C-X-C Motif ) Ligand Research Articles

PU.1 alleviates the inhibitory effects of cigarette smoke on endothelial progenitor cell function and lung-homing through Wnt/β-catenin and CXCL12/CXCR4 pathways.

Endothelial progenitor cells (EPCs) dysfunction is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The transcription factor PU.1 is essential for the maintenance of stem/progenitor cell homeostasis. However, the role of PU.1 in COPD and its effects on EPC function and lung-homing, remain unclear. This study aimed to explore the protective activity of PU.1 and the underlying mechanisms in a cigarette smoke extract (CSE)-induced emphysema mouse model. C57BL/6 mice were treated with CSE to establish a murine emphysema model and injected with overexpressed PU.1 or negative control adeno-associated virus. Morphometry of lung slides, lung function, and apoptosis of lung tissues were evaluated. Immunofluorescence co-localization was used to analyze EPCs homing into the lung. Flow cytometry was performed to detect EPC count in lung tissues and bone marrow (BM). The angiogenic ability of BM-derived EPCs cultured in vitro was examined by tube formation assay. We determined the expression levels of PU.1, β-catenin, C-X-C motif ligand 12 (CXCL12), C-X-C motif receptor 4 (CXCR4), stem cell antigen-1 (Sca-1), and stemness genes. CSE exposure significantly reduced the expression of PU.1 in mouse lung tissues, BM, and BM-derived EPCs. PU.1 overexpression attenuated CSE-induced emphysematous changes, lung function decline, and apoptosis. In emphysematous mice, PU.1 overexpression markedly reversed the decreased proportion of EPCs in BM and promoted the lung-homing of EPCs. The impaired angiogenic ability of BM-derived EPCs induced by CSE could be restored by the overexpression of PU.1. In addition, PU.1 upregulation evidently reversed the decreased expression of β-catenin, CXCL12, CXCR4, Scal-1, and stemness genes in mouse lung tissues, BM, and BM-derived EPCs after CSE exposure. PU.1 alleviates the inhibitory effects of CSE on EPC function and lung-homing via activating the canonical Wnt/β-catenin pathway and CXCL12/CXCR4 axis. While further research is needed, our research may indicate a potential therapeutic target for COPD patients.

Associations Between Metabolic Syndrome, Ulcerative Colitis, and Fecal sST2 and CXCL8 Levels: Unveiling New Inflammatory Pathways

Abstract Ulcerative Colitis (UC), a chronic inflammatory bowel disease, exhibits complex interactions with metabolic disorders such as Metabolic Syndrome (MetS), which can significantly impact disease progression and patient outcomes. Among the multitude of players in this intricate network, soluble ST2 (sST2) and Chemokine (C-X-C motif) ligand 8 (CXCL8) have emerged as critical mediators of immune responses, potentially modulating the disease course in UC patients with co-existing MetS. This study aimed to investigate the association between Metabolic Syndrome (MetS) and immune response modulation in patients with Ulcerative Colitis (UC). UC patients, stratified by the presence of MetS, underwent clinical, endoscopic, and histological evaluation, along with blood and fecal biochemical analyses. Serum and fecal concentrations of sST2 and CXCL8 were measured and compared between groups. UC patients with MetS exhibited lower white blood cell (WBC) count, higher levels of metabolic markers, and milder disease severity on clinical, endoscopic, and histological scales. Serum concentrations of sST2 and CXCL8 were similar between UC patients with and without MetS. However, fecal levels of these cytokines were significantly elevated in UC patients with MetS, suggesting a localized intensified immune response. Our findings indicate a potential dichotomy in the immune response of UC patients with MetS, characterized by a dampened systemic inflammation and heightened local immune response. The elevated fecal levels of sST2 and CXCL8 underscore a potentially unique immune modulation within the gut in the presence of MetS. These findings shed new light on the pathophysiological interplay between MetS and UC and may provide new avenues for targeted therapeutic strategies.

Association of postoperative delirium with serum and cerebrospinal fluid proteomic profiles: a prospective cohort study in older hip fracture patients.

Postoperative delirium (POD) is a common neuropsychiatric complication in geriatric inpatients after hip fracture surgery and its occurrence is associated with poor outcomes. The purpose of this study was to investigate the relationship between preoperative biomarkers in serum and cerebrospinal fluid (CSF) and the development of POD in older hip fracture patients, exploring the possibility of integrating objective methods into future predictive models of delirium. Sixty hip fracture patients were recruited. Blood and CSF samples were collected at the time of spinal anesthesia when none of the subjects had delirium. Patients were assessed daily using the 4AT scale, and based on these results, they were divided into POD and non-POD groups. The Olink® platform was used to analyze 45 cytokines. Twenty-one patients (35%) developed POD. In the subsample of 30 patients on whom proteomic analyses were performed, a proteomic profile was associated with the incidence of POD. Chemokine (C-X-C motif) ligand 9 (CXCL9) had the strongest correlation between serum and CSF samples in patients with POD (rho = 0.663; p < 0.05). Although several cytokines in serum and CSF were associated with POD after hip fracture surgery in older adults, there was a significant association with lower preoperative levels of CXCL9 in CSF and serum. Despite the small sample size, this study provides preliminary evidence of the potential role of molecular biomarkers in POD, which may provide a basis for the development of new delirium predictive models.

