Levels Of CD44 Research Articles

Differential immunophenotype and proviral composition in young adults with perinatally acquired HIV: Are special cure strategies needed?

To characterize the immune functionality and phenotype and the proviral composition of a cohort of young adults with perinatally-acquired HIV (p-YA) from Argentina. Cross-sectional study of 18 p-YA, 15 young adults with non-perinatally acquired HIV matched by age with p-YA and 14 adults with non-perinatally acquired HIV, matched by time from HIV diagnosis with p-YA, all from Argentina. Immune memory/effector phenotype, exhaustion, activation, PTK-7 and Ki-67 expression were evaluated by flow cytometry on NK and T-cells. Total, intact and defective proviral (TP, IP and DP) HIV-DNA were measured in CD4 T-cells by IPDA. Soluble markers were determined by ELISA. p-YA displayed lower expression of PD-1, higher levels of CD38+ CD4 T-cells and increased levels of naïve T-cells than control groups. Also, a trend of lower levels of IP HIV-DNA normalized to CD4 T-cell counts and to the proportion of naïve T-cells was found in p-YA. The higher frequency of naïve CD4 T-cells in p-YA cannot be explained by elevated thymic activity nor by a higher T-cell proliferation rate. This imbalance could have been generated early in life and persisted during adulthood. Naïve CD4 T-cells may not serve as a major viral reservoir in p-YA. Also, the lower PD-1+ CD4 T-cell count suggests that p-YA did not present higher levels of exhaustion. These findings suggest that acquiring HIV perinatally may imply different challenges for proviral eradication.

Impact of UPF2 on the levels of CD81 on extracellular vesicles

Extracellular vesicles (EVs) are involved in cell-to-cell communication. Following uptake, EV cargo molecules, including DNA, RNA, lipids, and proteins, influence gene expression and molecular signaling in recipient cells. Although various studies have identified disease-specific EV molecules, further research into their biogenesis and secretion mechanisms is needed for clinical application. Here, we investigated the role of UPF2 in regulating the biogenesis and components of EVs. Notably, UPF2 promoted the expression of CD81, a membrane protein marker of EVs, as UPF2 silencing decreased CD81 levels in EVs, both inside the cell and secreted. In contrast, the expression levels of CD63 increased, without altering the size or numbers of EVs. In addition, reducing UPF2 levels did not affect the total number of EVs but lowered production of CD81-positive EVs and reduced the efficiency of uptake by recipient cells. Collectively, our findings uncover a novel function for UPF2 in regulating the production of CD81 and changing EV properties.

CD74 promotes the formation of an immunosuppressive tumor microenvironment in triple-negative breast cancer in mice by inducing the expansion of tolerogenic dendritic cells and regulatory B cells.

CD74 is a cell-surface receptor for the cytokine macrophage migration inhibitory factor (MIF). MIF binding to CD74 induces a signaling cascade resulting in the release of its cytosolic intracellular domain (CD74-ICD), which regulates transcription in naïve B and chronic lymphocytic leukemia (CLL) cells. In the current study, we investigated the role of CD74 in the regulation of the immunosuppressive tumor microenvironment (TME) in triple-negative breast cancer (TNBC). TNBC is the most aggressive breast cancer subtype and is characterized by massive infiltration of immune cells to the tumor microenvironment, making this tumor a good candidate for immunotherapy. The tumor and immune cells in TNBC express high levels of CD74; however, the function of this receptor in the tumor environment has not been extensively characterized. Regulatory B cells (Bregs) and tolerogenic dendritic cells) tol-DCs (were previously shown to attenuate the antitumor immune response in TNBC. Here, we demonstrate that CD74 enhances tumor growth by inducing the expansion of tumor-infiltrating tol-DCs and Bregs. Utilizing CD74-KO mice, Cre-flox mice lacking CD74 in CD23+ mature B cells, mice lacking CD74 in the CD11c+ population, and a CD74 inhibitor (DRQ), we elucidate the mechanism by which CD74 inhibits antitumor immunity. MIF secreted from the tumor cells activates CD74 expressed on DCs. This activation induces the binding of CD74-ICD to the SP1 promotor, resulting in the up-regulation of SP1 expression. SP1 binds the IL-1β promotor, leading to the down-regulation of its transcription. The reduced levels of IL-1β lead to decreased antitumor activity by allowing expansion of the tol-DC, which induces the expansion of the Breg population, supporting the cross-talk between these 2 populations. Taken together, these results suggest that CD74+ CD11c+ DCs are the dominant cell type involved in the regulation of TNBC progression. These findings indicate that CD74 might serve as a novel therapeutic target in TNBC.

CD38 modulates cytokine secretion by NK cells through the Sirt1/NF-κB pathway, suppressing immune surveillance in colorectal cancer.

