Serglycin Research Articles

CCAAT/enhancer-binding protein α-dependent regulation of granule formation in mast cells by intestinal bacteria.

The antiallergic effects of gut microbiota have been attracting attention in recent years, but the underlying cellular and molecular mechanisms have not yet been fully understood. In this study, we aimed to investigate these mechanisms specifically focusing on mast cells. Mast cells retain intracellular granules containing various inflammatory mediators such as histamine, which are released outside the cells upon IgE and allergen stimulation. We previously reported that increased expression of the transcription factor, CCAAT/enhancer-binding protein α (C/EBPα), suppresses granule formation in mast cells and that Lacticaseibacillus casei JCM1134T (LC) upregulates C/EBPα levels. Here, granule formation in mouse bone marrow-derived mast cells was suppressed in a MyD88-dependent manner after LC treatment due to C/EBPα-dependent downregulation of the genes encoding serglycin (SRGN) and mast cell protease 4 (Mcpt4). Furthermore, C/EBPα expression was regulated by DNA methylation in the 5' region far upstream of the transcription start site. LC suppressed DNA methylation of specific CpG motifs in the 5' region of the C/EBPα gene. These results conclude that specific gut microbial components, such as those from LC, suppress granule formation in mast cells by inhibiting SRGN and Mcpt4 expression via reduced C/EBPα gene methylation.

SRGN promotes macrophage recruitment through CCL3 in osteoarthritis

ABSTRACT Background Osteoarthritis (OA) is a degenerative disease that affects synovial joints and leads to significant pain and disability, particularly in older adults. Infiltration of macrophages plays a key role in the progression of OA. However, the mechanisms underlying macrophage recruitment in OA are not fully understood. Methods The Serglycin (SRGN) expression pattern was analyzed, along with its association with macrophage infiltration in OA, using bioinformatic methods. SRGN expression in chondrocytes was altered by small interfering RNA (siRNA) and plasmids. Conditioned media (CM) was obtained from transfected chondrocytes to establish a co-culture model of chondrocytes and THP-1 derived macrophages. The impact of SRGN on macrophage recruitment was evaluated using a transwell assay. Furthermore, the regulatory effect of SRGN on CCL3 was validated through qPCR, WB, and ELISA experiments. Results In OA patients, the upregulation of SRGN positively correlated with K-L grade and macrophage infiltration. It was found that SRGN expression and secretion were up-regulated in OA and that it can promote macrophage migration in vitro. Further investigation showed that SRGN affects macrophage migration by regulating the expression of CCL3. Conclusion SRGN in chondrocytes plays a role in promoting the recruitment of THP-1 derived macrophages in vitro by regulating production of CCL3.

Serglycin controls megakaryocyte retention of platelet factor 4 and influences megakaryocyte fate in bone marrow

AbstractMegakaryocytes (MKs) produce platelets, and similar to other hematopoietic progenitors, they are involved in homeostatic aspects of their bone marrow niche. MKs release and endocytose various factors, such as platelet factor 4 (PF4)/CXCL4. Here, we show that the intra-α-granular proteoglycan, serglycin (SRGN), plays a key role in this process by retaining PF4, and perhaps other factors, during MK maturation. Immature, SRGN–/– MKs released ∼80% of their PF4, and conditioned media from these cells negatively affected wild-type MK differentiation in vitro. This was replicated in wild-type MKs by treatment with the polycation surfen, a known inhibitor of glycosaminoglycan (GAG)/protein interactions. In vivo, SRGN–/– mice had an interstitial accumulation of PF4, transforming growth factor β1, interleukin-1β, and tumor necrosis factor α in their bone marrow and increased numbers of immature MKs, consistent with their mild thrombocytopenia. SRGN–/– mice also had reduced numbers of hematopoietic stem cells and multipotent progenitors, reduced laminin, and increased collagen I deposition. These findings demonstrate that MKs depend on SRGN and its charged GAGs to balance the distribution of PF4 and perhaps other factors between their α-granules and their adjacent extracellular spaces. Disrupting this balance negatively affects MK development and bone marrow microenvironment homeostasis.

Tumour cell-derived serglycin promotes IL-8 secretion of CAFs in gastric cancer

BackgroundCancer-associated fibroblasts (CAFs)-derived IL-8 plays important roles in chemoresistance, immunosuppression, and lymph node metastasis of gastric cancer. However, the mechanisms underlying IL-8 production in CAFs remains unclear.MethodsDNA pulldown assay was performed to identify the transcription factors responsible for IL-8 expression in CAFs, which was further verified using CHIP-qPCR and DNA agarose gel electrophoresis assays. The cellular localisation of IL-8 was analysed using multiplex immunofluorescence (MxIF).ResultsMxIF demonstrated that IL-8 was mainly produced by CAFs in gastric cancer. Lysine[K]-specific demethylase 5B (KDM5B) was identified as an IL-8 transcription factor in CAFs, and the binding of KDM5B to phosphorylated RB1 limited the transcriptional regulation of IL-8 in gastric cancer cells. Serglycin (SRGN) secreted by tumour cells activated the CD44/c-Myc pathway to upregulate KDM5B expression, thereby promoting IL-8 production in CAFs. Furthermore, tumour-associated neutrophils (TANs)-derived regenerating family member 4 (REG4) upregulates SRGN expression by activating cAMP-responsive element binding protein 1 (CREB1) in gastric cancer cells. Thus, the SRGN-IL-8-TANs-SRGN loop, which facilitates tumour progression, has been explored in gastric cancer.ConclusionsThis study revealed the mechanisms of the preferential production of IL-8 by CAFs in gastric cancer, and paves the way for potential new therapeutic strategies for gastric cancer.

