Bioengineered Artificial Extracellular Vesicles Presenting PD-L1 and Gal-9 Ameliorate New-onset Type 1 Diabetes.

An important factor in the development of Type 1 diabetes (T1D) is the deficiency of inhibitory immune checkpoint ligands, specifically programmed cell death ligand 1 (PD-L1) and Galectin-9 (Gal-9), in ß-cells. Hence, modulation of the pancreas infiltrated T lymphocytes by exogenous PD-L1 or Gal-9 is an ideal approach for treating the new-onset T1D. Herein, we genetic engineered the macrophage cells to generate artificial extracellular vesicles (aEVs) overexpressing PD-L1 and Gal-9, which could restrict the islets autoreactive T lymphocytes and protect ß-cells from destruction. Intriguingly, overexpressing Gal-9 spurred macrophage polarization to M2 phenotype with immune suppressive attribute. Alternatively, both of PD-L1 and Gal-9 presenting aEVs (PD-L1-Gal-9 aEVs) favorably adhere to T cells via the interaction of programmed cell death protein 1 (PD-1)/PD-L1 or T cell immunoglobulin mucin 3 (TIM-3)/Gal-9. Moreover, PD-L1-Gal-9 aEVs prominently promoted effector T cell apoptosis and splenic regulatory T cells (Treg) cells differentiation in vitro. Virtually, PD-L1-Gal-9 aEVs efficaciously reversed the new-onset hyperglycemia in the NOD mice, prevented T1D progress, and declined the proportion and activation of CD4+ and CD8+ T cells infiltrating the pancreas notably, which together contributed to preserving the residual ß-cells survival and mitigating the hyperglycemia.

In Situ Synthesis of an Immune-Checkpoint Blocker from Engineered Bacteria Elicits a Potent Antitumor Response.

Immune-checkpoint blockade (ICB) reinvigorates T cells from exhaustion and potentiates T-cell responses to tumors. However, most patients do not respond to ICB therapy, and only a limited response can be achieved in a "cold" tumor with few infiltrated lymphocytes. Synthetic biology can be used to engineer bacteria as controllable bioreactors to synthesize biotherapeutics in situ. We engineered attenuated Salmonella VNP20009 with synthetic gene circuits to produce PD-1 and Tim-3 scFv to block immunosuppressive receptors on exhausted T cells to reinvigorate their antitumor response. Secreted PD-1 and Tim-3 scFv bound PD-1+ Tim-3+ T cells through their targeting receptors in vitro and potentiated the T-cell secretion of IFN-γ. Engineered bacteria colonized the hypoxic core of the tumor and synthesized PD-1 and Tim-3 scFv in situ, reviving CD4+ T cells and CD8+ T cells to execute an antitumor response. The bacteria also triggered a strong innate immune response, which stimulated the expansion of IFN-γ+ CD4+ T cells within the tumors to induce direct and indirect antitumor immunity.

PGRN inhibits CD8+T cell recruitment and promotes breast cancer progression by up-regulating ICAM-1 on TAM.

BACKGROUND: Tumor microenvironment actually reduces antitumor effect against the immune attack by exclusion of CD8+T cells. Progranulin (PGRN) is a multifunctional growth factor with significant pathological effects in multiple tumors; however, its role in immunity evasion of breast cancer (BCa) is not completely understood. METHODS: We depleted GRN (PGRN gene) genetically in mice or specifically in PY8119 murine BCa cell line, and mouse models of orthotopic or subcutaneous transplantation were used. Chimeric mice-deficient of PGRN (Grn-/-) in bone marrow (BM) compartment was also generated. Association of PGRN expression with chemokine production or BCa development was investigated by histological and immunological assays. RESULTS: We found PGRN was involved in exhaustion of cytotoxic CD8+T cell in BCa with the increasing expressions of M2 markers and intercellular cell adhesion molecule-1 (ICAM-1) on macrophages. Specifically, ablation of PGRN in PY8119 cells reduced tumor burden, accompanied by the infiltrating of cytotoxic CD8+T cells into tumor nests. Moreover, our result revealed that blockade of PD-1 in PGRN-depleted tumors exhibited better antitumor effect in vivo and significantly decreased tumor burden. CONCLUSION: These findings suggest that inhibition of PGRN may act as a potential immune-therapeutic strategy by recovering infiltration of CD8+T cell in BCa tissue and thereby enhancing the response to anti-PD-1 therapy.