Tregs and M2-type macrophages are essential for immune surveillance. CD38 + NK cells are involved in immunoregulation by modulating cytokine secretion. This study investigated how CD38 + NKs affect Tregs and macrophages in colorectal cancer (CRC). Higher proportions of CD38 + NKs and Tregs were detected in bloods and tumor tissues of CRC patients than that in the samples from healthy controls (HCs). Compared with CD38 + NKs from HCs, the NK cells from CRC promoted the differentiation of Tregs from CD4 + T cells, and secreted increased levels of IL-10, TGF-β and TNF-α and decreased levels of IFN-γ. CD38 + NKs from CRC expressed higher levels of CD38, NF-κB and acetyl-NF-κB and lower levels of Sirt1. When CRC CD38 + NK cells were treated with anti-CD38 monoclonal antibody, the above trends were reversed. CRC CD38 + NKs with treatment of NF-κB inhibitor also showed opposite effects on cytokine secretion and CD4 + T-cell differentiation. After treatment with a Sirt1 activator, NF-κB signaling was inhibited in these CD38 + NKs, whereas treatment with a Sirt1 inhibitor activated NF-κB signaling. The supernatants of CRC CD38 + NK culture promoted M0 macrophage polarization to M2-type. We suggest that CD38 modulates cytokine secretion by NK cells through Sirt1/NF-κB signaling pathway, thereby suppressing immune surveillance in tumorigenesis.

USP10 deubiquitinates and stabilizes CD44 leading to enhanced breast cancer cell proliferation, stemness and metastasis.

Despite extensive research, strategies to effectively combat breast cancer stemness and achieve a definitive cure remains elusive. CD44, a well-defined cancer stem cell marker is reported to promote breast cancer tumorigenesis, metastasis, and chemoresistance. However, mechanisms leading to its enhanced expression and function is poorly understood. Here, we demonstrate that USP10 positively regulates CD44 protein levels and its downstream actions. While USP10 depletion prominently downregulates CD44 protein levels and functions, its overexpression significantly enhances CD44 protein levels, leading to enhanced cluster tumor cell formation, stemness, and metastasis in breast cancer cells both in vitro and ex vivo in primary human breast tumor cells. USP10 interacts with CD44 and stabilizes it through deubiquitination both in breast cancer cell lines and human breast cancer-derived primary tumor cells. Stabilized CD44 shows enhanced interaction with cytoskeleton proteins Ezrin/Radixin/Moesin and potently activates PDGFRβ/STAT3 signalling which are involved in promoting CSC traits. Using USP10 stably expressing 4T1 cells, we further demonstrate that the USP10-CD44 axis potently promotes tumorigenicity in vivo in mice, while simultaneous depletion of CD44 in these cells renders them ineffective. In line with these findings, we further showed that inhibition of USP10 either through RNAi or the pharmacological inhibitor Spautin-1 significantly mitigated CD44 levels and its downstream function ex vivo in primary breast tumor cells. Finally, we demonstrated that primary breast tumor cells are more susceptible to chemotherapy when co-treated with USP10 inhibitor indicating that the USP10-CD44 axis could be an attractive therapeutic target in combination with chemotherapy in CD44 expressing breast cancers.

Inhibition of PCSK9 Protects against Cerebral Ischemia‒Reperfusion Injury via Attenuating Microcirculatory Dysfunction.

Proprotein convertase substilin/kexin type 9 (PCSK9), a pivotal protein regulating lipid metabolism, has been implicated in promoting microthrombotic formation and inflammatory cascades, thereby contributing to cardiovascular ischemia/reperfusion (I/R) injury. However, its involvement in cerebral I/R injury and its potential role in microcirculation protection remain unexplored. In this investigation, we utilized a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model to simulate ischemic stroke. Different concentrations of evolocumab (1, 5, 10mg/kg, i.v.), a PCSK9 inhibitor, were administered to assess its impact. Immunofluorescence staining was employed to analyze changes in the expression of occludin, claudin-5, thrombocyte, ICAM-1, VCAM-1, and CD45, providing insights into blood-brain barrier integrity, platelet adhesion, and immune cell infiltration. Moreover, the Morris water maze and elevated plus maze were utilized to evaluate neurological and behavioral functions in MCAO/R mice, shedding light on the effects of PCSK9 inhibition. Our findings revealed a surge in plasma PCSK9 levels post-MCAO/R, peaking at 24h post-reperfusion. Evolocumab (10mg/kg) treatment significantly mitigated brain infarction and neurological deficits, evidenced by enhanced locomotor function and reduced post-stroke anxiety. However, it did not ameliorate cognitive impairment following MCAO/R. Additionally, evolocumab administration led to diminished leakage of evans blue solution and upregulated expression of occludin and claudin-5. Thrombocyte, ICAM-1, VCAM-1, and CD45 levels were notably reduced in the penumbral area post-evolocumab treatment. These protective effects are speculated to be mediated through the inhibition of the ERK/NF-κB pathway. The PCSK9 inhibitor evolocumab holds promise as a therapeutic agent during the acute phase of stroke, exerting its beneficial effects by modulating the ERK/NF-κB signaling pathway.