The extracellular matrix proteoglycan serglycin is associated with human atherosclerotic plaque inflammation

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): This work was supported by grants from the Swedish Research Council, the Swedish Heart and Lung Foundation, Skåne University Hospital funds, Royal Physiographic Society in Lund and O.E. and Edla Johanssons Foundation. Background Atherosclerotic cardiovascular disease is caused by the formation of plaques in the arterial wall which contain lipids, cells, cell debris and a unique extracellular matrix (ECM) signature. These plaques may ultimately rupture or erode away causing thrombosis, thereby leading to myocardial infarction or stroke. Rupture-prone plaques generally bear a greater inflammatory activity, with ECM proteins suggested to influence the inflammatory activity. Among many ECM proteins, serglycin (SRGN) is highly expressed by inflammatory cells, and SRGN has been linked to modulation of the inflammatory response in disease such as cancer. Yet, if SRGN contributes to the inflammatory circuitry in human atherosclerotic plaques remains to be explored. Purpose We aimed to investigate if SRGN was associated with inflammation in atherosclerotic plaques. Methods Plaque protein levels of SRGN were measured by ELISA in human carotid plaques obtained from the Carotid Plaque Imaging Project (CPIP) biobank. Plaque RNA expression levels of SRGN and cell markers were assessed by plaque bulk RNA sequencing. SRGN expression and distribution were further examined in tissue plaque sections through histology and multispectral imaging. In vitro models were used to investigate the functional consequences of SRGN expression changes, using SRGN CRISPR knockout THP1 monocytic cells. Immunoprecipitation of SRGN and immunoblotting against SRGN, heparan and chondroitin sulfate chains were employed to structurally characterize plaque protein SRGN. Results SRGN protein levels were significantly higher in plaques from asymptomatic patients compared to symptomatic. Multispectral imaging showed that SRGN protein was observed in plaque areas rich in macrophages and lipids. Using immunoprecipitation and immunoblotting, we identified a unique low glycosylated plaque SRGN, with both heparan sulfate and chondroitin sulfate glycosaminoglycans decorating its protein core. This exclusive configuration suggests that the plaque cell origin of SRGN may be macrophages or mast cells. In support of this, plaque SRGN RNA expression correlated with RNA levels of pro-inflammatory macrophage cells markers like CD68, CD163, MRC1, ITGAX. Plaque SRGN protein levels correlated with plaque levels of oxidised lipoproteins, proatherogenic (CCL1, IL7, MCP-4 and TNFα) and antiatherogenic cytokines (IL33, IL16 and CXCL13), as well as with growth factors, such as TGF-β. On the one hand, in vitro suppression of SRGN expression in monocytes led to a reduction in the anti-inflammatory M2-polarized phenotype, while M1 pro-inflammatory macrophages were unskewed. Furthermore, our in vitro studies showed that upon SRGN knockdown M2 macrophages retained more oxidized lipoproteins. Conclusions Our results suggest that SRGN may be a crucial modulator of inflammation in human atherosclerotic plaques, potentially by affecting the bioavailability of effector molecules such as growth factors and cytokines.

The Expression of Serglycin Is Required for Active Transforming Growth Factor β Receptor I Tumorigenic Signaling in Glioblastoma Cells and Paracrine Activation of Stromal Fibroblasts via CXCR-2.

Serglycin (SRGN) is a pro-tumorigenic proteoglycan expressed and secreted by various aggressive tumors including glioblastoma (GBM). In our study, we investigated the interplay and biological outcomes of SRGN with TGFβRI, CXCR-2 and inflammatory mediators in GBM cells and fibroblasts. SRGN overexpression is associated with poor survival in GBM patients. High SRGN levels also exhibit a positive correlation with increased levels of various inflammatory mediators including members of TGFβ signaling pathway, cytokines and receptors including CXCR-2 and proteolytic enzymes in GBM patients. SRGN-suppressed GBM cells show decreased expressions of TGFβRI associated with lower responsiveness to the manipulation of TGFβ/TGFβRI pathway and the regulation of pro-tumorigenic properties. Active TGFβRI signaling in control GBM cells promotes their proliferation, invasion, proteolytic and inflammatory potential. Fibroblasts cultured with culture media derived by control SRGN-expressing GBM cells exhibit increased proliferation, migration and overexpression of cytokines and proteolytic enzymes including CXCL-1, IL-8, IL-6, IL-1β, CCL-20, CCL-2, and MMP-9. Culture media derived by SRGN-suppressed GBM cells fail to induce the above properties to fibroblasts. Importantly, the activation of fibroblasts by GBM cells not only relies on the expression of SRGN in GBM cells but also on active CXCR-2 signaling both in GBM cells and fibroblasts.