The role of visual rating and automated brain volumetry in early detection and differential diagnosis of Alzheimer's disease.

BACKGROUND: Medial temporal lobe atrophy (MTA) is a diagnostic marker for mild cognitive impairment (MCI) and Alzheimer's disease (AD), but the accuracy of quantitative MTA (QMTA) in diagnosing early AD is unclear. This study aimed to investigate the accuracy of QMTA and its related components (inferior lateral ventricle [ILV] and hippocampus) with MTA in the early diagnosis of MCI and AD. METHODS: This study included four groups: normal (NC), MCI stable (MCIs), MCI converted to AD (MCIs), and mild AD (M-AD) groups. Magnetic resonance image analysis software was used to quantify the hippocampus, ILV, and QMTA. MTA was rated by two experienced neurologists. Receiver operating characteristic area under the curve (AUC) analysis was performed to compare their capability in differentiating AD from NC and MCI, and optimal thresholds were determined using the Youden index. RESULTS: QMTA distinguished M-AD from NC and MCI with higher diagnostic accuracy than MTA, hippocampus, and ILV (AUCNC = 0.976, AUCMCI = 0.836, AUCMCIs = 0.894, AUCMCIc = 0.730). The diagnostic accuracy of QMTA was superior to that of MTA, the hippocampus, and ILV in differentiating MCI from AD. The diagnostic accuracy of QMTA was found to remain the best across age, sex, and pathological subgroups analyzed. The sensitivity (92.45%) and specificity (90.64%) were higher in this study when a cutoff value of 0.635 was chosen for QMTA. CONCLUSIONS: QMTA may be a better choice than the MTA scale or the associated quantitative components alone in identifying AD patients and MCI individuals with higher progression risk.

The impact of fear of childbirth on mode of delivery, postpartum mental health and breastfeeding: A prospective cohort study in Shanghai, China.

BACKGROUND: The prevalence and impact of fear of childbirth (FOC) has not been sufficiently understood. We aimed to investigate the prevalence of FOC among Chinese population and its impact on mode of delivery, postpartum mental health and breastfeeding. METHODS: We conducted a prospective cohort study, wherein pregnant women in their third trimester who underwent antenatal assessments at Shanghai Changning Maternity and Infant Health Hospital between September 2020 and March 2021 were recruited. Sociodemographic data of the participants were gathered by self-administered questionnaire, and their FOC was assessed using the Wijma Delivery Expectancy Questionnaire. Participants were followed up to 42 days postpartum. Information regarding their modes of delivery was retrieved from medical records, and data regarding postpartum mental health symptoms and one-month postpartum breastfeeding were obtained through self-administered questionnaires. RESULTS: Among 1287 participants, 461 (35.8 %) had high-level FOC (W-DEQ ≥ 66). Logistic regressions showed that women with high-level of FOC had higher rates of caesarean delivery on maternal request (CDMR) (aOR = 1.55, 95 % CI: 1.00-2.41, p = 0.049), a higher incidence of postpartum mental health symptoms (aOR = 1.68, 95 % CI: 1.09-2.59, p = 0.018), lower rates of one-month postpartum exclusive breastfeeding (aOR = 0.33, 95 % CI: 0.16-0.69, p = 0.003) and mixed feeding (aOR = 0.44, 95 % CI: 0.21-0.91, p = 0.028). LIMITATIONS: The long-term implications of FOC beyond the immediate postpartum period were not explored in the study. CONCLUSIONS: High-level FOC during the third trimester was associated with increased CDMR and postpartum mental health symptoms and reduced breastfeeding establishment. These results underscore the significance of FOC screening and tailored interventions for affected women.

Engineered Cell Membrane Vesicles Expressing CD40 Alleviate System Lupus Nephritis by Intervening B Cell Activation.

