CXCR4 Axis Research Articles

Research Progress on the SDF-1/CXCR4 Axis in Cartilage Injury

The chemokine CXCL12, also known as stromal cell-derived factor 1 (SDF-1), is a small pro-inflammatory chemokine that primarily binds to CXC receptor 4 (CXCR4; CD184), serving as a major regulatory factor for cell transport and adhesion. When SDF-1 binds to CXCR4, it triggers intracellular signaling through several pathways that initiate processes such as chemotaxis, cell survival, proliferation, calcium influx, and gene transcription. The SDF-1/CXCR4 axis plays a crucial role in the development of bone marrow stem cells and the process of tissue regeneration. Repairing cartilage tissue necessitates a sufficient number of chondrocytes, making bone marrow mesenchymal stem cells particularly suitable for this purpose. This signaling pathway is essential for cartilage injury repair. It promotes stem cell migration and localization, regulates cell proliferation and differentiation, and inhibits inflammatory responses. This article reviews the current research advancements related to the SDF-1/CXCR4 axis in cartilage tissue repair.

Impact of SDF-1 and AMD3100 on Hair Follicle Dynamics in a Chronic Stress Model.

Chronic stress is a common cause of hair loss, involving inflammatory responses and changes in cellular signaling pathways. This study explores the mechanism of action of the SDF-1/CXCR4 signaling axis in chronic stress-induced hair loss. The research indicates that SDF-1 promotes hair follicle growth through the PI3K/Akt and JAK/STAT signaling pathways. Transcriptome sequencing analysis was conducted to identify differentially expressed genes in the skin of normal and stressed mice, with key genes SDF-1/CXCR4 selected through machine learning and a protein-protein interaction network established. A chronic stress mouse model was created, with injections of SDF-1 and AMD3100 administered to observe hair growth, weight changes, and behavioral alterations and validate hair follicle activity. Skin SDF-1 concentrations were measured, differentially expressed genes were screened, and pathways were enriched. Activation of the PI3K/Akt and JAK/STAT signaling pathways was assessed, and siRNA technology was used in vitro to inhibit the expression of SDF-1 or CXCR4. SDF-1 promoted hair follicle activity, with the combined injection of SDF-1 and AMD3100 weakening this effect. The activation of the PI3K/Akt and JAK/STAT signaling pathways was observed in the SDF-1 injection group, confirmed by Western blot and immunofluorescence. Silencing SDF-1 through siRNA-mediated inhibition reduced cell proliferation and migration abilities. SDF-1 promotes hair growth in chronic stress mice by activating the PI3K/Akt and JAK/STAT pathways, an effect reversible by AMD3100. The SDF-1/CXCR4 axis may serve as a potential therapeutic target for stress-induced hair loss.

Overexpression of Nrf2 in bone marrow mesenchymal stem cells promotes B-cell acute lymphoblastic leukemia cells invasion and extramedullary organ infiltration through stimulation of the SDF-1/CXCR4 axis.

Tumor microenvironment (TME) represents the key factor inducing leukemia development. As stromal cells within the leukemia microenvironment, Bone Marrow Mesenchymal Stem Cells (BM-MSCs) can trigger leukemia progression under certain conditions. As a critical transcription factor, nuclear factor erythroid related factor 2 (Nrf2) can modulate antioxidant response and antioxidant enzyme gene expression, and prevent various oxidative changes. We previously identified a novel mechanism by which Nrf2 promotes leukemia resistance, providing a potential therapeutic target for the treatment of drug-resistant/refractory leukemias. However, the role of Nrf2 in BM-MSCs from B-cell acute lymphoblastic leukemia (B-ALL) patients has not been clearly reported. The present work focused on investigating the effect of Nrf2 overexpression within MSCs on leukemia cell invasion, extramedullary infiltration and proliferation as well as its downstream pathway. Through clinical sample detection, in vitro cell experiments and in vivo animal experiments, the role of Nrf2 within MSCs within adult B-ALL cell migration and invasion and its potential molecular mechanism was explored through transcriptome sequencing analysis, RT-PCR, Western blot, cell migration, cell invasion, lentivirus transfection and other experiments. Nrf2 was highly expressed in BM-MSCs from patients with B-ALL as well as in BM-MSCs co-cultured with leukemia cells. Overexpression of Nrf2 within MSCs significantly promoted leukemia cell migration, invasion and proliferation. The extramedullary organ infiltration rate in B-ALL model mice receiving the combined infusion of both cell types dramatically increased relative to that of leukemia cells alone, accompanied by the significantly shortened survival time. Mechanism study found that Nrf2 overexpression within MSCs promoted PI3K-AKT/ERK1/2 phosphorylation in the downstream pathway by activating SDF-1/CXCR4 axis, ultimately leading to extramedullary infiltration of leukemia cells. High Nrf2 expression with in MSCs enhances leukemia cell invasion and migration, which then accelerates infiltration in leukemic extramedullary organs. Targeting Nrf2 or inhibiting its downstream signal molecules may be the effective interventions for B-ALL patients treatment.