Blood CD45+/CD3+ lymphocyte-released extracellular vesicles and mortality in hospitalized patients with coronavirus disease 2019.

The global pandemic of coronavirus disease 2019 (COVID-19) represented a major public health concern. Growing evidence shows that plasma of COVID-19 patients contains large numbers of circulating extracellular vesicles (cEVs) that correlate with disease severity and recovery. In this study, we sought to characterize the longitudinal cEV signature in critically ill COVID-19 patients during hospitalization and its relation to mortality risk. cEVs were quantitatively and phenotypically analysed in hospitalized non-surviving COVID-19 patients at baseline (n = 42) and before exitus (n = 40) and in 40 healthy volunteers as a reference group by high sensitivity nano flow cytometry using specific markers for parental cell sources and activation. Levels of cEV subtypes differed between patients with severe COVID-19 and healthy subjects, specifically those from platelets and endothelial, inflammatory and viral infected cells, which associate to high mortality risk. In the longitudinal analysis from baseline to the time point immediately preceding death, no changes were found for platelet, pan-leukocyte, and lung epithelial cell-shed cEVs, while endothelial cell releases of EVs (eEVs) significantly differed. Vascular endothelial growth factor receptor 2-positive eEVs were significantly increased before death compared to admission whereas endoglin and E-selectin-containing eEVs did not change. Conversely, lymphocyte (ℓEV), monocyte, macrophage, pericyte and progenitor cell-derived cEVs displayed significant reductions before exitus. Noteworthy, levels of CD45+/CD3+-ℓEVs were significantly associated to the patient's survival time. An evolving cEV profile able to discriminate prompt risk of death during hospitalization has been defined suggesting a role for circulating and vascular cell-derived EVs in COVID-19 pathogenesis.

Abstract A007: Characterization of miR-141 and miR-145 expression in cancer stem cells of metastatic prostate cancer patients

Abstract Background: Prostate cancer (PCA) is the second most prevalent cancer globally and the fifth leading cause of cancer-related mortality. However, effective prognostic biomarkers for metastatic PCA remain lacking. Recent research highlights the role of microRNAs (miRNAs) in regulating cancer stem cells (CSCs) and influencing tumour progression across various cancers. Therefore, in this we investigated the effects of miR-141 and miR-145 expression on the metastatic PCA of CSCs and assessed their potential role as biomarkers for metastatic PCA prognosis. Methods: The expression levels of miR-141 and miR-145 were quantified using real-time qPCR. Flow cytometry was employed to assess the levels of prostate cancer stem cells (PCSCs) in tissue samples from patients with localized (non-metastatic) and metastatic PCA. Results: Our findings revealed a significant increase in miR-141 expression, accompanied by elevated levels of CD44^high/CD24^low and CD133^high, in metastatic PCA compared to localized PCA. Conversely, reduced miR-145 expression was associated with enhanced stemness characteristics, as indicated by increased CD44^high/CD24^low and CD133^high levels, in metastatic PCA compared to localized PCA. Additionally, prostate-specific antigen (PSA) and testosterone levels exhibited a significant rise (p<0.001), while apoptosis rates decreased in both localized and metastatic PCA compared to localized PCA. Conclusions: The collective findings of this study suggest that miR-141 and miR-145, in conjunction with CSC markers, hold promise as diagnostic and prognostic tools for PCA. They may also be valuable for detecting minimal residual disease and improving therapeutic strategies for managing metastatic PCA. Citation Format: Kamla Kant Shukla, Gautam Ram Choudhary. Characterization of miR-141 and miR-145 expression in cancer stem cells of metastatic prostate cancer patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A007.

Rituximab Administration to Treat Nephrotic Syndrome in Children: 2-Year Follow-Up.