Immune intervention of B cell activation to blockade the production of autoantibodies provokes intense interest in the field of systemic lupus erythematosus (SLE) therapy development. Although the survival rate for SLE is improved, many patients die untimely. Engineered cell membrane vesicles manifest remarkable capacity of targeted drug delivery and immunomodulation of immune cells such as B cells. Herein, this work engineered cellular nanovesicles (NVs) presenting CD40 (CD40 NVs) that can blunt B cells and thus alleviate SLE. CD40 NVs disrupt the CD40/CD40 ligand (CD40L) costimulatory signal axis through the blockade of CD40L on CD4+ T cells. Therefore, the CD40 NVs restrain the generation of the germinal center structure and production of antibodies from B cells. Furthermore, immunosuppressive drug mycophenolate mofetil (MMF) is also encapsulated in the vesicles (MMF-CD40 NVs), which is employed to deplete immunocytes including B cells, T cells, and dendritic cells. Together, CD40 NVs are promising formulations for relieving autoimmunity and lupus nephritis in MRL/lpr mice.

Highly sensitive Curcumin-conjugated nanotheranostic platform for detecting amyloid-beta plaques by magnetic resonance imaging and reversing cognitive deficits of Alzheimer's disease via NLRP3-inhibition.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disorder without effective therapy and lack diagnosis strategy for preclinical AD patients. There is an urgent need for development of both early diagnosis and therapeutic intervention of AD. RESULTS: Herein, we developed a nanotheranostics platform consisting of Curcumin (Cur), an anti-inflammatory molecule, and superparamagnetic iron oxide (SPIO) nanoparticles encapsulated by diblock 1,2-dio-leoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethylene glycol)] (DSPE-PEG) that are modified with CRT and QSH peptides on its surface. Furthermore, we demonstrated that this multifunctional nanomaterial efficiently reduced ß-amyloid plaque burden specifically in APP/PS1 transgenic mice, with the process noninvasively detected by magnetic resonance imaging (MRI) and the two-dimensional MRI images were computed into three-dimension (3D) plot. Our data demonstrated highly sensitive in vivo detection of ß-amyloid plaques which more closely revealed real deposition of Aß than previously reported and we quantified the volumes of plaques for the first time based on 3D plot. In addition, memory deficits of the mice were significantly rescued, probably related to inhibition of NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasomes. CONCLUSIONS: Gathered data demonstrated that this theranostic platform may have both early diagnostic and therapeutic potential in AD.

The Correlation Between White Matter Hyperintensity Burden and Regional Brain Volumetry in Patients With Alzheimer's Disease.

Background: White matter hyperintensities (WMHs) and regional brain lobe atrophy coexist in the brain of patients with Alzheimer's disease (AD), but the association between them in patients with AD still lacks comprehensive investigation and solid imaging data support. Objective: We explored whether WMHs can promote the pathological process of AD by aggravating atrophy in specific brain regions and tried to explain the regional specificity of these relationships. Methods: A sample of 240 adults including 180 normal controls (NCs) and 80 cases with AD were drawn from the ADNI database. T1-weighted magnetic resonance imaging (MRI) and T2-weighted fluid-attenuated MRI of the participants were downloaded and were analyzed using AccuBrain® to generate the quantitative ratio of WMHs (WMHr, WMH volumes corrected by intracranial volume) and regional brain atrophy. We also divided WMHr into periventricular WMHr (PVWMHr) and deep WMHr (DWMHr) for the purpose of this study. The Cholinergic Pathways Hyperintensities Scale (CHIPS) scores were conducted by two evaluators. Independent t-test, Mann-Whitney U test, or χ2 test were used to compare the demographic characteristics, and Spearman correlation coefficient values were used to determine the association between WMHs and different regions of brain atrophy. Results: Positive association between WMHr and quantitative medial temporal lobe atrophy (QMTA) (r s = 0.281, p = 0.011), temporal lobe atrophy (r s = 0.285, p = 0.011), and insular atrophy (r s = 0.406, p < 0.001) was found in the AD group before Bonferroni correction. PVWMHr contributed to these correlations. By separately analyzing the relationship between PVWMHr and brain atrophy, we found that there were still positive correlations after correction in QMTA (r s = 0.325, p = 0.003), temporal lobe atrophy (r s = 0.298, p = 0.007), and insular atrophy (r s = 0.429, p < 0.001) in AD group. Conclusion: WMH severity tends to be associated with regional brain atrophy in patients with AD, especially with medial temporal lobe, temporal lobe, and insular lobe atrophy. PVWMHs were devoted to these correlations.