Role of the IL-8/CXCR2 axis in promoting vasculogenic mimicry in triple-negative breast cancer through epithelial-mesenchymal transition

Vasculogenic mimicry (VM) — a phenomenon where tumor cells form de novo vascular networks independent of endothelial cells — supports the growth of highly aggressive tumors, including triple-negative breast cancer (TNBC). Evidence indicates that the interleukin 8 (IL-8)/CXCR2 axis and epithelial-mesenchymal transition (EMT) independently promote VM in TNBC. This study tested the hypothesis that the IL-8/CXCR2 axis promotes VM via EMT. Tissue samples from 71 female breast cancer cases (age range: 50 – 55 years), comprising 37 TNBC and 34 non-TNBC cases, were retrieved from the Department of Pathology at the University of Ghana Medical School. Out of the 37 TNBC tissues, 10 expressed either IL-8, CXCR2, or vimentin exclusively; three expressed all three markers; and 14 did not express any of these markers. For the non-TNBC tissues, 31 expressed CXCR2 (alone or co-expression with IL-8 and/or vimentin), 27 co-expressed IL-8 with CXCR2 and/or vimentin, and two expressed all three markers. Notably, 24 of the 37 non-TNBC tissues co-expressed IL-8 and CXCR2. A phi coefficient analysis displayed an inverse relationship between CXCR2 or IL-8 expression and TNBC and a positive correlation between vimentin expression and TNBC. Inhibiting the IL-8/CXCR2 axis with the CXCR2 antagonist SB225002 suppressed VM in MDA-MB-231 breast cancer cells. In addition, inhibiting the IL-8/CXCR2 axis using SB225002 and siRNA-mediated CXCR2 knockdown suppressed EMT by upregulating E-cadherin expression and downregulating N-cadherin and vimentin expression. Our findings suggest that the IL-8/CXCR2 signaling axis promotes VM in TNBC by facilitating EMT. Therefore, therapies targeting IL-8/CXCR2 may be used to suppress EMT and VM in TNBC simultaneously.

Single-cell atlas of human gingiva unveils a NETs-related neutrophil subpopulation regulating periodontal immunity

IntroductionExaggerated neutrophil recruitment and activation are the major features of pathological alterations in periodontitis, in which neutrophil extracellular traps (NETs) are considered to be responsible for inflammatory periodontal lesions. Despite the critical role of NETs in the development and progression of periodontitis, their specific functions and mechanisms remain unclear. ObjectivesTo demonstrate the important functions and specific mechanisms of NETs involved in periodontal immunopathology. MethodsWe performed single-cell RNA sequencing on gingival tissues from both healthy individuals and patients diagnosed with periodontitis. High-dimensional weighted gene co-expression network analysis and pseudotime analysis were then applied to characterize the heterogeneity of neutrophils. Animal models of periodontitis were treated with NETs inhibitors to investigate the effects of NETs in severe periodontitis. Additionally, we established a periodontitis prediction model based on NETs-related genes using six types of machine learning methods. Cell-cell communication analysis was used to identify ligand-receptor pairs among the major cell groups within the immune microenvironment. ResultsWe constructed a single-cell atlas of the periodontal microenvironment and obtained nine major cell populations. We further identified a NETs-related subgroup (NrNeu) in neutrophils. An in vivo inhibition experiment confirmed the involvement of NETs in gingival inflammatory infiltration and alveolar bone absorption in severe periodontitis. We further screened three key NETs-related genes (PTGS2, MME and SLC2A3) and verified that they have the potential to predict periodontitis. Moreover, our findings revealed that gingival fibroblasts had the most interactions with NrNeu and that they might facilitate the production of NETs through the MIF-CD74/CXCR4 axis in periodontitis. ConclusionThis study highlights the pathogenic role of NETs in periodontal immunity and elucidates the specific regulatory relationship by which gingival fibroblasts activate NETs, which provides new insights into the clinical diagnosis and treatment of periodontitis.