Steroid-sensitive nephrotic syndrome (SSNS) and steroid-resistant nephrotic syndrome (SRNS) significantly affect children's quality of life. There are frequent relapses in SSNS and progression in SRNS. IPNA guidelines suggest that monoclonal antibodies like rituximab (RTX) are promising treatments. This study aims to evaluate the long-term efficacy and safety of rituximab administration in children with SSNS, encompassing FRNS and SDNS, and SRNS over a two-year follow-up period, facilitating individualized management. We conducted an open-label, multicenter, randomized, and patient-oriented study (RICHNESS), involving children aged 3-18 with SRNS (18) and SSNS (11) undergoing 2 years continuous RTX therapy. The primary outcome was complete/partial remission (CR/PR), as defined by IPNA/KDIGO guidelines, at 6, 12, 18, and 24 months on RTX; secondary outcomes included adverse events. Key endpoints included the estimated glomerular filtration rate (eGFR), the albumin-to-creatinine ratio (ACR), CD20 levels, IgG levels, and the incidence of infections. Kidney biopsies were performed in 94% of SRNS patients. RTX was administered every 6-9 months, depending on CD20 levels, IgG levels, and the presence of infections. The eGFR and ACR were assessed every 6 months. Some 31 children were selected for RTX treatment. Overall, 2 experienced severe allergic reactions, leading to their exclusion from the final analysis of 29 children. In the SSNS group, all children achieved and maintained complete remission within 2 years. Remission rates in the SRNS group ranged from 39% (RR 0.78; 95% CI: 16.4-61.4%, NNT 9) at the 6th month to 72% (RR 1.44; 95% CI: 51.5-92.9%) over the 2-year follow-up period due to continuous RTX therapy. The median duration of RTX use was 26.1 months, with a median cumulative dose of 1820 mg/m2. Adverse reactions and complications were presented by mild infusion-related reactions in 3 children (10.3%), severe allergic reactions in 2 children (6.2%), hypogammaglobulinemia in 7 children (24%), infections in 3 children (10.3%), severe destructive pneumonia in 1 child, recurrent respiratory infections in 2 children, and neutropenia in 1 child (3.44%). RTX was tolerated well, and proved highly effective as a steroid-sparing agent, offering potential in terms of stopping relapses and minimizing steroid-related side effects. It also demonstrated efficacy in slowing progression in SRNS, indicating potential for use in ACR reduction and renal function restoration, but requires careful use given potential severe allergic reactions and infectious complications. Further studies should focus on long-term cost-effectiveness and deferred side effects.

Abstract 4119262: Anti-atherosclerotic Potential of M2 Macrophage-Derived Exosomes by Promoting Cholesterol Efflux of Foam Cell-Like Vascular Smooth Muscle Cells via the PPARγ-LXRα-ABCA1/ ABCG1 Pathway

Background: Impediment in the excretion of lipid deposits within SMC-derived foam cells (SMC-FCs) is one of the reasons for the continuous expansion of the plaque necrotic core. This study aims to explore the effect and underlying mechanimsm of exosomes secreted by M2 macrophage (M2-exos) on SMC-FCs lipid metabolism and plaque stability. Methods: First, immunofluorescence was used to detect the expression levels of CD45 (a recognized differentially expressed molecule of myeloid and SMC-FCs) and the key proteins of cholesterol efflux pathway, ABCA1 and ABCG1, in human early and late plaques. Exosomes derived from M0 and M2 macrophages were purified by sucrose density gradient centrifugation, and characterized based on specific morphology and surface markers. Western blot, Oil red O staining and cell total cholesterol assay were used to assess the effects of M2-exos on the lipid mechanism of SMC-FCs. RNA-seq was used to detect the miRNA profiles of M2-exos. Quantitative real-time PCR was used to identify candidate miRNAs. The dual-luciferase reporting system was utilized to assess the regulatory effect of candidate miRNA on target gene. Then, the effect of M2-exos on the progression and stability of plaques in ApoE -/- mice was evaluated using Oil red O, H&E, Masson, Movat and immunohistochemistry. Results: Immunofluorescence revealed that compared with early plaques, VSMC-FCs (CD45 - ) were significantly increased in late plaques, and the expression levels of ABCG1 and ABCA1 were marked lower than those in macrophage-derived foam cells (CD45 + ). Purified M2-exos treatment significantly promoted the cholesterol efflux of SMC-FCs in vitro. In high-fat-fed ApoE -/- mice, M2-exos significantly reduced the VSMC-FCs, delayed the progression of plaques, decreased necrotic core area and enhanced plaque stability. MiRNA profiling and comprehensive analysis of signaling pathways showed that M2-exos were rich in miR-7683-3p, which played a key role in regulating SMC-FCs lipid metabolism through PPARγ-LXRα-ABCA1/ABCG1 pathway. Dual-luciferase reporting assay showed that miR-7683-3p can specifically bind to the promoter region of homeobox genes A1(HoxA1), an inhibitor of PPARγ-LXRα-ABCA1/ABCG1 pathway. Conclusion: M2-exos exerted an obvious atherosclerotic protective effect, and the underlying mechanism was closely related to MiR-7683-3p, which targeted the 3’UTR of HoxA1 mRNA and activated the PPARγ-LXRα-ABCA1/ABCG1 mediated cholesterol efflux in SMC-FCs.