AD Resemblance Atrophy Index of Brain Magnetic Resonance Imaging in Predicting the Progression of Mild Cognitive Impairment Carrying Apolipoprotein E-ε4 Allele.

Background and Objective: Early identification is important for timely Alzheimer's disease (AD) treatment. Apolipoprotein E ε4 allele (APOE-ε4) is an important genetic risk factor for sporadic AD. The AD-Resemblance Atrophy Index (RAI)-a structural magnetic resonance imaging-derived composite index-was found to predict the risk of progression from mild cognitive impairment (MCI) to AD. Therefore, we investigated whether the AD-RAI can predict cognitive decline and progression to AD in patients with MCI carrying APOE ε4. Methods: We included 733 participants with MCI from the Alzheimer's Disease Neuroimaging Initiative Database (ADNI). Their APOE genotypes, cognitive performance, and levels of AD-RAI were assessed at baseline and follow-up. Linear regression models were used to test the correlations between the AD-RAI and baseline cognitive measures, and linear mixed models with random intercepts and slopes were applied to investigate whether AD-RAI and APOE-ε4 can predict the level of cognitive decline. Cox proportional risk regression models were used to test the association of AD-RAI and APOE status with the progression from MCI to AD. Results: The baseline AD-RAI was higher in the MCI converted to AD group than in the MCI stable group (P < 0.001). The AD-RAI was significantly correlated with cognition, and had a synergistic effect with APOE-ε4 to predict the rate of cognitive decline. The AD-RAI predicted the risk and timing of MCI progression to AD. Based on the MCI population carrying APOE-ε4, the median time to progression from MCI to AD was 24 months if the AD-RAI > 0.5, while the median time to progression from MCI to AD was 96 months for patients with an AD-RAI ≤ 0.5. Conclusion: The AD-RAI can predict the risk of progression to AD in people with MCI carrying APOE ε4, is strongly correlated with cognition, and can predict cognitive decline.

Self-assembled tetrahedral framework nucleic acid mediates tumor-associated macrophage reprogramming and restores antitumor immunity.

There is increasing interest in depleting or repolarizing tumor-associated macrophages (TAMs) to generate a proinflammatory effect. However, TAMs usually display an immunosuppressive M2-like phenotype in the tumor microenvironment. Apparently, developing a macrophage-targeting delivery system with immunomodulatory agents is urgent. In this study, an efficient siRNA and CpG ODNs delivery system (CpG-siRNA-tFNA) was prepared with nucleic acid stepwise self-assembled. The tFNA composed of CpG ODNs and siRNA showed a higher stability and an enhanced cellular uptake efficiency. Moreover, the CpG-siRNA-tFNA effectively reprogrammed TAMs toward M1 phenotype polarization with increased proinflammatory cytokine secretion and NF-κB signal pathway activation, which triggers dramatic antitumor immune responses. Additionally, the CpG-siRNA-tFNA exhibited superior antitumor efficacy in a breast cancer xenograft mouse model without obvious systemic side effects. Taken together, CpG-siRNA-tFNA displayed greatly antitumor effect by facilitating TAM polarization toward M1 phenotypes in favor of immunotherapy. Hence, we have developed an efficient therapeutic strategy with immunomodulatory agents for clinical applications.

Study protocol for the Sino-Canadian Healthy Life Trajectories Initiative (SCHeLTI): a multicentre, cluster-randomised, parallel-group, superiority trial of a multifaceted community-family-mother-child intervention to prevent childhood overweight and obesity.