Alleviation of Angiotensin II-Induced Vascular Endothelial Cell Injury Through Long Non-coding RNA TUG1 Inhibition.

Hypertension damages endothelial cells, causing vascular remodelling. It is caused by Ang II-induced endothelial cell (EC) destruction. The long noncoding RNA (lncRNAs) are emerging regulators of endothelium homeostasis. Injured endothelium expresses lncRNA taurine-upregulated gene 1 (TUG1), which may mediate endothelial cell damage, proliferation, apoptosis, and autophagy and contribute to cardiovascular disease. However, uncertainty surrounds the function of lncRNA TUG1, on arterial endothelium cell damage. This research aimed to investigate the role and mechanism of lncRNA TUG1 in vascular endothelial cell injury. A microarray analysis of lncRNA human gene expression was used to identify differentially expressed lncRNAs in human umbilical vein endothelial cell (HUVEC) cultures. The viability, apoptosis, and migration of Ang II-treated HUVECs were then evaluated. In order to investigate the role of lncRNA TUG1 in hypertension, qRT-PCR, western blotting, and RNA-FISH were used to examine the expression of TUG1 in SHR mice. Ang II-activated HUVECs and SHR rats' abdominal aortas highly express the lncRNA TUG1. LncRNA TUG1 knockdown in HUVECs could increase cell viability, reduce apoptosis, and produce inflammatory factors. In SHR rat abdominal aortas, lncRNA TUG1 knockdown promoted proliferation and inhibited apoptosis. HE spotting showed that lncRNA TUG1 knockdown improved SHR rats' abdominal aorta shape. lncRNA TUG1 knockdown promotes miR-9- 5p, which inhibits CXCR4 following transcription. The lncRNA TUG1/miR-9-5p/CXCR4 axis and vascular cell injury were also examined. MiR-9-5p silencing or CXCR4 overexpression lowered cell survival, apoptosis, and lncRNA TUG1-induced IL-6 and NO expression. lncRNA TUG1 suppression could reduce Ang II-induced endothelial cell damage by regulating and targeting miR-9-5p to limit CXCR4 expression and open new vascular disease research pathways.

Astragalus polysaccharides augment BMSC homing via SDF-1/CXCR4 modulation: a novel approach to counteract peritoneal mesenchymal transformation and fibrosis

PurposeThis study aimed to evaluate the potential of astragalus polysaccharide (APS) pretreatment in enhancing the homing and anti-peritoneal fibrosis capabilities of bone marrow mesenchymal stromal cells (BMSCs) and to elucidate the underlying mechanisms.MethodsForty male Sprague-Dawley rats were allocated into four groups: control, peritoneal dialysis fluid (PDF), PDF + BMSCs, and PDF + APSBMSCs (APS-pre-treated BMSCs). A peritoneal fibrosis model was induced using PDF. Dil-labeled BMSCs were administered intravenously. Post-transplantation, BMSC homing to the peritoneum and pathological alterations were assessed. Stromal cell-derived factor-1 (SDF-1) levels were quantified via enzyme-linked immunosorbent assay (ELISA), while CXCR4 expression in BMSCs was determined using PCR and immunofluorescence. Additionally, a co-culture system involving BMSCs and peritoneal mesothelial cells (PMCs) was established using a Transwell setup to examine the in vitro effects of APS on BMSC migration and therapeutic efficacy, with the CXCR4 inhibitor AMD3100 deployed to dissect the role of the SDF-1/CXCR4 axis and its downstream impacts.ResultsIn vivo and in vitro experiments confirmed that APS pre-treatment notably facilitated the targeted homing of BMSCs to the peritoneal tissue of PDF-treated rats, thereby amplifying their therapeutic impact. PDF exposure markedly increased SDF-1 levels in peritoneal and serum samples, which encouraged the migration of CXCR4-positive BMSCs. Inhibition of the SDF-1/CXCR4 axis through AMD3100 application diminished BMSC migration, consequently attenuating their therapeutic response to peritoneal mesenchyme-to-mesothelial transition (MMT). Furthermore, APS upregulated CXCR4 expression in BMSCs, intensified the activation of the SDF-1/CXCR4 axis’s downstream pathways, and partially reversed the AMD3100-induced effects.ConclusionAPS augments the SDF-1/CXCR4 axis’s downstream pathway activation by increasing CXCR4 expression in BMSCs. This action bolsters the targeted homing of BMSCs to the peritoneal tissue and amplifies their suppressive influence on MMT, thereby improving peritoneal fibrosis.