Abstract C014: CXCR4 inhibition enhances anti-tumor immune response in an ex vivo autologous patient-derived organoids and PBMC model system of pancreatic ductal adenocarcinoma

Abstract Introduction Immune checkpoint blockade either alone or in combination with chemotherapy has been ineffective in pancreatic ductal adenocarcinoma (PDAC) likely due to underlying immunosuppressive pathways. The CXC chemokine receptor type 4 (CXCR4)/chemokine (CXC motif) ligand 12 (CXCL12) axis is one key immunosuppressive pathway. CXCL12 (released by cancer-associated fibroblasts (CAF)) binds to CXCR4 on CD8+ T cells (CTL) and decreases its infiltration into the tumor. Encouraging preclinical studies from murine PDAC models and human PDAC tumor sections have demonstrated infiltration of T cells with tumors after co-inhibition of CXCR4 and PD1/PDL1, however with limited tumor regressions in mouse model. We established an innovative ex vivo autologous patient-derived immune/organoid (PDIO) co-culture system that allows studying the interactions between distinct immune cells and tumor cells. Methods Patient-derived tumor organoids (PDTOs) generated from tumors resected from patients diagnosed with pancreas cancer were grown to confluence in Matrigel domes. We conducted ex vivo migration assays with autologous peripheral blood mononuclear cells (PBMCs) which were placed in the surrounding liquid phase of the matrigels. We separately treated PBMCs and PDTOs with AMD3100, an inhibitor of CXCR4, and tested the role of CXCR4 inhibition on PBMCs and PDTOs in T cell exclusion and immune cell activation. Results Pre-treatment of PBMCs with AMD3100 increased CD8+ T cell mobilization towards autologous PDTOs in matrigel dome migration assays by up to 11-fold. After co-culture in the liquid interphase of PDTOs matrigel domes, AMD3100 pre-treated PBMCs exhibit a more robust proliferative response compared to untreated PBMCs and a significant increase in the number of reactive T cells migrated inside the PDTO matrigel dome and expressing activation markers (p=0.02). Nearly 50% of T cells in proximity of tumor cells produce the cytotoxic factor Granzyme B (p=0.002), with AMD3100 treatment of PBMC increasing it by up to 3-fold compared to untreated PBMC. CXCR4 inhibition of PDTOs resulted in modulation of immune checkpoint ligands involved in the tumor immune response and tumor cell immunogenicity. Combination treatment of PBMCs and PDTOs with AMD3100 and anti-PD1 antibody ultimately resulted in increased tumor cell death with up to 45% of PDTOs exhibiting apoptosis markers after coculture with AMD3100-treated PBMC, implicating a role of this combination in the clinic. Conclusions We demonstrate that inhibition of CXCR4, not only increases migration of autologous T cells towards PDTOs, but also results in proliferation of PBMCs and activation of T cells. Furthermore, CXCR4 inhibition of PDTOs increases antigen presentation and tumor cell death. Inhibition of CXCR4 is an attractive therapeutic target as it enhances anti-tumor immune response via dual targeting of immune and neoplastic compartments. We are testing this combination in a multicenter investigator-initiated clinical trial in treatment-naïve metastatic PDAC patients. Citation Format: Ilenia Pellicciotta. CXCR4 inhibition enhances anti-tumor immune response in an ex vivo autologous patient-derived organoids and PBMC model system of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C014.

BRCA1 mutation promotes sprouting angiogenesis in inflammatory cancer-associated fibroblast of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with inferior outcomes owing to its low treatment response and high invasiveness. Based on abundant cancer-associated fibroblasts (CAFs) and frequent mutation of breast cancer-associated 1 (BRCA1) in TNBC, the characteristics of CAFs in TNBC patients with BRCA1 mutation compared to wild-type were investigated using single-cell analysis. Intriguingly, we observed that characteristics of inflammatory CAFs (iCAFs) were enriched in patients with BRCA1 mutation compared to the wild-type. iCAFs in patients with BRCA1 mutation exhibited outgoing signals to endothelial cells (ECs) clusters, including chemokine (C-X-C motif) ligand (CXCL) and vascular endothelial growth factor (VEGF). During CXCL signaling, the atypical chemokine receptor 1 (ACKR1) mainly interacts with CXCL family members in tumor endothelial cells (TECs). ACKR1-high TECs also showed high expression levels of angiogenesis-related genes, such as ANGPT2, MMP1, and SELE, which might lead to EC migration. Furthermore, iCAFs showed VEGF signals for FLT1 and KDR in TECs, which showed high co-expression with tip cell marker genes, including ZEB1 and MAFF, involved in sprouting angiogenesis. Moreover, BRCA1 mutation patients with relatively abundant iCAFs and tip cell gene expression exhibited a limited response to neoadjuvant chemotherapy, including cisplatin and bevacizumab. Importantly, our study observed the intricate link between iCAFs-mediated angiogenesis and chemoresistance in TNBC with BRCA1 mutation.