INTRODUCTION: Childhood overweight and obesity (OWO) is a primary global health challenge. Childhood OWO prevention is now a public health priority in China. The Sino-Canadian Healthy Life Trajectories Initiative (SCHeLTI), one of four trials being undertaken by the international HeLTI consortium, aims to evaluate the effectiveness of a multifaceted, community-family-mother-child intervention on childhood OWO and non-communicable diseases risk. METHODS AND ANALYSIS: This is a multicentre, cluster-randomised, controlled trial conducted in Shanghai, China. The unit of randomisation is the service area of Maternal Child Health Units (N=36). We will recruit 4500 women/partners/families in maternity and district level hospitals. Participants in the intervention group will receive a multifaceted, integrated package of health promotion interventions beginning in preconception or in the first trimester of pregnancy, continuing into infancy and early childhood. The intervention, which is centred on a modified motivational interviewing approach, will target early-life maternal and child risk factors for adiposity. Through the development of a biological specimen bank, we will study potential mechanisms underlying the effects of the intervention. The primary outcome for the trial is childhood OWO (body mass index for age ≥85th percentile) at 5 years of age, based on WHO sex-specific standards. The study has a power of 0.8 (α=0.05) to detect a 30% risk reduction in the proportion of children with OWO at 5 years of age, from 24.4% in the control group to 17% in the intervention group. Recruitment was launched on 30 August 2018 for the pilot study and 10 January 2019 for the formal study. ETHICS AND DISSEMINATION: The study has been approved by the Medical Research Ethics Committee of the International Peace Maternity and Child Health Hospital in Shanghai, China, and the Research Ethics Board of the Centre Intégré Universitaire de Santé et Services Sociaux de l'Estrie-CHUS in Sherbrooke, Canada. Data sharing policies are consistent with the governance policy of the HeLTI consortium and government legislation. TRIAL REGISTRATION NUMBER: ChiCTR1800017773. PROTOCOL VERSION: November 11, 2020 (Version #5).

[Progranulin (PGRN) promotes invasion and migration of mouse breast cancer 4T1 cells by promoting epithelial-mesenchymal transition of cancer cells and activating ERK1/2 pathway].

Objective To investigate the effect of progranulin (PGRN) on the invasion and migration of mouse breast cancer 4T1 cells and its mechanism. Methods After treated with PGRN (1 µg/mL) for 24 hours, the invasion ability of breast cancer 4T1 cells was detected by TranswellTM invasion assay, the migration ability was detected by scratch test, and the epithelial cadherin (E-cadherin), vimentin mRNA expression was detected by real-time fluorescent quantitative PCR. Western blot assay was used to detect the expression of E-cadherin, vimentin, extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2). After treated with 1 µg/mL PGRN and ERK1/2 signaling pathway inhibitor U0126 (10 µmol/L) simultaneously, the migration and invasion ability of 4T1 cells and the changes in the expression of E-cadherin, vimentin and p-ERK proteins were detected again. Results After treated with PGRN, the migration and invasion capabilities of breast cancer 4T1 cells were significantly enhanced; E-cadherin expression decreased; vimentin and p-ERK1/2 expression increased. After treated with ERK1/2 signaling pathway inhibitor, the ability of PGRN to promote breast cancer 4T1 cell migration, invasion and epithelial-mesenchymal transition (EMT) was significantly inhibited. Conclusion PGRN can promote the migration and invasion of breast cancer 4T1 cells by promoting EMT and activating the ERK1/2 pathway.

AD Resemblance Atrophy Index as a Diagnostic Biomarker for Alzheimer's Disease: A Retrospective Clinical and Biological Validation.