SRGN amplifies microglia-mediated neuroinflammation and exacerbates ischemic brain injury

BackgroundMicroglia is the major contributor of post-stroke neuroinflammation cascade and the crucial cellular target for the treatment of ischemic stroke. Currently, the endogenous mechanism underlying microglial activation following ischemic stroke remains elusive. Serglycin (SRGN) is a proteoglycan expressed in immune cells. Up to now, the role of SRGN on microglial activation and ischemic stroke is largely unexplored.MethodsSrgn knockout (KO), Cd44-KO and wild-type (WT) mice were subjected to middle cerebral artery occlusion (MCAO) to mimic ischemic stroke. Exogenous SRGN supplementation was achieved by stereotactic injection of recombinant mouse SRGN (rSRGN). Cerebral infarction was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Neurological functions were evaluated by the modified neurological severity score (mNSS) and grip strength. Microglial activation was detected by Iba1 immunostaining, morphological analysis and cytokines’ production. Neuronal death was examined by MAP2 immunostaining and FJB staining.ResultsThe expression of SRGN and its receptor CD44 was significantly elevated in the ischemic mouse brains, especially in microglia. In addition, lipopolysaccharide (LPS) induced SRGN upregulation in microglia in vitro. rSRGN worsened ischemic brain injury in mice and amplified post-stroke neuroinflammation, while gene knockout of Srgn exerted reverse impacts. rSRGN promoted microglial proinflammatory activation both in vivo and in vitro, whereas Srgn-deficiency alleviated microglia-mediated inflammatory response. Moreover, the genetic deletion of Cd44 partially rescued rSRGN-induced excessed neuroinflammation and ischemic brain injury in mice. Mechanistically, SRGN boosted the activation of NF-κB signal, and increased glycolysis in microglia.ConclusionSRGN acts as a novel therapeutic target in microglia-boosted proinflammatory response following ischemic stroke.

Identification of Candidate Biomarkers of Alzheimer's Disease via Multiplex Cerebrospinal Fluid and Serum Proteomics.

Alzheimer's disease (AD) is the most prevalent form of dementia among elderly people worldwide. Cerebrospinal fluid (CSF) is the optimal fluid source for AD biomarkers, while serum biomarkers are much more achievable. To search for novel diagnostic AD biomarkers, we performed a quantitative proteomic analysis of CSF and serum samples from AD and normal cognitive controls (NC). CSF and serum proteomes were analyzed via data-independent acquisition quantitative mass spectrometry. Our bioinformatic analysis was based on Gene Ontology (GO) functional annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. In comparison to the controls, 8 proteins were more abundant in AD CSF, and 60 were less abundant in AD CSF, whereas 55 proteins were more and 10 were less abundant in the serum samples. ATPase-associated activity for CSF and mitochondrial functions for CSF and serum were the most enriched GO terms of the DEPs. KEGG enrichment analysis showed that the most significant pathways for the differentially expressed proteins were the N-glycan biosynthesis pathways. The area under the curve (AUC) values for CSF sodium-/potassium-transporting ATPase subunit beta-1 (AT1B1), serglycin (SRGN), and thioredoxin-dependent peroxide reductase, mitochondrial (PRDX3) were 0.867 (p = 0.004), 0.833 (p = 0.008), and 0.783 (p = 0.025), respectively. A panel of the above three CSF proteins accurately differentiated AD (AUC = 0.933, p = 0.001) from NC. The AUC values for serum probable phospholipid-transporting ATPase IM (AT8B4) and SRGN were moderate. The AUC of the CSF SRGN + serum SRGN was 0.842 (p = 0.007). These novel AD biomarker candidates are mainly associated with inflammation, ATPase activity, oxidative stress, and mitochondrial dysfunction. Further studies are needed to investigate the molecular mechanisms by which these potential biomarkers are involved in AD.

Decellularised spinal cord matrix manipulates glial niche into repairing phase via serglycin-mediated signalling pathway.

Astrocytes are the most abundant and widespread glial cells in the central nervous system. The heterogeneity of astrocytes plays an essential role in spinal cord injury (SCI) repair. Decellularised spinal cord matrix (DSCM) is advantageous for repairing SCI, but little is known regarding the exact mechanisms and niche alterations. Here, we investigated the DSCM regulatory mechanism of glial niche in the neuro-glial-vascular unit using single-cell RNA sequencing. Our singlecellsequencing,molecular and biochemical experiments validated that DSCM facilitated the differentiation of neural progenitor cells through increasing the number of immature astrocytes. Upregulation of mesenchyme-related genes, which maintained astrocyte immaturity, causing insensitivity to inflammatory stimuli. Subsequently, we identified serglycin (SRGN) as a functional component of DSCM, which involves inducing CD44-AKT signalling to trigger human spinal cord-derived primary astrocytes (hspASCs) proliferation and upregulation of genes related to epithelial-mesenchymal transition, thus impeding astrocyte maturation. Finally, we verified that SRGN-COLI and DSCM had similar functions in the human primary cell co-culture system to mimic the glia niche. In conclusion, our work revealed that DSCM reverted astrocyte maturation and altered the glia niche into the repairing phase through the SRGN-mediated signalling pathway.