CXCL1 promotes cell migration in hepatocellular carcinoma by regulating the miR-30b-5p/ICAM-1 axis.

Hepatocellular carcinoma (HCC) is a highly lethal cancer with a growing global incidence and is often associated with poor prognosis due to its tendency to metastasize. Intercellular adhesion molecule (ICAM) 1 is a transmembrane protein found in various cancer cells and is associated with the spread of cancer and poor prognosis. Chemokine (C-X-C motif) ligand 1 (CXCL1) is a chemokine that significantly affects the cell motility of various cancers. However, the role of CXCL1 in ICAM-1 expression and in metastasis of hepatocellular carcinoma remains unclear. We determined that CXCL1 expression is positively and significantly associated with advanced-stage tumors in the HCC tissue array. Kaplan-Meier analysis revealed worse overall survival rates in the high CXCL1 expression group, suggesting its potential as a biomarker for cancer progression and stimulating hepatocellular carcinoma cells with CXCL1 enhanced migration abilities by upregulating ICAM-1 expression. CXCL1 was shown to enhance ICAM-1-dependent cell motility by inhibiting miR-30b-5p. This study provides novel evidence that CXCL1 could serve as a therapeutic target for metastasis in hepatocellular carcinoma.

Hepatocyte-specific Smad4 deficiency inhibits hepatocarcinogenesis by promoting CXCL10/CXCR3-dependent CD8+- T cell-mediated anti-tumor immunity.

Rationale: Sma mothers against decapentaplegic homologue 4 (Smad4) is a key mediator of the transforming growth factor β (TGF-β) pathway and plays complex and contradictory roles in hepatocellular carcinoma (HCC). However, the specific role of Smad4 in hepatocytes in regulating hepatocarcinogenesis remains poorly elucidated. Methods: A diethylnitrosamine/carbon tetrachloride-induced HCC model was established in mice with hepatocyte-specific Smad4 deletion (AlbSmad4-/-) and liver tumorigenesis was monitored. Immune cell infiltration was examined by immunofluorescence and fluorescence activated cell sorting (FACS). Cytokine secretion, glycolysis, signal pathway, and single-cell RNA sequencing were analysed for mechanism. Results: AlbSmad4-/- mice exhibited significantly fewer and smaller liver tumor nodules, less fibrosis, reduced myeloid-derived suppressor cell infiltration and increased CD8+ T cell infiltration. Smad4 deletion in hepatocytes enhanced C-X-C motif ligand 10 (CXCL10) secretion, promoting tumor necrosis factor-α (TNF-α) production in CD8+ T cells. The loss of Smad4 activated the CXCL10/mammalian target of rapamycin (mTOR)/lactate dehydrogenase A (LDHA) pathway, which increased glycolytic activity in CD8+ T cells. HCC patients with high Smad4 expression exhibited decreased CD8+ T cell infiltration and altered glycolysis. Conclusion: Our results demonstrate that Smad4 in hepatocytes promotes hepatocarcinogenesis and is a potential and candidate target for the prevention and therapy of HCC.

Hsa_circ_0136666 mediates the antitumor effect of curcumin in colorectal carcinoma by regulating CXCL1 via miR-1301-3p.

This study investigate the anti-tumor effect of curcumin and whether its mediated by hsa_circ_0136666 through miR-1301-3p/CXCL1 in colorectal carcinoma (CRC). Through multiple experiments, we have drawn the conclusion that curcumin inhibited CRC development through the hsa_circ_0136666/miR-1301-3p/CXCL1 axis, hinting at a novel treatment option for curcumin to prevent CRC development. To determine whether hsa_circ_0136666 involvement in curcumin-triggered CRC progression was mediated by sponging miR-1301-3p. Cell counting kit-8, colony-forming cell, 5-ethynyl-2'-deoxyuridine, and flow cytometry assays were carried out to determine cell proliferation, apoptosis, and cell cycle progression. Real-time quantitative polymerase chain reaction quantified hsa_circ_0136666, miR-1301-3p, and chemokine (C-X-C motif) ligand 1 (CXCL1), and western blot analysis determined CXCL1, B-cell lymphoma-2 (Bcl-2), and Bcl-2 related X protein (Bax) protein levels. CircBank or starbase software was first used for the prediction of miR-1301-3p binding with hsa_circ_0136666 and CXCL1, followed by RNA pull-down, RNA immunoprecipitation, and dual-luciferase reporter assay validation. In vivo experiments were implemented in a murine xenograft model. Curcumin blocked CRC cell proliferation but boosted apoptosis. Moreover, elevated hsa_circ_0136666 Levels were observed in CRC cells, which were reduced by curcumin. In vitro, hsa_circ_0136666 overexpression abolished the antitumor activity of CRC cells. Mechanical analysis revealed the ability of hsa_circ_0136666 to sponge miR-1301-3p to modulate CXCL1 levels. Curcumin inhibited CRC development through the hsa_circ_0136666/miR-1301-3p/CXCL1 axis, hinting at a novel treatment option for curcumin to prevent CRC development.

