD22−/− mice, wherein blood IgG titers did not significantly differ between treatment and control groups  and co-stimulation , as well as the potential for antigens themselves to harbor glycans, it is worth noting that total blood IgG levels may represent the final outcome of perturbed glycosylation in a number of cell types , review, and editing. PP contributed to the conceptualization, methodology, and formal analysis. JL contributed to the conceptualization, resources, writing (review and editing), supervision, projection administration, and funding acquisition.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Alterations in microRNA (miRNA) profiles, induced by tumor microenvironment stressors, like hypoxia, allow cancer cells to acquire immune-resistance phenotypes. Indeed, hypoxia-induced miRNAs have been implicated in cancer progression through numerous cancer cell non-autonomous mechanisms, including the direct transfer of hypoxia-responsive miRNA from cancer to immune cells via extracellular vesicles. Connexin-43 (Cx43)-constituted gap junctions (GJs) have also been involved in miRNA intercellular mobilization, in other biological processes. In this report, we aimed to evaluate the involvement of Cx43-GJs in the shift of miRNAs induced by hypoxia, from hypoxic melanoma cells to dendritic cells and melanoma-specific cytotoxic T lymphocytes (CTLs). Using qRT-PCR arrays, we identified that miR-192-5p was strongly induced in hypoxic melanoma cells. Immune cells acquired this miRNA after co-culture with hypoxic melanoma cells. The transfer of miR-192-5p was inhibited when hypoxic melanoma cells expressed a dominant negative Cx43 mutant or when Cx43 expression was silenced using specific short-hairpin RNAs. Interestingly, miR-192-5p levels on CTLs after co-culture with hypoxic melanoma cells were inversely correlated with the cytotoxic activity of T cells and with ZEB2 mRNA expression, a validated immune-related target of miR-192-5p, which is also observed in vivo. Altogether, our data suggest that hypoxic melanoma cells may suppress CTLs cytotoxic activity by transferring hypoxia-induced miR-192-5p through a Cx43-GJs driven mechanism, constituting a resistance strategy for immunological tumor escape. Briefly synapse . Finallygranules ,6.Unfortunately, cancer cells may develop numerous immuno-evasive mechanisms that allow them to resist natural or therapy-induced immune attacks. Through these mechanisms, tumor cells are capable of modulating themselves and their surroundings in order to promote their survival, growth and invasion, even under a persistent immune pressure. Indeed, stressors present in the TME, such as chronic hypoxia, play determinant roles in promoting tumor cell plasticity and heterogeneity, which finally leads into the acquisition of immune tolerance and tumor progression . For exaMicroRNAs (miRNAs) are 19–22 nucleotide long non-coding RNAs that regulate gene expression at a post-transcriptional level. The profile and levels of miRNAs are frequently altered in cancer cells, affecting tumorigenesis and progression, via the induction of sustained proliferation, resistance to cell death, acquisition of invasive phenotypes, and regulating anti-tumor immune responses . AlteratInterestingly, the mobility of miRNAs between cells is not limited only to an exosome-mediated mechanism. Indeed, recent data suggests that miRNAs could be transferred between contacting cells via gap junctions (GJs), and it has been proposed as a novel pathway for intercellular regulation of gene expression ,18. GJs 2 tension (1% O2) or in presence of CoCl2, a chemical simulator of hypoxia. In addition to Cx43, melanoma cells mostly express Cx26 [2-stimulated B16F10 cells showed increased levels of both Cx43 and nuclear HIF-1α as compared with control normoxic cells , described in The Cancer Genome Atlas (TCGA) database (We observed that hypoxia induced Cx43 expression in human (Mel3) and murine (B16F10) melanoma cell lines, both at mRNA and protein levels A–D, suppess Cx26 , whose eess Cx26 . Accordiic cells E. Moreover cells . Importadatabase F. Associdatabase F.