BACKGROUND: Magnetic resonance imaging (MRI) provides objective information about brain structural atrophy in patients with Alzheimer's disease (AD). This multi-structural atrophic information, when integrated as a single differential index, has the potential to further elevate the accuracy of AD identification from normal control (NC) compared to the conventional structure volumetric index. OBJECTIVE: We herein investigated the performance of such an MRI-derived AD index, AD-Resemblance Atrophy Index (AD-RAI), as a neuroimaging biomarker in clinical scenario. METHOD: Fifty AD patients (19 with the Amyloid, Tau, Neurodegeneration (ATN) results assessed in cerebrospinal fluid) and 50 age- and gender-matched NC (19 with ATN results assessed using positron emission tomography) were recruited in this study. MRI-based imaging biomarkers, i.e., AD-RAI, were quantified using AccuBrain®. The accuracy, sensitivity, specificity, and area under the ROC curve (AUC) of these MRI-based imaging biomarkers were evaluated with the diagnosis result according to clinical criteria for all subjects and ATN biological markers for the subgroup. RESULTS: In the whole groups of AD and NC subjects, the accuracy of AD-RAI was 91%, sensitivity and specificity were 88% and 96%, respectively, and the AUC was 92%. In the subgroup of 19 AD and 19 NC with ATN results, AD-RAI results matched completely with ATN classification. AD-RAI outperforms the volume of any single brain structure measured. CONCLUSION: The finding supports the hypothesis that MRI-derived composite AD-RAI is a more accurate imaging biomarker than individual brain structure volumetry in the identification of AD from NC in the clinical scenario.

An MRI-based strategy for differentiation of frontotemporal dementia and Alzheimer's disease.

BACKGROUND: The differential diagnosis of frontotemporal dementia (FTD) and Alzheimer's disease (AD) is difficult due to the overlaps of clinical symptoms. Structural magnetic resonance imaging (sMRI) presents distinct brain atrophy and potentially helps in their differentiation. In this study, we aim at deriving a novel integrated index by leveraging the volumetric measures in brain regions with significant difference between AD and FTD and developing an MRI-based strategy for the differentiation of FTD and AD. METHODS: In this study, the data were acquired from three different databases, including 47 subjects with FTD, 47 subjects with AD, and 47 normal controls in the NACC database; 50 subjects with AD in the ADNI database; and 50 subjects with FTD in the FTLDNI database. The MR images of all subjects were automatically segmented, and the brain atrophy, including the AD resemblance atrophy index (AD-RAI), was quantified using AccuBrain®. A novel MRI index, named the frontotemporal dementia index (FTDI), was derived as the ratio between the weighted sum of the volumetric indexes in "FTD dominant" structures over that obtained from "AD dominant" structures. The weights and the identification of "FTD/AD dominant" structures were acquired from the statistical analysis of NACC data. The differentiation performance of FTDI was validated using independent data from ADNI and FTLDNI databases. RESULTS: AD-RAI is a proven imaging biomarker to identify AD and FTD from NC with significantly higher values (p < 0.001 and AUC = 0.88) as we reported before, while no significant difference was found between AD and FTD (p = 0.647). FTDI showed excellent accuracy in identifying FTD from AD (AUC = 0.90; SEN = 89%, SPE = 75% with threshold value = 1.08). The validation using independent data from ADNI and FTLDNI datasets also confirmed the efficacy of FTDI (AUC = 0.93; SEN = 96%, SPE = 70% with threshold value = 1.08). CONCLUSIONS: Brain atrophy in AD, FTD, and normal elderly shows distinct patterns. In addition to AD-RAI that is designed to detect abnormal brain atrophy in dementia, a novel index specific to FTD is proposed and validated. By combining AD-RAI and FTDI, an MRI-based decision strategy was further proposed as a promising solution for the differential diagnosis of AD and FTD in clinical practice.

Progranulin induces immune escape in breast cancer via up-regulating PD-L1 expression on tumor-associated macrophages (TAMs) and promoting CD8+ T cell exclusion.