New Cardiovascular Biomarkers in Breast Cancer Patients Undergoing Doxorubicin-Based Chemotherapy.

Central Illustration : New Cardiovascular Biomarkers in Breast Cancer Patients Undergoing Doxorubicin-Based Chemotherapy. Cardiovascular diseases (CVDs) are relevant to the management of breast cancer treatment since a substantial number of patients develop these complications after chemotherapy. This study aims to evaluate new cardiovascular biomarkers, namely CXCL-16 (C-X-C motif ligand 16), FABP3 (fatty acid binding protein 3), FABP4 (fatty acid binding protein 4), LIGHT (tumor necrosis factor superfamily member 14/TNFS14), GDF-15 (Growth/differentiation factor 15), sCD4 (soluble form of CD14), and ucMGP (uncarboxylated Matrix Gla-Protein) in breast cancer patients treated with doxorubicin (DOXO). This case-control study was conducted in an oncology clinic that included 34 women diagnosed with breast cancer and chemotherapy with DOXO and 34 control women without cancer and CVD. The markers were determined immediately after the last cycle of chemotherapy. The statistical significance level adopted was 5%. The breast cancer group presented higher levels of GDF-15 (p<0.001), while control subjects had higher levels of FABP3 (p=0.038), FABP4 (p=0003), sCD14, and ucMGP (p<0.001 for both). Positive correlations were observed between FABPs and BMI in the cancer group. GDF15 is an emerging biomarker with potential clinical applicability in this scenario. FABPs are proteins related to adiposity, which are potentially involved in breast cancer biology. sCD14 and ucMGP engage in inflammatory and vascular calcification. The evaluation of these novel cardiovascular biomarkers could be useful in the management of breast cancer chemotherapy with DOXO.

Blockade of C5a receptor unleashes tumor-associated macrophage antitumor response and enhances CXCL9-dependent CD8+ T cell activity

Macrophages play a crucial role in shaping the immune state within the tumor microenvironment (TME) and are often influenced by tumors to hinder antitumor immunity. However, the underlying mechanisms are still elusive. Here, we observed abnormal expression of complement 5a receptor (C5aR) in human ovarian cancer (OC), and identified high levels of C5aR expression on tumor-associated macrophages (TAMs), which led to the polarization of TAMs toward an immunosuppressive phenotype. C5aR knockout or inhibitor treatment restored TAM antitumor response and attenuated tumor progression. Mechanistically, C5aR deficiency reprogrammed macrophages from a protumor state to an antitumor state, associating with the upregulation of immune response and stimulation pathways, which in turn resulted in the enhanced antitumor response of cytotoxic Tcells in a manner dependent on chemokine (C-X-C motif) ligand 9 (CXCL9). The pharmacological inhibitionof C5aR also improved the efficacy of immune checkpoint blockade therapy. In patients, C5aR expression associated with CXCL9 production and infiltration of CD8+ Tcells, and a high C5aR level predicted poor clinical outcomes and worse benefits from anti-PD-1 therapy. Thus, our study sheds light on the mechanisms underlying the modulation of TAM antitumor immune response by the C5a-C5aR axis andhighlights the potential of targeting C5aR for clinical applications.

Mechanism of Wendan decoction in preventing obesity by regulating multiple signal pathway networks based on gene promoter methylation