Clinical Significance of Complement and Coagulation Cascades Genes for Patients With Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the second most common acute leukemia in adults and the 5-year survival remains low. We analyzed the gene expression profiles of the complement and coagulation cascades pathway (CCCP) in 998 bone marrow (BM) and 122 peripheral blood (PB) samples of ALL patients and healthy individuals obtained from the TCGA database and evaluated their clinical significance in terms of being diagnostic and prognostic biomarkers. We identified 18 CCCP genes (SERPINA1, C5AR1, F5, CD55, PLAUR, C3AR1, THBD, CD59, PLAU, VWF, CFD, F13A1, C1QA, C1QB, C1QC, A2M, SERPINE1 and CR2) differentially expressed in the BM samples of ALL patients compared to healthy individuals. The expression levels of CD55, F13A1 and CR2 in BM were linked with the overall survival of ALL patients. While in PB only 11 CCCP genes (e.g., SERPINA1, C5AR1, F5, PLAUR, C3AR1, THBD, CFD, F13A1, C1QA, SERPINE1, and CR2) were differentially expressed and F13A1 was significantly associated with ALL patient survival. Machine learning enabled us to predict ALL using the CCCP genes and the accuracy can reach 0.9701 and 0.9167 using the BM and PB, respectively. Furthermore, using single-cell RNA sequencing, we found that the differential expression of CCCP genes was found with diversity in the BM-derived immune cells of ALL patients. Our findings suggest that the CCCP genes may play a key role in the progression of ALL and can be used as potential therapeutic targets and diagnostic markers.

Study on the correlation between viral load and activation and exhaustion levels of CD8+T cells in HIV/AIDS patients

Objective: To investigate and analyze the correlation between the expression levels of CD38, HLA-DR and programmed cell death-1 (PD-1) on peripheral blood CD8+T cells and HIV-1 RNA viral load, immune activation and exhaustion in patients with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS). Methods: A total of 81 HIV/AIDS patients (64 without antiretroviral therapy and 17 with therapy) and 40 healthy donors in the same period were enrolled as the control group. Flow cytometry was used to analyze the CD4+T lymphocyte count and the expression levels of activation markers CD38 and HLA-DR and apoptosis marker PD-1 on CD8+T cells. HIV-1 RNA in the plasma of HIV-1 infected patients was quantitatively detected by real-time fluorescence quantitative polymerase chain reaction. Variance analysis was used to compare the expression levels of CD38, HLA-DR and PD-1 on CD8+T cells between HIV/AIDS patients and healthy controls. Spearman correlation analysis was used to analyze the correlation between different T lymphocyte counts and HIV RNA viral load, and the correlation between HIV RNA viral load and peripheral blood CD8+T cell CD38, HLA-DR and PD-1. Results: Among the 81 HIV/AIDS patients, 69 (85.19%) were males and 12 (14.81%) were females, with an age M (Q1, Q3) of 58 (36.5, 65.0) years. There were 60 HIV/AIDS patients over 55 years old (74.07%) and 21 HIV/AIDS patients between 18 and 55 years old (25.93%). The results of variance analysis showed that compared with the healthy control group, the expression levels of CD38, HLA-DR and PD-1 on CD8+T cells in HIV/AIDS patients increased, and the differences were statistically significant (all P<0.05). In addition, the expression of CD38, HLA-DR and PD-1 increased significantly in patients with CD4+T cell count less than 350 cells/μl, and the differences were statistically significant (all P<0.05). Spearman correlation analysis showed that CD4+and CD4+/CD8+were negatively correlated with viral load in HIV/AIDS patients (r=-0.407 and -0.378, respectively, both P<0.05), and CD8+was positively correlated with viral load (r=0.356, P<0.05). When the HIV RNA level was≤105 CPs/ml, there was no correlation between the HIV RNA level and the expression levels of CD38, HLA-DR and PD-1 on CD8+T cells (all P>0.05). However, when the level of HIV RNA was>105 CPs/ml, the level of HIV RNA was positively correlated with the expression levels of CD38, HLA-DR and PD-1 on CD8+T cells (r=0.412, 0.387, 0.395, respectively, all P<0.05). Conclusions: The activation levels of CD38 and HLA-DR and the expression of PD-1 on CD8+T cells in the peripheral blood of HIV/AIDS patients are increased. When the viral load is high, the HIV RNA viral load is positively correlated with the activation and exhaustion levels of CD8+T cells.