M6PR- and EphB4-Rich Exosomes Secreted by Serglycin-Overexpressing Esophageal Cancer Cells Promote Cancer Progression.

Accumulating evidence shows that exosomes participate in cancer progression. However, the functions of cancer cell exosome-transmitted proteins are rarely studied. Previously, we reported that serglycin (SRGN) overexpression promotes invasion and metastasis of esophageal squamous cell carcinoma (ESCC) cells. Here, we investigated the paracrine effects of exosomes from SRGN-overexpressing ESCC cells (SRGN Exo) on ESCC cell invasion and tumor angiogenesis, and used mass spectrometry to identify exosomal proteins involved. Cation-dependent mannose-6-phosphate receptor (M6PR) and ephrin type-B receptor 4 (EphB4) were pronouncedly upregulated in SRGN Exo. Upregulated exosomal M6PR mediated the pro-angiogenic effects of SRGN Exo both in vitro and in vivo, while augmented exosomal EphB4 mediated the pro-invasive effect of SRGN Exo on ESCC cells in vitro. In addition, in vitro studies showed that manipulation of M6PR expression affected the viability and migration of ESCC cells. Both M6PR and EphB4 expression levels were positively correlated with that of SRGN in the serum of patients with ESCC. High level of serum M6PR was associated with poor overall survival rates. Taken together, this study presents the first proof that exosomal M6PR and EphB4 play essential roles in tumor angiogenesis and malignancy, and that serum M6PR is a novel prognostic marker for ESCC patients.

Cathepsin L promotes chemresistance to neuroblastoma by modulating serglycin

Cathepsin L (CTSL), a lysosomal acid cysteine protease, is found to play a critical role in chemosencitivity and tumor progression. However, the potential roles and molecular mechanisms of CTSL in chemoresistance in neuroblastoma (NB) are still unclear. In this study, the correlation between clinical characteristics, survival and CTSL expression were assessed in Versteeg dataset. The chemoresistant to cisplatin or doxorubicin was detected using CCK-8 assay. Western blot was employed to detect the expression of CTSL, multi-drug resistance proteins, autophagy-related proteins and apoptosis-related proteins in NB cells while knocking down CTSL. Lysosome staining was analyzed to access the expression levels of lysosomes in NB cells. The expression of apoptosis markers was analyzed with immunofluorescence. Various datasets were analyzed to find the potential protein related to CTSL. In addition, a subcutaneous tumor xenografts model in M-NSG mice was used to assess tumor response to CTSL inhibition in vivo. Based on the validation dataset (Versteeg), we confirmed that CTSL served as a prognostic marker for poor clinical outcome in NB patients. We further found that the expression level of CTSL was higher in SK-N-BE (2) cells than in IMR-32 cells. Knocking down CTSL reversed the chemoresistance in SK-N-BE (2) cells. Furthermore, combination of CTSL inhibition and chemotherapy potently blocked tumor growth in vivo. Mechanistically, CTSL promoted chemoresistance in NB cells by up-regulating multi-drug resistance protein ABCB1 and ABCG2, inhibiting the autophagy level and cell apoptpsis. Furthermore, we observed six datasets and found that Serglycin (SRGN) expression was positively associated with CTSL expresssion. CTSL could mediate chemoresistance by up-regulating SRGN expression in NB cells and SRGN expression was positively correlated with poor prognosis of NB patients. Taken together, our findings indicate that the CTSL promotes chemoresistance to cisplatin and doxorubicin by up-regulating the expression of multi-drug resistance proteins and inhibiting the autophagy level and cell apoptosis in NB cells. Thus, CTSL may be a therapeutic target for overcoming chemoresistant to cisplatin and doxorubicin in NB patients.

MiR-26b-5p suppresses chemoresistance in breast cancer by targeting serglycin.