ObjectiveTo investigate the potential mechanism of Wendan decoction in obesity by screening target genes with promoter region methylation changes and constructing a multiple signaling pathways network based on promoter methylation. MethodsThe methylation degree of Itgad, Col8a1, Adra2b, Jund, Rab2a, Wnt8b, Fzd9, B4galt7, Pik3cd, Creb1, Stard8, and Mmp1 in the abdominal adipose tissue of obese rats was determined using the Agena MassARRAY system. Western blot was performed to assess protein expression levels. Target genes were identified based on the methylation degree in the promoter region and protein expression. Enrichment analysis of signaling pathways was conducted to identify relevant target genes and obtain a multiple signaling pathway network associated with obesity. Core and terminal effector molecules in the pathway networks were selected as research targets for reverse transcription-polymerase chain reaction (RT-PCR) analysis. ResultsFour genes (Adra2b, Creb1, Itgad, and Pik3cd) showed a degree of promoter methylation consistent with their respective protein expression levels. Among them, Adra2b, Creb1, and Pik3cd expression increased, while that of Itgad decreased. Enrichment analysis revealed that Creb1 and Pik3cd were involved in 6 signaling pathways related to obesity: tumor necrosis factor (TNF) signaling pathway, growth hormone synthesis/secretion and action, adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling pathway, relaxin signaling pathway, cyclic nucleotide (cAMP) signaling pathway, and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Subsequently, a multiple signaling pathways network was constructed based on promoter methylation. Key molecules including protein kinase B (AKT), mechanistic target of rapamycin complex 1 (mTORC1), and unc-51 like autophagy activating kinase 1 (ULK1), as well as terminal effector molecules interleukin-1β (IL-1β), interleukin-6 (IL-6), and chemokine (C-X-C motif) ligand 2 (CXCL2) were selected as research targets. Wendan decoction decreased the expressions of AKT, mTORC1, IL-1β, IL-6, and CXCL2 while up-regulating ULK1 expression. ConclusionThe mechanism of Wendan decoction in preventing obesity involves the regulation of multiple signaling pathways through the control of Creb1 and Pik3cd gene promoter methylation. However, the associated multi-path gene regulation mechanism in preventing obesity is complex. Thus, further exploration is needed to elucidate the role of methylation changes in this mechanism.

Identification of FOXP3+ epithelial cells contributing to pancreatic proliferation and angiogenesis.

Forkhead box protein 3 (FOXP3), traditionally recognized as a specific transcription factor for regulatory T cells (Tregs), has also been identified in various tumor epithelial cells (named as cancer-FOXP3, c-FOXP3). However, the natural state and functional role of FOXP3 positive tumor epithelial cells remain unknown. Monoclonal cells expressing varying levels of c-FOXP3 were isolated from established PANC-1 cells using limited dilution. Whole transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) were conducted on these subsets, followed by in vitro and in vivo functional investigations. In addition, we identified c-FOXP3+E-cadherin- epithelial cells in human pancreatic cancer tissues after radical resection by immunofluorescence co-staining. We also investigated the connection between c-FOXP3+E-cadherin- epithelial cells and their clinicopathological features. Our study uncovered a distinct subset of c-FOXP3+ tumor epithelial cells characterized by reduced E-cadherin expression. C-FOXP3+E-cadherin- cells displayed significant proliferation potential and pro-angiogenic effect through the expression of chemokines, including C-X-C motif ligand 1 (CXCL1), C-X-C motif ligand 5 (CXCL5), and C-X-C motif ligand 8 (CXCL8). Notably, higher counts of c-FOXP3+E-Cadherin- cells correlated with poorer prognosis, lower tumor differentiation, lymph node metastasis, and vascular invasion in pancreatic ductal adenocarcinoma (PDAC). In conclusion, this work revealed the stable expression of FOXP3 in tumor epithelial cells, marking a distinct subset. C-FOXP3+E-cadherin- epithelial cells exhibit active proliferation and promote angiogenesis in a vascular endothelial growth factor A (VEGFA) independent manner. These findings provide novel insights into PDAC prognosis and therapeutic avenues.NEW & NOTEWORTHY In this study, we revealed a novel c-FOXP3+ tumor epithelial cell subset marked by diminished E-cadherin and stable FOXP3 expression. These subpopulations not only show robust proliferation and drive angiogenesis via CXCL1, CXCL5, and CXCL8, bypassing VEGFA pathways, but their heightened presence also correlates with adverse PDAC outcomes. By challenging traditional epithelial cell definitions and extending lymphocyte markers to these cells, our findings present innovative targets for PDAC treatment and enrich our understanding of cell biology.

Pathophysiology and Treatment Opportunities of CX3CL1 in Liver Acute Gvhd

Acute graft-versus-host disease (aGVHD) is one of the most familiar and severe complications of hematopoietic stem cell transplantation (HSCT). Liver aGVHD, which has limited response to treatment and high mortality, is a critical concern among clinicians in the field. Chemokine (C-X3-C motif) ligand 1 (CX3CL1), also called fractalkine, as a chemokine and adhesion molecule, plays an important role in the pathogenesis of rheumatoid arthritis, primary biliary cirrhosis, and multiple sclerosis. Whether CX3CL1 is involved in liver aGVHD has not been investigated. We collected peripheral blood samples from 10 patients with liver aGVHD during the onset phase and 15 patients without aGVHD after HSCT. The results of the enzyme-linked immunosorbent assay (ELISA) indicated a significant increase in serum CX3CL1 levels among patients with liver aGVHD. Similarly, we observed elevated levels of CX3CL1 in the serum of a mouse model with aGVHD, and found a positive correlation between elevated bilirubin levels and higher serum CX3CL1 levels. The expression of CX3CL1 was markedly elevated in the liver of aGVHD mice by real-time quantitative polymerase chain reaction (RT-qPCR), western blotting (WB). The results of immunohistochemistry showed that the expression of CX3CL1 in the bile duct epitheliums of aGVHD mice was significantly increased compared with those of control mice, but not in hepatocytes. To further analyze CX3CL1 expression, we isolated hepatocytes and bile duct epitheliums from mice and measured the levels using RT-qPCR and WB. Similar to the results of immunohistochemistry, the expression level of CX3CL1 in bile duct epitheliums from aGVHD mice was increased significantly, while the expression level in hepatocytes was unchanged. The prominent bile duct damage observed in liver aGVHD may be attributed to the high CX3CL1 expression in bile duct epitheliums instead of hepatocytes. Anti-CX3CL1 monoclonal antibody (mAb) significantly improved the clinical symptoms and survival rates of liver aGVHD mice, and the liver enzymes and bilirubin levels were decreased remarkably after treatment. Additionally, HE staining showed that the application of anti-CX3CL1 mAb obviously reduced the infiltration of inflammatory cells in bile duct epithelium of liver aGVHD mice. In conclusion, these results provide evidence for the close relationship between CX3CL1 and the pathogenesis of liver aGVHD, suggesting anti-CX3CL1 mAb as a promising strategy for the management of liver aGVHD. Keywords: Hematopoietic stem cell transplantation, Liver aGVHD, CX3CL1, Bile duct