+ immune cells were determined by flow cytometry. Our results showed that hypoxic melanoma cells transferred more than twice the amount of RNA molecules to mo-DCs or CTLs than normoxic melanoma cells  for 2 h and then separated by CD11c magnetic cell sorting. The levels of the top-six HRMs were determined in each cell type before and after co-cultures A. After Our results also suggest that the transfer of miRNAs between hypoxic B16F10 and BM-DCs not only depends on their differential levels between the interacting cells, given that although miR-504-3p levels were higher in hypoxic B16F10 cells, there were no increase in its levels in BM-DCs after co-culture B. In add+ T cells [A search in the miRTarBase 2020 database  showed t T cells ,37. AccoTo determine whether hypoxic B16F10 cells can transfer miR-192-5p to gp100-specific CTLs by a Cx43-mediated mechanism, normoxic and hypoxic shCtrl- or shCx43-expressing B16F10 cells were co-cultured with pMEL-1 CTLs for 2 h, separated by CD8 magnetic cell sorting and miR-192-5p levels were determined in each cell type. After co-culture with hypoxic B16F10 cells, CTLs showed significantly increased miR-192-5p levels, compared with pre-co-culture CTLs B. Importp = 0.002) and LUAD (p = 0.0002)  , we foun 0.0002) B–D. More T cells , we obse+ T cells could occur in the TME, we challenged C57BL6 mice with B16F10 cells expressing wild type (Cx43WT) or dominant negative (Cx43DN) mutant Cx43 that causes closed channels [DN established lower levels of GJIC with CTLs as compared with control cells (+ T cells and in non-immune (CD45−) cells. Tumor infiltrating CD8+ T cells showed higher levels of miR-192-5p than CD8+ T cells from the spleens and, interestingly, these levels were significantly lower in CD8+ T cells isolated from Cx43DN-expressing than from Cx43WT-expressing melanoma tumors  or shRNAs ,58. Simi+ exosomes in the transfer of this (and/or others) miRNA in different experimental conditions. The deregulation of miRNA expression has been implicated in tumor initiation, progression and resistance to therapy or immune attack in most cancers, including in melanoma . Many of+ T cells [+ T follicular helper cell differentiation by targeting CXCR5 [Our results also suggest that the identity of the transferred miRNAs depends on the nature of the interacting cells more than the levels of miRNAs in those cells. In our experimental conditions, miR-192-5p was transferred from hypoxic melanoma cells to DCs, as well as to CTLs. The hypothetical functional relevance of these observations was suggested by the analysis of the expression of a strongly validated miR-192-5p target, ZEB2, a transcription factor that regulates the expression of many important genes (like GZMA) necessary for the formation of the terminal effector a state in CD8 T cells ,37. Acco T cells . In adding CXCR5 . WhetherWild-type C57BL6 and transgenic pMEL-1 (C57BL6 background) mice  were bre® CRL-6475™) and Mel1, Mel2 and Mel3 [2 and 95% relative humidity.B16F10  at 37 °C. In some experiments, CoCl2 , a chemical inductor of hypoxia, was used at 150 μM final concentration. Hypoxia induction was routinely tested by flow cytometry using Hypoxia Green Reagent  according to the manufacturer instructions. HGR is a membrane-permeant probe that releases rhodamine as O2 levels decrease, resulting in a fluorogenic response.For the induction of hypoxia, melanoma cell cultures were incubated for 24 (B16F10) to 72 h (Mel3) in a Modulator Incubator Chamber  in a humidified atmosphere containing 5% COpMEL-1 CTLs were differentiated as described before . BrieflyMurine BM-DCs were differentiated from femurs and tibias of C57BL/6 mice by culturing bone marrow cells in the presence of 20 ng/mL mouse recombinant GM-CSF (Biolegend) for 8 days. Fresh culture medium with cytokine was added on day 3, and, on day 6, the medium was refreshed. The phenotype of the cells was analyzed by flow cytometry as described before . Human p2) or hypoxic (1% O2) conditions for 72 (Mel3) or 24 h (B16F10). Intracellular total RNA molecules were stained by incubating the melanoma cells with 5 μM Syto RNASelect  [+ recipient cells was determined by flow cytometry.Mel3 or B16F10 melanoma cells were cultured in normoxic   for 20 m5 melanoma cells were co-culture with BM-DC, pMEL-1 CTLs or moDC at 1:2 cell ratios for 2 h. After co-culture, CD11c+ cells  or CD8+ cells (CTL) were isolated