BACKGROUND: Progranulin (PGRN), as a multifunctional growth factor, is overexpressed in multiple tumors, but the role of PGRN on tumor immunity is still unclear. Here, we studied the effect of PGRN on breast cancer tumor immunity and its possible molecular mechanism. METHODS: The changes of macrophage phenotypes after PGRN treatment were detected by western blot, quantitative polymerase chain reaction (PCR) and flow cytometry. Western blot was used to study the signal molecular mechanism of PGRN regulating this process. The number and localization of immune cells in Wild-type (WT) and PGRN-/- breast cancer tissues were analyzed by immunohistochemical staining and immunofluorescence techniques. The activation and proliferation of CD8+ T cells were measured by flow cytometry. RESULTS: After being treated with PGRN, the expressions of M2 markers and programmed death ligand 1 (PD-L1) on macrophages increased significantly. Signal transducer and activator of transcription 3 (STAT3) signaling pathway inhibitor Stattic significantly inhibited the expression of PD-L1 and M2 related markers induced by PGRN. In WT group, CD8 were co-localized with macrophages and PD-L1, but not tumor cells. The number of immune cells in PGRN-/- breast cancer tissue increased, and their infiltration into tumor parenchyma was also enhanced. Moreover, in the co-culture system, WT peritoneal macrophages not only reduced the ratio of activated CD8+ T cells but also reduced the proportion of proliferating CD8+ T cells. The addition of programmed death receptor 1 (PD-1) and PD-L1 neutralizing antibodies effectively reversed this effect and restored the immune function of CD8+ T cells. CONCLUSION: These results demonstrate that PGRN promotes M2 polarization and PD-L1 expression by activating the STAT3 signaling pathway. Furthermore, through PD-1/PD-L1 interaction, PGRN can promote the breast tumor immune escape. Our research may provide new ideas and targets for clinical breast cancer immunotherapy.

PGRN-/- TAMs-derived exosomes inhibit breast cancer cell invasion and migration and its mechanism exploration.

Breast cancer is one of the most malignant diseases world-wide and ranks the first among female cancers. Progranulin (PGRN) plays a carcinogenic role in breast cancer, but its mechanisms are not clear. In addition, there are few reports on the relationship between PGRN and tumor-associated macrophages (TAMs). AIMS: To investigate the effects of exosomes derived from PGRN-/- TAMs on invasion and migration of breast cancer cells. MAIN METHODS: Mouse breast cancer xenograft model was constructed to explore the effect of PGRN-/- tumor environment (TME) on breast cancer. Flow cytometry was used to compare TAMs of wild type (WT) and PGRN-/- tumor tissue. Transwell assay, wound healing assay and western blot were used to explore the effect of WT and PGRN-/- TAMs and their exosomes on invasion, migration and epithelial-mesenchymal transition (EMT) of breast cancer cells. MicroRNA (miRNA) assay was used to find out the differentially expressed miRNA of negative control (NC) and siPGRN-TAMs exosomes. Quantitative PCR and luciferase report assay were used to explore the target gene. KEY FINDINGS: The lung metastasis of breast cancer of PGRN-/- mice was inhibited. PGRN-/- TAMs inhibited invasion, migration and EMT of breast cancer cells through their exosomes. MiR-5100 of PGRN-/- TAMs-derived exosomes was up-regulated, which might regulate expression of CXCL12, thereby inhibiting the CXCL12/CXCR4 axis, and ultimately inhibiting the invasion, migration and EMT of breast cancer cells. SIGNIFICANCE: Our study elucidates a new molecular mechanism of lung metastasis of breast cancer, so it may contribute to efficient prevention and therapeutic strategies.

Nucleus-located PDK1 regulates growth, invasion and migration of breast cancer cells.

AIMS: It is well known that pyruvate dehydrogenase kinase 1 (PDK1) is highly expressed in breast cancer (BC) tissues and promotes tumor growth, but the underlying mechanisms of this process are unclear. Here, we investigated the effects of nuclear PDK1 on growth, migration and invasion in human BC cells. MAIN METHODS: The sub-cellular localization of PDK1 in BC cells was performed with subcellular fractionation followed by Western blot and immunofluorescence. The localization of PDK1 in breast normal tissue and breast duct carcinoma was detected by Immunohistochemistry. Then the protein-protein interaction between PDK1 and Importin ß was verified by co-immunoprecipitation assay. Finally, the effects of nuclear PDK1 on cell proliferation, apoptosis, migration and invasion of BC cells were assessed. KEY FINDINGS: In addition to its well-known sub-cellular localization, PDK1 was present in the nucleus of BC cells, and EGF treatment increased nucleus distribution of PDK1. Moreover, the level of nuclear PDK1 accumulation facilitated the growth of BC cells. We also found that the entry of PDK1 into nucleus mainly relied on the nuclear localization signal (NLS), and NLS mutation inhibited the entry of PDK1 into nucleus; as a result, the migration and invasion abilities of BC cells were impaired, and the number of apoptotic cells was significantly increased. SIGNIFICANCE: Our findings provided a new supplement to the sub-cellular localization of PDK1 in BC cells and uncovered the function of nuclear PDK1 in facilitating BC cells growth, migration and invasion.