Cell-Specific Regulation of Inflammatory Cytokines and Acute-Phase Proteins by the Glucocorticoid Receptor

Background. Literature and data mining found abnormal induction of chemokine (C-X-C motif) ligand 1 (CXCL1) and CXCL8 and down-regulation of CXCL2 in inflammatory liver diseases. This study was performed to understand the glucocorticoid receptor’s (GR’s) effects on chemokine and acute-phase protein expression in human liver, in settings of bacterial infection (modeled using LPS) or inflammation (modeled using TNFα). Methods. Primary human hepatocytes (PHH) were treated with combinations of tumor necrosis factor alpha (TNFα), lipopolysaccharide (LPS), and dexamethasone (DEX) for 24 h, following which chemokine mRNA and protein expression were analyzed using qPCR and enzyme-linked immunosorbent assay assays. Dual luciferase assays were performed on transfected cell lines. Mutant CXCL2 promoters were used in dual luciferase assays to identify specific regions of the CXCL2 promoter affected by GR, TNFα, or hepatocyte nuclear factor 4α (HNF4α, a liver-enriched transcription factor). Results. In PHH from donor 1, GR strongly inhibited LPS-induced CXCL1 and CXCL8 translation and transcription, whereas CXCL2 transcription tended to increase with DEX treatment. In PHH from donor 2, DEX treatment inhibited protein expression and secretion of CXCL1 and CXCL8 induced by TNFα and/or LPS, whereas CXCL2 upregulation was largely unaffected by DEX treatment. In nonliver HEK293T cells GR activity inhibited CXCL2 promoter activity. However, in liver-derived HEPG2 cells, GR induced CXCL2 promoter activity. A 407-base pair region upstream of CXCL2 promoter is necessary for full GR functionality in HEPG2 cells. TNFα synergized with HNF4α in inducing CXCL2 promoter activity in HEPG2 cells. Conclusions. GR’s effects on chemokine expression are cell-type specific and chemokine specific. GR down-regulated CXCL1 and CXCL8 in different cell types, whereas the specific activation of CXCL2 in hepatocytes and down-regulation of CXCL2 in nonhepatocytes by GR appears due to cell-specific utilization of CXCL2 promoter. By specifically increasing GR activity in the liver, we may normalize chemokine imbalances and prevent sepsis in inflammatory liver diseases.

Aitongping patch could alleviate cancer pain via suppressing microglia activation and modulating the miR-150-5p/CXCL12 signaling.

We aimed to investigate the pharmacological effects and mechanisms of the Aitongping formula for treating cancer pain. We enrolled 60 cancer patients with Numeric Rating Scale above 4 and grouped them randomly as a Control group (N= 30) and a Patch group (N= 30). We also established bone cancer mice models via tumor implantation. And the animal groups were established as a Sham group, a tumor cell implantation (TCI) group, a TCI + Patch group, and a Patch group. After the validation of successful tumor implantation, we identified candidate miRNAs and genes that were dysregulated in TCI mice and compared their expressions between different mice groups. We also observed the effect of Aitongping patch in vitro in mice primary microglia. The time to disease progression and cancer stability were prolonged by Aitongping patch in cancer patients. And the daily morphine dose was lower, and patients' quality of life was improved in the Patch group. Moreover, Aitongping patch alleviated cancer pain and inhibited microglia activation after the successful implantation of bone tumor in TCI mice. We also observed the dysregulation of miR-150-5p and chemokine CXC motif ligand 12 (CXCL12) mRNA in TCI mice. And CXCL12 was found to be targeted by miR-150-5p. Aitongping patch was found to upregulate miR-150-5p and downregulate CXCL12 in vivo and in vitro. Aitongping patch could alleviate cancer pain via suppressing microglia activation, and the downregulation of miR-150-5p, as well as the upregulation of CXCL12 mRNA and protein, induced by tumor implantation or lipopolysaccharide stimulation, was restored by Aitongping treatment.