from CD11c- or CD8- cells (melanoma) using specific magnetic cell sorting according to manufacturer’s instructions . Total RNA samples, including miRNA, were isolated from pre-co-culture (named culture) and post-co-culture (named co-culture) using miRNeasy Mini kit according to manufacturer’s instructions . Quality and concentration of RNA samples were determined by Agilent 2200 TapeStation analysis using RNA ScreenTapes according to manufacturer instructions . Only RNA samples with RNA integrity number (RIN) values >8 were used. cDNAs were generated by reverse transcription using miScript II RT Kit and the miScript HiFlex buffer according to manufacturer instructions (Qiagen). Expressions of different mature miRNAs were determined using miScript SYBR Green PCR Kit and specific mouse or human miScript primer assays according to the manufacturer’s protocols (Qiagen). Levels of each miRNA were expressed relative to the level of small nuclear RNA RNU6-2 (miScript primer assays) and analyzed according to the Δ/ΔCt method. PCRs were performed in a LightCycler 480 Instrument and LightCycler 480 Software . As controls of mature miRNA transfer or cell isolation, precursor miRNA expression levels or CD8 and gp100 mRNA expressions were determined in culture and co-cultured cells, using specific miScript primer assays (for precursor miRNAs) or designed primers for mRNA  or 72 h (Mel3). Then, 1 × 10mRNA see   or a dominant negative Cx43 mutant (Cx43DN). Fourteen days after tumor challenge, spleens and tumors were collected and treated as described before [−) and CD45+ CD3+ CD8+ T cells were isolated using FACS sorting. RNA samples were obtained and miR-192-5p expression analyzed as described before.For in vivo determination of miR-192-5p expression, 6–12 weeks old C57BL6 mice were subcutaneously challenged with 1.5 × 10d before  and non-WT) or a dominant negative Cx43 mutant (Cx43DN) were cultured for 24 h upon normoxic or hypoxic conditions. Then, pre-conditioned melanoma cells were co-cultured for 2 h with pMEL-1 CTLs at a 1:2 cell ratio, allowing or not  cell-to-cell contact. After co-culture, the cytotoxic activity of the CTLs against B16F10 cells was determined by flow cytometry using the Pantoxilux®  granzyme and upstream caspase activity kit, following manufacturer’s instructions.B16F10 cells expressing wild type , CD8-PE , CD45-PerCP-Cy5.5 , all from BioLegend. 7-AAD  staining was used for dead cell exclusion.Flow cytometry experiments were performed as previously described ,74. Intr2 on poly-l-lysine-coated slides, using 5 × 104 cells per slide in 100 μL of complete culture medium. Cells were then cultured in absence (N) or presence (H) of 150 μM CoCl2 (Sigma-Aldrich) for additional 48 h and gently washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min at room temperature in the dark. After gentle washing with PBS, cells were incubated in ammonium chloride (50 mM) for 10 min. Then, cells were permeabilized for 10 min (0.5% Triton X-100 and 0.5% FBS) and blocked with 0.5% bovine serum albumin (BSA) for 15 min. Cells were stained with 1:500 dilutions of FITC-coupled monoclonal antibody anti-Cx43  or a PE-coupled monoclonal antibody anti-HIF-1α  for 1.25 h at room temperature, and then stained with 1:1000 dilution of Hoechst 33,342 (Invitrogen) for 15 min. Cells were analyzed with a C2+ confocal microscope  and a Spinning Disk Olympus BX61WI microscope . Cx43 and HIF-1α expression and nucleus areas were quantified using ImageJ software .Confocal microscopy of fixed B16F10 cells was performed as previously described . BrieflyFor knocking down Cx43 expression, B16F10 and Mel3 melanoma cells were stably transfected with lentiviral vector plasmids encoding a pool of 4 Gja1 (sc-35091-SH) or GJA1 (sc-29276-SH)-specific shRNAs (plus hairpin), using plasmid transfection reagent (sc-108061) and shRNA plasmid transfection medium (sc-108062) according to the manufacturer’s instructions (Santa Cruz Biotechnology). A control shRNA plasmid  was used for generating stable transfection in the control cells. Puromycin  was added to the culture medium for selection of transfected cells.B16F10 cells were stably transfected with pcDNA3.1 + NeGFP plasmids containing wild type or mutant dominant negative Cx43 (T154A)  