Chemoresistance is a crucial barrier to limit the therapeutic outcome of breast cancer (BC), and the mechanism underlying chemoresistance development in BC is not fully understood. In this study, we aimed to investigate the potential involvement of miR-26b-5p/serglycin (SRGN) axis in BC drug resistance. The expression level of SRGN in drug-resistant BC cells was investigated by western blotting analysis, real-time quantitative PCR (qRT-PCR), immunohistochemical staining, and ELISA. Its expression between chemoresistant and sensitive patient samples was compared by qRT-PCR. Bioinformatics tool and dual-luciferase reporter assay were employed to identify miR-26b-5p as a regulator of SRGN. Functional assays were performed to examine cell proliferation, cell viability, apoptosis, migration, and invasion ability in vitro. Xenograft tumorigenesis experiment was conducted to evaluate the tumor suppressor effect of miR-26b-5p on chemoresistant BC cells. SRGN expression was significantly upregulated in both chemoresistant BC cell lines and chemoresistant patient samples. miR-26b-5p was identified as an upstream regulator of SRGN. Overexpression of miR-26b-5p downregulated SRGN expression, overcame chemoresistance, and suppressed cell proliferation, migration, and invasion in BC cells. Overexpression of miR-26b-5p also suppressed the tumorigenesis of chemoresistant BC cells in vivo. Mechanistically, the downregulation of SRGN by miR-26b-5p decreased the expression of breast cancer drug-resistant protein and multidrug-resistant protein 1 in chemoresistant BC cells. Our study identified miR-26b-5p as a tumor suppressor which targets SRGN to sensitize BC cells to chemotherapeutics. These results suggest that miR-26b-5p and SRGN may serve as potential biomarkers and targets for BC chemotherapy.

Identification of serum peptide biomarker for ovarian cancer diagnosis by Clin-TOF-II-MS combined with magnetic beads technology

Objective: To explore the serum cyclic polypeptide biomarkers for ovarian cancer diagnosis. Methods: A total of 54 patients with epithelial ovarian cancer confirmed by pathology in Cancer Hospital, Chinese Academy of Medical Sciences from March 2018 to September 2018 were selected as the study subjects, and 40 healthy women with normal examination results in the cancer screening center were selected as the control. All of the samples were randomly divided into training set and validation set at the ratio of 1∶1 with a random number. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) combined with magnetic bead technology was used for detecting peptide profiling in serum samples to screen significantly differently expressed peptides between ovarian cancer group and control group of the training set (score>5). Receiver operating characteristic (ROC) curve analysis was used to screen differential peptide peaks with area under curve (AUC) ≥0.8, sensitivity and specificity>90% in the training set and validation set. Liquid chromatography-mass spectrometry (LC-MS/MS) was further used to determine the composition of differentially expressed peptides. Results: By comparing the peptide profiles of the two groups, 102 differential peptide peaks were initially detected in the mass-to-charge ratio range of 1 000 to 10 000. ROC curve analysis showed that there were 42 differential peptide peaks with AUC ≥0.8 in both training set and validation set, 19 of which were highly expressed in ovarian cancer group, and 23 were lowly expressed. There were 15 different peptide peaks in highly expressed ovarian cancer group with sensitivity and specificity over 90%. The mass-to-charge ratios were 7 744.27, 5 913.41, 5 329.87, 4 634.21, 4 202.02, 3 879.26, 3 273.35, 3 253.79, 3 234.34, 2 950.33, 2 664.51, 2 018.38, 1 893.37, 1 498.69 and 1 287.55. There were 15 different peptide peaks in lowly expressed ovarian cancer group with sensitivity and specificity over 90%, the mass-to-charge ratios were 9 288.46, 7 759.77, 5 925.24, 4 652.77, 4 210.42, 3 887.02, 3 279.90, 3 240.82, 2 962.15, 2 932.70, 2 022.42, 1 897.16, 1 501.69, 1 337.38 and 1 290.13. No protein composition was identified in 15 different peptide peaks in lowly expressed ovarian cancer group. The two protein compositions identified in 15 different peptide peaks in highly expressed ovarian cancer group were recombinant serglycin (SRGN) and fibinogen alpha chain (FGA), the mass-to-charge ratios of which were 1 498.696 and 5 913.417, respectively. The sensitivity and specificity of the two proteins for ovarian cancer diagnosis were 100%, 100% and 90.9%, 100%, respectively. Conclusion: SRGN and FGA are highly expressed in the serum of ovarian cancer patients, which may be potential diagnostic markers for ovarian cancer.

Single-Cell Multi-Omic Analysis Uncovers Comprised Immune Function and Primary Resistance Mechanism in Acute Myeloid Leukemia