EXTH-81. RE-EXPRESSION OF CXCL9 FOLLOWING ADENO-ASSOCIATED VIRUS (AAV) TRANSGENE DELIVERY TRANSFORMS THE IMMUNOLOGICAL LANDSCAPE OF GLIOBLASTOMA, SENSITIZING TUMORS TO PD-1 IMMUNE CHECKPOINT BLOCKADE (ICB)

Abstract BACKGROUND To date, few glioblastoma (GBM) patients have demonstrated clinical benefit from treatment with anti-PD-1 ICB. GBM is notoriously devoid of lymphocytes, signifying that a possible obstacle for effective ICB response may be the limited ability of cytotoxic T-lymphocytes (CTLs) to home to these tumors. CTLs are recruited via long-range signaling mediated by the diffusion of chemokines present in inflammatory environments. Following a comprehensive chemokine screen of clinical tumor specimens, we identified that CXC motif ligand 9 (CXCL9) is notably absent in GBM. To test if GBM reconstitution with CXCL9 could transform these intractable tumors into ICB-responsive, we leveraged AAV gene therapy to generate durable production of this potent lymphotactic signal. HYPOTHESIS AAV mediated re-expression of CXCL9 will improve lymphotaxis in GBM, sensitizing these tumors to PD-1 ICB. METHODS 3-dimensional whole brain fluorescent imaging was performed to characterize the distribution, tropism, and durability of AAV transgene expression in preclinical GBM models. Single-cell RNA sequencing (scRNAseq) and multiparameter flow cytometry was utilized to immunologically profile tumors in response to single and combination treatment. Survival studies with and without lymphocyte depletion were performed to evaluate immunogenic responsiveness of therapy. RESULTS Intra-tumor delivery of AAV6 yields stable transduction of tumor-reactive astrocytes, producing a localized, peri-tumoral pattern of transgene signal distribution. Combination therapy increases tumor infiltration by lymphocytes. scRNAseq analyses reveal widespread innate and adaptive immune activation in response to each CXCL9 and PD-1 ICB treatment. Infiltrating CD8 T-lymphocytes demonstrate inflammatory signatures associated with increased effector function, including antigen presentation, cytotoxicity, and TCR signaling. Combination treatment considerably improved overall survival in two distinct GBM models, with durable responses dependent on CD8 T-lymphocyte infiltration. CONCLUSIONS This work suggests direct conditioning of the tumor microenvironment by AAV-CXCL9 could potentially overcome a key resistance factor in GBM, sensitizing tumors to immune mediated therapies such as PD-1 ICB.

Unraveling temporal and spatial biomarkers of epithelial-mesenchymal transition in colorectal cancer: insights into the crucial role of immunosuppressive cells

The occurrence and progression of tumors can be established through a complex interplay among tumor cells undergoing epithelial-mesenchymal transition (EMT), invasive factors and immune cells. In this study, we employed single-cell RNA sequencing (scRNA-seq) and spatially resolved transcriptomics (ST) to evaluate the pseudotime trajectory and spatial interactive relationship between EMT-invasive malignant tumors and immune cells in primary colorectal cancer (CRC) tissues at different stages (stage I/II and stage III with tumor deposit). Our research characterized the spatiotemporal relationship among different invasive tumor programs by constructing pseudotime endpoint-EMT-invasion tumor programs (EMTPs) located at the edge of ST, utilizing evolution trajectory analysis integrated with EMT-invasion genes. Strikingly, the invasive and expansive process of tumors undergoes remarkable spatial reprogramming of regulatory and immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), regulatory T cells (Treg), and exhausted T cells (Tex). These EMTP-adjacent cell are linked to EMT-related invasion genes, especially the C-X-C motif ligand 1 (CXCL1) and CXCL8 genes that are important for CRC prognosis. Interestingly, the EMTPs in stage I mainly produce an inflammatory margin invasive niche, while the EMTPs in stage III tissues likely produce a hypoxic pre-invasive niche. Our data demonstrate the crucial role of regulatory and immunosuppressive cells in tumor formation and progression of CRC. This study provides a framework to delineate the spatiotemporal invasive niche in CRC samples.Graphical

Hypothalamic FTO promotes high-fat diet-induced leptin resistance in mice through increasing CX3CL1 expression