using Lipofectamine (Invitrogen) and G418 neomycin antibiotic as selection pressure (Sigma-Aldrich).RNA samples were isolated and treated as described in ® West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific) according to manufacturer’s instructions and analyzed in an ImageQuant LAS 500 . Images were analyzed using ImageJ software . Unedited image of the western blot is shown in Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 5 mM EDTA and protease/phosphatase inhibitors cocktail  for 30 min on ice. Western blotting was conducted as described previously , loadingt-test where appropriate. Differences were considered statistically significant at p < 0.05.Data were analyzed using GraphPad Prism 7 . Statistical analyses were performed using one-way ANOVA, Tukey’s multiple comparison tests, or a two-tailed Student’s


Furthermore, we validated the predictive potential of KMT2A/C mutations in an expanded ICI-treated cohort (n = 1395). KMT2A/C-mutant patients achieved better OS compared with KMT2A/C-wildtype patients ; and the survival advantages appeared in the majority of cancer subtypes. Our study suggests that KMT2A/C mutations function as a novel and potential predictive biomarker for ICI treatment in multiple solid tumors and the underlying mechanism is worth investigating.Epigenetic factors play important roles in tumor immunology. Histone-lysine N-methyltransferase 2 (KMT2) family genes exert histone H3 methylation, but its role in immunotherapy remains unclear. Our study is the first to investigate the correlation between KMT2 gene mutations and the clinical benefit of immune checkpoint inhibitors (ICI) treatment. We firstly collected a primary ICI-treated cohort ( To the Editor,Recent years have witnessed the great success of immune checkpoint inhibitors (ICIs) in treating multiple advanced tumors . HoweverGrowing evidence suggests that epigenetic factors play important roles in immuno-oncology , 3. For The KMT2 family genes, one of the important epigenetic regulator genes, were initially recognized in mixed-lineage leukemia (MLL) caused by the rearrangement of KMT2A on chromosome 11q23 , 8. Recen = 546) with annotated response and mutational data as well as survival data from seven published studies (see P = 0.007) and KMT2C (P = 0.041) mutations significantly correlated with better progression-free survival (PFS) in patients receiving ICI treatment, while KMT2B (P = 0.964) and KMT2D (P = 0.200) did not. Then we combined KMT2A and KMT2C mutation as KMT2A/C mutations, and found that patients harboring KMT2A/C mutations correlated with longer PFS most significantly , durable clinical benefit (DCB) and overall survival (OS) in patients receiving ICI treatment. Compared with KMT2A/C-wildtype patients, KMT2A/C-mutant patients achieved significantly higher ORR  from Memorial Sloan Kettering Cancer Center on the basis of Samstein and his colleagues’ work , confirming that the improvement of OS after ICI treatment in KMT2A/C-mutant patients compared with that in KMT2A/C-wildtype patients was indeed transformed from its higher response rate, not contributed by its general prognostic impact.To further validate the predictive value of KMT2A/C mutations, we employed an expanded ICI-treated cohort  in the TMB-low subgroup .Additional file 3: Figure S1. Kaplan-Meier curves comparing the overall survival (OS) between the KMT2A/C-Mut group and the KMT2A/C-Wt group in the non-ICI-treated cohort (n = 2252).Additional file 4: Figure S2. The mutational frequency of KMT2A/C across 33 cancer types in the TCGA cohort.Additional file 5: Figure S3. Boxplot comparing the tumor mutational burden (TMB) between KMT2A/C-Mut tumors and the KMT2A/C-Wt tumors in the primary ICI-treated cohort.Additional file 6: Figure S4. Barplot comparing the objective response rate (ORR) between KMT2A/C-Mut patients and the KMT2A/C-Wt patients in the TMB-low subgroup of the primary ICI-treated cohort.


Scientific Reports 10.1038/s41598-019-38607-6, published online 14 February 2019Correction to: In the Supplementary Information file originally published with this Article, Supplementary Tables (Tables S1-4) were omitted. These errors have been corrected in the Supplementary Information that now accompanies the Article.