Background: Approximately 20% of AML patients do not respond to induction chemotherapy (primary resistance) and 40-60% of patients develop secondary resistance, eventually leading to relapse followed by refractory disease (RR-AML). Diversified molecular mechanisms have been proposed for drug resistance and RR phenotype. However, we still cannot predict when relapse will occur, nor which patients will become resistant to therapy.Single-cell multi-omic (ScMo) profiling may provide new insights into our understanding of hematopoietic stem cell (HSC) differentiation trajectories, tumor heterogeneity and clonal evolution. Here we applied ScMo to profile bone marrow (BM) from AML patients and healthy controls.Methods: AML samples were collected at diagnosis with institutional IRB approval. Cells were stained with a panel of 62 DNA barcoded antibodies and 10x Genomics Single Cell 3' Library Kit v3 was used to generate ScMo data. After normalization, clusters were identified using Uniform Manifold Approximation and Projection (UMAP) and annotated using MapCell (Koh and Hoon, 2019). We analyzed 23,933 cells from 4 adult AML BM samples, and 39,522 cells from 2 healthy adults and 3 sorted CD34+ normal BM samples. Gene set enrichment analysis (GSEA) and Enrichr program were used to examine underlying pathways among differentially expressed genes between healthy and AML samples.Results: We identified 16 cell types between the AML and normal samples (Fig 1a) amongst 45 clusters in the UMAP projection (Fig 1b). Comparative analysis of the T cell clusters in AML samples with healthy BM cells identified an “AML T-cell signature” with over-expression of genes such as granzymes, NK/T cell markers, chemokine and cytokine, proteinase and proteinase inhibitor (Fig 2a). Among them, IL32 is known to be involved in activation-induced cell death in T cells and has immunosuppressive role, while CD8+ GZMB+ and CD8+ GZMK+ cells are considered as dysfunctional or pre-dysfunctional T cells. Indeed, Enrichr analysis showed the top rank of phenotype term - “decreased cytotoxic T cell cytolysis”.We next examined whether NK cells, are similarly dysfunctional in the AML ecosystem. The “AML NK cell signature” includes Fc Fragment family, IFN-stimulated genes (ISGs), the effector protein-encoding genes and other genes when compared to normal NK cells (Fig 2b). GSEA analysis revealed “PD-1 signalling” among the top 5 ranked pathways in AML-NK cells, though no increase in PD-1 protein nor PDCD1 gene were identified in these cells. Inhibitory receptor CD160 was expressed higher in AML samples along with exhaustion (dysfunction) associated genes TIGIT, PRF1 and GZMB (Fig 2c). Enrichr analysis uncovered enrichment of “abnormal NK cell physiology and ”impaired natural killer cell mediated cytotoxicity“. Similarly, the ”AML monocyte signature“ was significantly enriched with genes in ”Tumor Infiltrating Macrophages in Cancer Progression and Immune Escape“ and ”Myeloid Derived Suppressor Cells in Cancer Immune Escape“.We also analyzed HSPC component in one pair of cytogenetically matched, untreated complete remission (CR) /RR AML pair (Fig 2d). Notably, half of the 10 genes overexpressed in RR-AML, CXCR4, LGALS1, S100A8, S100A9, SRGN (Serglycin), regulate cell-matrix interaction and play pivotal roles in leukemic cells homing bone marrow niche. The first 4 of these genes have been demonstrated as prognostic indicators of poor survival and associated with chemo-resistance and anti-apoptotic function. Furthermore, single-cell trajectory analysis of this CR/RR pair illustrated a change in differentiation pattern of HSPCs in CR-AML to monocytes in RR-AML. We are currently analyzing more AML samples to validate these findings.Conclusions: Our ScMo analysis demonstrates that the immune cells are systematically reprogrammed and functionally comprised in the AML ecosystem. Upregulation of BM niche factors could be the underlying mechanism for RR-AML. Thus, reversing the inhibited immune system is an important strategy for AML therapy and targeting leukemic cell-BM niche interaction should be considered for cases with high expression of these molecules on AML HSPCs.Note: J.Z. and J.A.S. share co-first authorship. [Display omitted] DisclosuresScolnick: Proteona Pte Ltd: Current holder of individual stocks in a privately-held company. Xu: Proteona Pte Ltd: Current Employment. Ooi: Jansen: Honoraria; Teva Pharmaceuticals: Honoraria; GSK: Honoraria; Abbvie: Honoraria; Amgen: Honoraria. Lovci: Proteona Pte Ltd: Current Employment. Chng: Aslan: Research Funding; Takeda: Honoraria; Johnson & Johnson: Honoraria, Research Funding; BMS/Celgene: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Antengene: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; AbbVie: Honoraria.

Serglycin induces osteoclastogenesis and promotes tumor growth in giant cell tumor of bone

Giant cell tumor of bone (GCTB) is an aggressive osteolytic bone tumor characterized by the within-tumor presence of osteoclast-like multinucleated giant cells (MGCs), which are induced by the neoplastic stromal cells and lead to extensive bone destruction. However, the underlying mechanism of the pathological process of osteoclastogenesis in GCTB is poorly understood. Here we show that the proteoglycan Serglycin (SRGN) secreted by neoplastic stromal cells plays a crucial role in the formation of MGCs and tumorigenesis in GCTB. Upregulated SRGN expression and secretion are observed in GCTB tumor cells and patients. Stromal-derived SRGN promotes osteoclast differentiation from monocytes. SRGN knockdown in stromal cells inhibits tumor growth and bone destruction in a patient-derived orthotopic xenograft model of mice. Mechanistically SRGN interacts with CD44 on the cell surface of monocytes and thus activates focal adhesion kinase (FAK), leading to osteoclast differentiation. Importantly, blocking CD44 with a neutralizing antibody reduces the number of MGCs and suppresses tumorigenesis in vivo. Overall, our data reveal a mechanism of MGC induction in GCTB and support CD44-targeting approaches for GCTB treatment.