SPL-108 mitigates metastasis and chemoresistance in tubo-ovarian carcinoma

BackgroundOvercoming the heterogeneous mechanisms of metastasis and chemoresistance will improve outcomes for women with tubo-ovarian carcinomas (TOCs). CD44 expression has been shown to be associated with poor prognosis and advanced disease in TOCs. In addition, studies have shown a link between chemoresistance and CD44 pathways. Given the therapeutic implications of targeting CD44, this manuscript examines the biologic effects of a novel CD44 modulator, SPL-108, in TOCs. Materials and MethodsWe assessed the effects of SPL-108 on chemosensitivity and migration in a panel of ovarian cancer cell lines with varied CD44 and MDR1 expression. In vitro experiments (cell viability assay, Western blot analysis, Calcein AM fluorescence assay, and migration assay) were carried out to determine the functional effects of SPL-108 in TOCs. FindingsOvarian cancer cell lines OVCAR5 and OVCAR8 expressed higher protein levels of CD44 as demonstrated through Western Blot analysis. SPL-108 treatment significantly decreased the number of migrating cells in OVCAR8, OVCAR5 and OVCAR3 cell lines and migratory response was independent of CD44 expression. Treatment with SPL-108 led to significant accumulation of the MDR1 substrate Calcein in OVCAR5, OVCAR8 and OVCAR3 cells lines compared to verapamil treated positive control cells. Retention of Calcein after SPL-108 treatment was seen in cell lines with high MDR1 protein expression and no Calcein retention was seen in cells lacking MDR1 expression, suggesting SPL-108 inhibits MDR1. ConclusionsSPL-108 treatment has anti-metastatic properties and may play a role in chemoresistance in preclinical models of TOCs independent of CD44 expression. Ongoing in vitro and in vivo studies will help guide further clinical development of SPL-108.

Identification of senescent cell subpopulations by CITE-seq analysis.

Cellular senescence, a state of persistent growth arrest, is closely associated with aging and age-related diseases. Deciphering the heterogeneity within senescent cell populations and identifying therapeutic targets are paramount for mitigating senescence-associated pathologies. In this study, proteins on the surface of cells rendered senescent by replicative exhaustion and by exposure to ionizing radiation (IR) were identified using mass spectrometry analysis, and a subset of them was further studied using single-cell CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) analysis. Based on the presence of proteins on the cell surface, we identified two distinct IR-induced senescent cell populations: one characterized by high levels of CD109 and CD112 (cluster 3), the other characterized by high levels of CD112, CD26, CD73, HLA-ABC, CD54, CD49A, and CD44 (cluster 0). We further found that cluster 0 represented proliferating and senescent cells in the G1 phase of the division cycle, and CITE-seq detection of cell surface proteins selectively discerned those in the senescence group. Our study highlights the heterogeneity of senescent cells and underscores the value of cell surface proteins as tools for distinguishing senescent cell programs and subclasses, paving the way for targeted therapeutic strategies in disorders exacerbated by senescence.

Conditioned medium of human umbilical cord-mesenchymal stem cells cultivated with human cord blood serum enhances stem cell stemness and secretome profiles.

The proteins secreted by human umbilical cord mesenchymal stem cells (hUC-MSCs) may enhance tissue regeneration and wound healing. Traditional hUC-MSC cultures may not be enough since they undergo recurrent cellular senescence during large-scale production. This decreases the therapeutic ability of hUC-MSCs by altering genes and proteins that control stemness, proliferation, and protein release. Human cord blood serum (CBS) and the middle-density technique were used to evaluate hUC-MSC regeneration ability. To evaluate early-passage hMSCs for secretome-based therapies, they were expanded and secreted in vitro. After 4 days, hUC-MSCs cultivated at 3000 cells/cm2 and supplemented with 1 ng/ml CBS showed increased growth, cell proliferation, and a much lower population doubling time. CBS treatment reduced CD34, CD45, and HLA-DR levels in human umbilical cord mesenchymal stem cells (hUC-MSCs) by less than 2 %. Positive markers such CD73, CD90, and CD105 were found at >97 %, like control hUC-MSCs. Over extended culture, this combination culture can increase survival, proliferation, and stemness and postpone cell death and hUC-MSC senescence. The protein profile and hUC-MSC secretion were improved to make MSC secretion protein therapeutic. This improves cell-free treatment, proliferation, and wound healing in human skin cells. To improve cell-based transplantation or cosmeceutical manufacturing, this technique can boost hUC-MSC regeneration capacity.

Monocarboxylate transporter dependent mechanism is involved in proliferation, migration, and invasion of human glioblastoma cell lines via activation of PI3K/Akt signaling pathway.

Glioblastoma multiforme is one of the most common primary tumors of the central nervous system, with a very poor prognosis. Cancer cells have been observed to upregulate pH regulators, such as monocarboxylate transporters (MCTs), with an increase in MCT4 expression being observed in several malignancies. MCT4/ recombinant cluster of differentiation 147 (CD147) transporter complex was reported to stimulate vascular endothelial growth factor (VEGF) via the phosphatidylinositol 3 kinase (PI3K) /protein kinase B (Akt) pathway, which has been proven to mediate glioblastoma invasion and migration. The present study aimed to clarify the role of the MCT4/CD147 transporter complex in glioblastoma cell proliferation, migration, and invasion. In this work, lentiviral vectors were used to overexpress MCT4/CD147 and small interfering RNA (siRNA) was used to silence MCT4/CD147 in the human glioma cell lines U87 and U251, respectively. The effects on cell proliferation, migration and invasiveness, as well as the protein expression levels of MCT4 and CD147, extracellular lactate content and Akt activation were assessed by MTT, wound-healing and invasion assays, western blotting and colorimetric method, respectively. The analysis results suggested that cell proliferation, migration, invasion, and Akt activation were decreased by siRNA in all cell lines, but were increased by lentivirus-mediated MCT4 overexpression. These findings suggest that inhibiting the activity and expression of the MCT4/CD147 transporter complex via metabolic-targeting drugs, particularly in cells with a high rate of glycolysis, should be explored as a novel strategy for glioblastoma treatment.