Honokiol inhibits the growth of SKBR3 cells.

BACKGROUND: Breast cancer is one of the most malignant tumors in the reproductive system and has a poor prognosis. Finding drugs with high efficiency, low side-effects, and low cost has become a research hotspot. METHODS: In the present study, we treated SK-BR-3 cells with different doses of honokiol. Crystal violet staining method was used to detect changes in the total number of living cells; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to detect the effect of honokiol on SK-BR-3 cell proliferation. Cell migration ability change was determined by wound healing assay. Cell invasion ability change was determined by Transwell migration assay. Flow cytometry was used to detect the apoptotic rate of SK-BR-3 cells, and Western blot was used to detect the expression levels of proliferation-associated protein (PCNA); migration- and invasion-related protein matrix metalloproteinase-2 (MMP-2); vimentin; apoptosis-related proteins Bcl-xl, caspase 3, and cleaved caspase 3 (CC3); and ß-catenin and its downstream target molecule c-Myc. RESULTS: Compared with the control group, different doses of honokiol have different degrees of inhibitory effects on cells, including proliferation and invasion and migration (P<0.01). After treatment with 50 or 60 µmol·L-1 honokiol, the apoptotic rate of SK-BR-3 cells increased (both P<0.01); PCNA expression was significantly downregulated (P<0.01). Intracellular accumulation of apoptosis-related proteins Bcl-xl and caspase-3 decreased but C-C3 increased. We also found downregulation of MMP-2 expression, a protein related to invasion and migration (P<0.01), and a decrease in the expression levels of the Wnt/ß-catenin signaling pathway-related proteins ß-catenin and c-Myc (P<0.01). CONCLUSIONS: Honokiol can promote the apoptosis of SK-BR-3 cells and can inhibit the proliferation, migration, and invasion of human breast cancer SK-BR-3 cells. The underlying mechanism may be through inhibiting the activation of the Wnt signaling pathway.

[Molecular mechanism of macrophages derived from PGRN gene knockout mice inhibit invasion and migration of breast cancer cells].

Objective To explore the functions and mechanisms of macrophages derived from PGRN gene knockout (PGRN-/- ) C57BL/6 mice in the invasion and migration of breast cancer cells. Methods Breast cancer cells were cultured in conditioned medium of macrophages derived from WT and PGRN-/- mice. TranswellTM assay and scratch assay were used to detect the invasion and migration ability of cancer cells. Western blot analysis was used to detect the expression of E-cadherin and N-cadherin in cancer cells. Cytokine array, real-time quantitative PCR and ELISA were performed to investigate the differences of cytokines secreted by macrophages derived from WT and PGRN-/- mice. Breast cancer cells were treated by the differentially expressed cytokine interleukin-6 (IL-6), and then the above methods were used to investigate its effect on cancer cells. Western blot analysis was used to verify the roles of NF-κB and JAK/STAT3 signaling pathways. Results The macrophages derived from PGRN-/- mice blocked NF-κB signaling pathway, reduced IL-6 secretion, and inhibited the invasion and migration of breast cancer cells. IL-6 activated JAK/STAT3 signaling pathway to promote the invasion and migration of breast cancer cells. Conclusion The macrophages derived from PGRN-/- mice can block the NF-κB and JAK/STAT3 signaling pathways, down-regulate IL-6 expression, and inhibit the invasion and migration of breast cancer cells.