Serglycin promotes proliferation, migration, and invasion via the JAK/STAT signaling pathway in osteosarcoma.

Background: Osteosarcoma (OS) is a common disease in the world, and its pathogenesis is still unclear. This study aims to identify the key genes that promote the proliferation, invasion, and metastasis of osteosarcoma cells.Method: GSE124768 and GSE126209 were downloaded from the Gene Expression Omnibus (GEO) database. The gene ontology and enrichment pathway were analyzed by FunRich software. qPCR and Western blot were used to detect the gene expression. After gene knockdown, Transwell and wound healing assays were conducted on osteosarcoma cells to detect whether the genes were defined before enhancing the invasion of osteosarcoma.Results: Totally, 341 mRNAs were found to be regulated differentially in osteosarcoma cells compared to osteoblasts. In addition, the expression level of Serglycin (SRGN) in osteosarcoma cells was higher than that in human osteoblasts. The invasion and proliferation ability of osteosarcoma cells with upregulated Serglycin was significantly increased, and on the contrary, decreased after Serglycin knockdown. Moreover, we preliminarily found that Serglycin may associate with the JAK/STAT signaling pathway.Conclusions: By using microarray and bioinformatics analyses, differently expressed mRNAs were identified and a complete gene network was constructed. To our knowledge, we describe for the first time Serglycin as a potential biomarker.

Serglycin-Deficiency Causes Reduced Weight Gain and Changed Intestinal Cytokine Responses in Mice Infected With Giardia intestinalis.

The proteoglycan serglycin (SG) is expressed by different innate and adaptive immune cells, e.g. mast cells, macrophages, neutrophils, and cytotoxic T lymphocytes, where SG contributes to correct granule storage and extracellular activity of inflammatory mediators. Here the serglycin-deficient (SG−/−) mouse strain was used to investigate the impact of SG on intestinal immune responses during infection with the non-invasive protozoan parasite Giardia intestinalis. Young (≈11 weeks old) oral gavage-infected congenic SG−/− mice showed reduced weight gain as compared with the infected SG+/+ littermate mice and the PBS-challenged SG−/− and SG+/+ littermate mice. The infection caused no major morphological changes in the small intestine. However, a SG-independent increased goblet cell and granulocyte cell count was observed, which did not correlate with an increased myeloperoxidase or neutrophil elastase activity. Furthermore, infected mice showed increased serum IL-6 levels, with significantly reduced serum IL-6 levels in infected SG-deficient mice and decreased intestinal expression levels of IL-6 in the infected SG-deficient mice. In infected mice the qPCR analysis of alarmins, chemokines, cytokines, and nitric oxide synthases (NOS), showed that the SG-deficiency caused reduced intestinal expression levels of TNF-α and CXCL2, and increased IFN-γ, CXCL1, and NOS1 levels as compared with SG-competent mice. This study shows that SG plays a regulatory role in intestinal immune responses, reflected by changes in chemokine and cytokine expression levels and a delayed weight gain in young SG−/− mice infected with G. intestinalis.

Cancer-associated fibroblasts secrete hypoxia-induced serglycin to promote head and neck squamous cell carcinoma tumor cell growth in vitro and in vivo by activating the Wnt/β-catenin pathway.

The tumor microenvironment (TME) is known to play a prominent role in the pathology of head and neck squamous cell carcinoma (HNSCC). Cancer-associated fibroblasts (CAFs) have been reported to regulate tumor progression, and serglycin (SRGN), one of the paracrine cytokines of CAFs, has been reported to play an important role in various signaling pathways. Hypoxia is a distinct feature of the HNSCC TME. Here, we investigated the mechanism underlying CAF-secreted SRGN leading to HNSCC progression under hypoxia. Immunohistochemical staining was used to detect SRGN expression in clinical HNSCC samples, after which its relation with patient survival was assessed. CAFs were isolated and SRGN expression and secretion by CAFs under normoxia and hypoxia were confirmed using qRT-PCR and ELISA assays, respectively. HNSCC sphere-forming abilities, stemness-related gene expression, and chemoresistance were assessed with or without SRGN treatment. A Wnt/β-catenin pathway inhibitor (PNU-75,654) was used to block its activation, after which nuclear translocation of β-catenin in the presence of SRGN with or without PNU-75,654 was evaluated. shRNAs were used to stably knock down SRGN expression in CAFs. HNSCC tumor cells with or without (SRGN silenced) CAFs were inoculated submucosally in nude mice after which tumor weights and sizes were determined to assess the effects of CAFs and SRGN on tumor growth. We found that SRGN was expressed in both HNSCC tumor and stroma cells, and that high SRGN expression in the stroma cells, but not in the tumor cells, was significantly related to a poor patient survival. After the extraction of CAFs and normal fibroblasts (NFs) from paired tumor samples and adjacent normal tissues, respectively, we found that the expression of CAF-specific genes, including fibroblast activation protein (FAP) and alpha-smooth muscle actin (α-SMA), was clearly upregulated compared to the expression in NFs. The hypoxia marker HIF-1α was found to be expressed in tumor stroma cells. Hypoxyprobe immunofluorescence staining confirmed stromal hypoxia in an orthotopic tongue cancer mouse model. Using qRT-PCR and ELISA we found that a hypoxic TME upregulated SRGN expression and secretion by CAFs. SRGN markedly enhanced the sphere-forming ability, stemness-related gene expression and chemoresistance of HNSCC tumor cells. SRGN activated the Wnt/β-catenin pathway and promoted β-catenin nuclear translocation. An in vivo study confirmed that CAFs can accelerate HNSCC tumor growth, and that this effect can be counteracted by SRGN silencing. Our data indicate that a hypoxic tumor stroma can lead to upregulation of SRGN expression. SRGN secreted by CAFs can promote β-catenin nuclear translocation to activate downstream signaling pathways, leading to enhanced HNSCC cell stemness, chemoresistance and accelerated tumor growth.