CD47 and Calreticulin Expression in Breast Cancer Subtypes and Anti-CD47 Inhibitory Effects in Macrophage-mediated Phagocytosis.

Macrophage-mediated cancer immune evasion is modulated by the balance between "the cluster of differentiation 47 (CD47), an anti-phagocytic signal" and "calreticulin (CALR), a pro-phagocytic signal". CD47 is highly expressed in various types of cancer. However, the expression profiles of CD47 and CALR in breast cancer, especially in different hormone receptor subtypes, and the effects of CD47 blockade in macrophage-mediated therapy are not well understood. The expression levels of CD47 and CALR were investigated in breast cancer and adjacent normal tissues using immunohistochemistry. To study the effects of CD47 blockade therapy, CD47 and CALR expression in breast cancer cell lines were determined. In vitro macrophage-mediated phagocytosis of breast cancer upon treatment with a monoclonal CD47 antibody (B6H12) were performed. CD47 and CALR were overexpressed in breast cancer tissues and their up-regulation was associated with hormone receptor subtypes. Patients with Luminal A breast cancer had higher levels of CD47, while patients with triple-negative breast cancer (TNBC) had higher levels of CALR. The levels of CD47 and CALR were also elevated in breast cancer cell lines of Luminal A (MCF-7) and TNBC (MDA-MB-231) subtypes. Interestingly, the expression ratio of surface CD47/CALR was significantly higher in MCF-7. Moreover, in vitro phagocytosis assays revealed that blockage of CD47 enhanced macrophage-mediated activity in both cancer cells with dramatically higher degrees of phagocytosis in MCF-7. The expression profiles of CD47 and CALR in breast cancer subtypes and the benefit of CD47 blocking-based immunotherapy are herein provided.

Malondialdehyde-specific natural IgM inhibit toll-like receptor 4 and peptidyl-arginine deiminase 4-dependent NETosis triggered by coronary extracellular vesicles of myocardial infarction patients

Abstract Background Neutrophil extracellular traps (NETs) are critical mediators of thromboinflammation during acute myocardial infarction (AMI). However, triggers and signalling pathways of NETosis in AMI remain incompletely understood. Levels of extracellular vesicles (EV) carrying oxidation-specific epitopes (OSE) originating from lipid peroxidation are increased at the culprit site in AMI. Importantly, natural IgM antibodies with specificity for OSE, such as malondialdehyde (MDA), have been shown to modulate functional effects of EV, and are associated with reduced cardiovascular risk. Purpose We investigated the stimulatory capacity of EV on neutrophil effector functions and specifically targeted key mediators of NET formation using pharmacological inhibitors and atheroprotective natural IgM to inhibit this process. Methods Patients were included after diagnosis of ST-segment elevation myocardial infarction and blood was aspirated from the culprit site and peripheral arterial site (n=28) during primary percutaneous coronary intervention (pPCI). Myocardial function was documented by cardiac magnetic resonance imaging 4±2 days and 195±15 days after pPCI. EV were isolated from cell culture supernatants and culprit site plasma for neutrophil stimulation in vitro. Pharmacological inhibitors were used to map NET signalling pathways of EV. Isolated EV were used for neutrophil stimulation in a murine injection model in the presence of the MDA-specific IgM LR04 or an isotype control. NET formation and other neutrophil functions were assessed by flow cytometry, ELISA and fluorescence microscopy. Results Levels of NET surrogate markers and CD45+ MDA+ EV were higher at the culprit site than in the peripheral circulation. EV generated by LPS-activated THP-1 monocytic cells induced in vitro NET formation, release of neutrophil elastase and IL-8, and degranulation of MPO and NGAL in primary human neutrophils, but not reactive oxygen species. Toll-like receptor 4 (TLR4) and peptidyl-arginine deiminase 4 (PAD4) were identified as key mediators of NET formation induced by in vitro-generated EV and EV isolated from the culprit site of AMI patients. Functionally, the MDA-specific monoclonal IgM antibody LR04, but not an isotype control, inhibited the ability of patient-derived and in vitro-generated EV to trigger release of NETs in primary human neutrophils and in mice. Titres of MDA-specific IgM antibodies were inversely associated with the NET marker citH3 in blood of AMI patients. Finally, we found that the concentration of CD45+ MDA+ EV per protective MDA-specific IgM at the culprit site showed an inverse association with left ventricular ejection fraction 72 hours and 6 months after AMI. Conclusions We show that EV can trigger several neutrophil effector functions. EV-induced NET formation is TLR4- and PAD4-dependent and can be inhibited by OSE-specific natural IgM potentially influencing cardiovascular outcomes after an acute event.