Long-term consumption of a high-fat diet (HFD) disrupts energy homeostasis and leads to weight gain. The fat mass and obesity-associated (FTO) gene has been consistently identified to be associated with HFD-induced obesity. The hypothalamus is crucial for regulating energy balance, and HFD-induced hypothalamic leptin resistance contributes to obesity. FTO, an N6-methyladenosine (m6A) RNA methylation regulator, may be a key mediator of leptin resistance. However, the exact mechanisms remain unclear. Therefore, the present study aims to investigate the association between FTO and leptin resistance. After HFD or standard diet (SD) feeding in male mice for 22 weeks, m6A-sequencing and western blotting assays were used to identify target genes and assess protein level, and molecular interaction changes. CRISPR/Cas9 gene knockout system was employed to investigate the potential function of FTO in leptin resistance and obesity. Our data showed that chemokine (C-X3-C motif) ligand 1 (CX3CL1) was a direct downstream target of FTO-mediated m6A modification. Furthermore, upregulation of FTO/CX3CL1 and suppressor of cytokine signaling 3 (SOCS3) in the hypothalamus impaired leptin-signal transducer and activator of transcription 3 signaling, resulting in leptin resistance and obesity. Compared to wild-type (WT) mice, FTO deficiency in leptin receptor-expressing neurons of the hypothalamus significantly inhibited the upregulation of CX3CL1 and SOCS3, and partially ameliorating leptin resistance under HFD conditions. Our findings reveal that FTO involved in the hypothalamic leptin resistance and provides novel insight into the function of FTO in the contribution to hypothalamic leptin resistance and obesity.

FRI530 Immune-Modulation By Rapamycin, Or Mycophenolic Acid, Of Th1 And Th2 Chemokines Secretion In Retro-orbital Cells Of Patients With Graves’ Ophthalmopathy

Abstract Disclosure: G. Elia: None. F. Ragusa: None. S. Paparo: None. V. Mazzi: None. A. Patrizio: None. P. Fallahi: None. A. Antonelli: None. S. Ferrari: None. Objectives: Cytokines and chemokines contribute to the inflammation of Graves’ ophthalmopathy (GO). Indeed cytokines stimulate the T helper (Th)1 and Th2 chemokines release from orbital cells (fibroblasts, preadipocytes and myocytes).We aim to investigate the effects of rapamycin or mycophenolic acid on the secretion of these chemokines in GO orbital cells. Methods: We obtained primary cultures of fibroblasts, preadipocytes and myoblasts from the orbits of GO patients, and we tested increasing concentrations of rapamycin and/or mycophenolic acid evaluating the secretion of the prototype Th1 [chemokine (C-X-C motif) ligand 10 (CXCL10)] and Th2 [chemokine (C-C motif) ligand 2 (CCL2)] chemokines. Results: We observed that CXCL10 secretion in the retro-orbital cells of GO was undetectable in the supernatants, whereas interferon (IFN)-gamma induce its release in a dose-dependently manner. No effect was observed under the tumor necrosis factor (TNF)-alpha stimuli, while the combination of both cytokines had a significant synergistic effect on CXCL10 secretion. As regards the effect on the CCL2 secretion, it was released in low amounts in basal condition; TNF-alpha dose-dependently induced its release, whereas IFN-gamma alone had no effect. The combination of TNF-alpha plus IFN-gamma had a significant synergistic effect on CCL2 secretion. By adding mycophenolic acid, or rapamycin, (in a pharmacological range) at the time of IFN-gamma and TNF-alpha stimulation, we observed a dose-dependently inhibitory effect on the chemokines release in the retro-orbital cells of GO. Furthermore, the combination of mycophenolic acid with rapamycin, have a synergistic inhibitory effect on the release of chemokines. Conclusion: Our data showed an immune-modulating effect of mycophenolic acid or rapamycin on the Th1 and Th2 chemokines secretion, in GO orbital cells. These findings suggest the therapeutic role of these drugs that could obtained, at least in part, through this mechanism. Presentation: Friday, June 16, 2023

Identifying Effective Biomarkers for Accurate Pancreatic Cancer Prognosis Using Statistical Machine Learning.

Pancreatic cancer (PC) has one of the lowest survival rates among all major types of cancer. Consequently, it is one of the leading causes of mortality worldwide. Serum biomarkers historically correlate well with the early prognosis of post-surgical complications of PC. However, attempts to identify an effective biomarker panel for the successful prognosis of PC were almost non-existent in the current literature. The current study investigated the roles of various serum biomarkers including carbohydrate antigen 19-9 (CA19-9), chemokine (C-X-C motif) ligand 8 (CXCL-8), procalcitonin (PCT), and other relevant clinical data for identifying PC progression, classified into sepsis, recurrence, and other post-surgical complications, among PC patients. The most relevant biochemical and clinical markers for PC prognosis were identified using a random-forest-powered feature elimination method. Using this informative biomarker panel, the selected machine-learning (ML) classification models demonstrated highly accurate results for classifying PC patients into three complication groups on independent test data. The superiority of the combined biomarker panel (Max AUC-ROC = 100%) was further established over using CA19-9 features exclusively (Max AUC-ROC = 75%) for the task of classifying PC progression. This novel study demonstrates the effectiveness of the combined biomarker panel in successfully diagnosing PC progression and other relevant complications among Egyptian PC survivors.