Significance of serglycin and its binding partners in autocrine promotion of metastasis in esophageal cancer.

Rationale: Little is known about the roles of proteoglycans in esophageal cancer. This study aims to investigate the roles and mechanisms of serglycin (SRGN) proteoglycan in promoting metastasis of esophageal squamous cell carcinoma (ESCC).Methods: Reverse phase protein array analysis was used to identify activated signaling pathways in SRGN-overexpressing cells. Chemokine array was used to identify differentially secreted factors from SRGN-overexpressing cells. Binding between SRGN and potential interacting partners was evaluated using proximity ligation assay and co-immunoprecipitation. The glycosaminoglycan (GAG) chains of SRGN were characterized using fluorophore-assisted carbohydrate electrophoresis. Tissue microarray and serum samples were used to determine the correlation of SRGN expression with clinicopathological parameters and patient survival.Results: In vitro and in vivo experiments showed that SRGN promoted invasion and metastasis in ESCC via activating ERK pathway, stabilizing c-Myc and upregulating the secretion of matrix metalloproteinases. SRGN-knockdown suppressed tumorigenic hallmarks. These SRGN-elicited functions were carried out in an autocrine manner by inducing the secretion of midkine (MDK), which was further identified as a novel binding partner of SRGN for the formation of a SRGN/MDK/CD44 complex. In addition, SRGN interacted with MDK and matrix metalloproteinase 2 in ESCC via its GAG chains, which were mainly decorated with chondroitin sulfate comprising of ∆di-4S and ∆di-6S CS. Clinically, high expression of serum SRGN in serum of patients with ESCC was an independent prognostic marker for poor survival.Conclusions: This study provides the first evidence that elevated serum SRGN has prognostic significance in patients with ESCC, and sheds light on the molecular mechanism by which elevated circulating SRGN in cancer patients might promote cancer progression.

The relationship between plasma serglycin levels and the diagnosis of diabetic retinopathy.

BackgroundDiabetic retinopathy (DR), a microvascular complication which is closely related to diabetes, remains the leading cause of vision loss around the world among older adults. Serglycin (SRGN) was known as a hematopoietic cell granule proteoglycan, exerting its function in the formation of mast cell secretory granules and mediates the storage of various compounds in secretory vesicles. The present study illustrates the potential clinical value and experimental mechanisms of SRGN in the DR.MethodsFirstly, the mRNA expression and protein expression of SRGN in plasma samples from NPDR, PDR patients, type 2 diabetes mellitus (T2Dm) cases, and healthy controls were measured by qPCR and Western blotting assays, respectively. Then, the potentials of SRGN functioning as a diagnostic indicator in DR were verified by the receiver operating characteristic (ROC) analysis. We established in vitro DR model of human retinal endothelial cells through high‐glucose treatment. The expression of SRGN and its mechanisms of regulating cellular processes were illustrated subsequently.ResultsOur data revealed that SRGN was dramatically upregulated in NPDR and PDR cases compared with healthy controls and T2DM patients; meanwhile, the expression of SRGN was further increased in the PDR group with regard to the NPDR group. Furthermore, the ROC analysis demonstrated that SRGN could distinguish the DR cases from type 2 diabetes mellitus (T2DM) patients and healthy controls with the area under the curve (AUC) of 0.7680 (95% CI = 0.6780 ~ 0.8576, sensitivity = 47.27%, specificity = 100%, cutoff value = 1.4727) and 0.8753 (95% CI = 0.8261 ~ 0.9244, sensitivity = 69.23%, specificity = 100%, cutoff value = 1.6923), respectively. In vitro high‐glucose treatment showed that the SRGN expressions were dramatically increased. The loss of SRGN could partially counteract the inhibition of HREC proliferation caused by high‐glucose stimulation. Meanwhile, SRGN knockdown could reverse the promotion of HREC apoptosis induced by high glucose as well.ConclusionsConsequently, our study implied that SRGN might serve as a promising biomarker with high specificity and sensitivity in the DR diagnosis and progression.