Combined administration of atorvastatin and mesenchymal stem cells synergizes to attenuate inflammation and promote efferocytosis in atherosclerotic mice

Abstract Background Atherosclerosis (AS), characterized by chronic systemic and lesional inflammation, is by far the most frequent underlying cause of cardio-cerebrovascular diseases. Recently, impaired efferocytosis, referring to defective removal of dead cells, was identified as another essential factor contributing to the development of AS, which is partly attributed to upregulation of CD47 and explain the benefits of atorvastatin (ATV). Mesenchymal stem cells (MSCs) were reported to protect against AS by inhibiting inflammation; while whether ATV and MSCs could synergize to attenuate inflammation and promote efferocytosis and consequently, reduce AS burden, remains unclear. Methods To establish the AS model, 6-week-old male ApoE-/- mice fed with Western diet for 12 weeks were used and grouped randomly (n=15 per group). The scheme of administration of ATV and human umbilical cord MSCs was shown in Figure 1A. After 6-week treatment, mice were sacrificed, and hearts and aortas were harvested. Oil Red O staining was used to evaluate the lesional burden. Immunofluorescence staining was used to assess the inflammation and efferocytosis in the plaque. A high level of CD11b+ cells (macrophages) infiltration was considered an indicator of inflammation; a high level of cleaved caspase 3 puncta unmerged with macrophages (Mac3+ area) was considered an indicator of impaired efferocytosis. Results Human umbilical cord MSCs were identified as CD73+CD90+CD105+CD45-CD14- cells. Evoked inflammation and defective clearance of apoptotic cells in the plaque were observed (Figure 1B, C). ATV and MSC treatment inhibited macrophage infiltration and activated efferocytosis. Given the mechanistic idea that upregulated CD47 blocks the Signal regulatory protein α (SIRPα) / src homology-2 (SH2)-domain-containing protein tyrosine phosphatases (SHP) signal and leads to efferocytosis impairment, we evaluated the expression levels of CD47 in plaque and phosphorylated SHP (p-SHP) in lesional macrophages (Figure 2A, B). The elevation of CD47 expression and consequent downregulation of p-SHP was reversed by both ATV and MSCs, with a lower lesion burden observed in NS+MSC, ATV+PBS and ATV+MSC groups. All benefits were more pronounced in the group received combined treatment (ATV+MSC). Conclusions The results indicated ATV and MSC administration could synergizes to attenuate inflammation and promote efferocytosis in AS plaque, and the combined treatment led to the lowest lesion burden. CD47/p-SHP signal plays an essential role in the activation of efferocytosis. The mechanisms underlying the inhibition of CD47 are currently under investigation.

Receptor for Hyaluronan Mediated Motility (RHAMM)/Hyaluronan Axis in Breast Cancer Chemoresistance.

Background/Objectives: Receptor for hyaluronan-mediated motility (RHAMM) is a hyaluronan (HA) receptor, which exerts diverse biological functions in not only physiological but also pathological conditions in human malignancies, including breast cancer. Although chemoresistance is a significant clinical challenge in breast cancer, a possible contribution of RHAMM and hyaluronan to breast cancer chemoresistance has remained unclear. Methods: We immunolocalized RHAMM and HA in breast carcinoma tissues. Also, we utilized epirubicin-sensitive (parental) and rpirubicin-resistant (EPIR) breast cancer cell lines to explore the role of RHAMMM in breast cancer progression. Results: We found out that RHAMM and HA were cooperatively correlated with breast cancer aggressiveness and recurrence after chemotherapy. In vitro studies demonstrated that RHAMM was overexpressed in EPIR cells compared to parental cells. In addition, the knockdown of RHAMM significantly suppressed proliferation and migration of both parental and EPIR cells. On the other hand, the expression level of cancer stem cell marker CD44, which was overexpressed in M-EPIR (epirubicin-resistant MCF-7 subline) compared to MCF-7, was significantly suppressed by knockdown of RHAMM. In addition, the knockdown of RHAMM significantly altered the expression of N-cadherin and E-cadherin, leading to an epithelial phenotype. Conclusions: Aberrant RHAMM signaling were considered to cause chemoresistance related to cancer stemness and epithelial to mesenchymal transition, and increased cell proliferation and migration of both chemo-sensitive and chemo-resistant breast cancer cells.