The effect of CXCL12 on survival outcomes of patients with viral hepatitis-associated hepatocellular carcinoma after hepatectomy

BackgroundThe CXCL12-CXCR4/CXCR7 axis is garnering growing attention. But the comprehension of its function in the progression of HCC remains controversial. The purpose of this study was to investigate the effects of CXCL12 and its receptor on the prognosis of patients with viral hepatitis-associated HCC after hepatectomy. MethodsA total of 86 patients had been enrolled who had undergone hepatectomy for HCC and followed up to July 31, 2019, and their clinicopathological and follow-up data were recorded. Tumor and peritumoral tissues were obtained to detect the expression of CXCL12, CXCR4, and CXCR7 using immunohistochemistry. Real-time polymerase chain reaction was utilized to detect hepatitis B or C virus loads, while survival analysis was performed using the Kaplan–Meier method. Furthermore, the Cox proportional hazards regression model was employed to analyze the factors affecting the prognosis. ResultsThe results revealed that the CXCL12, CXCR4, and CXCR7 expression in tumor tissues was lower than in the corresponding non-tumor tissues in 20.93 %, 22.09 %, and 23.26 % of the patients, respectively, and that only CXCL12 was found to be related to the extrahepatic invasion of HCC. The survival analysis and Cox regression showed that only CXCL12 was associated with the postoperative survival of patients with HCC, and that it was an independent prognostic risk factor in the CXCL12-CXCR4/CXCR7 axis. The CXCL12low group represented shorter progression-free survival and lower overall survival rates. However, the subgroup analysis displayed that the survival difference associated with CXCL12 was only manifested in patients with higher expression of CXCR4 or CXCR7 in HCC, as compared to the surrounding tissues. ConclusionsOur findings suggest that, when assessing the prognostic significance of CXCL12 in HCC, it is essential to consider the expression level of its receptor. Nevertheless, CXCL12 can potentially serve as a promising prognostic marker for HCC.

Recent developments in targeting breast cancer stem cells (BCSCs): a descriptive review of therapeutic strategies and emerging therapies.

Despite recent advancements in the diagnosis and treatment of breast cancer (BC), patient outcomes in terms of survival, recurrence, and disease progression remain suboptimal. A significant factor contributing to these challenges is the cellular heterogeneity within BC, particularly the presence of breast cancer stem cells (BCSCs). These cells are thought to serve as the clonogenic nexus for new tumor growth, owing to their hierarchical organization within the tumor. This descriptive review focuses on the evolving strategies to target BCSCs, which have become a pivotal aspect of therapeutic development. We explore a variety of approaches, including targeting specific tumor surface markers (CD133 and CD44), transporters, heat shock proteins, and critical signaling pathways like Notch, Akt, Hedgehog, KLF4, and Wnt/β-catenin. Additionally, we discuss the modulation of the tumor microenvironment through the CXCR-12/CXCR4 axis, manipulation of pH levels, and targeting hypoxia-inducible factors, vascular endothelial growth factor, and CXCR1/2 receptors. Further, this review focuses on the roles of microRNA expression, strategies to induce apoptosis and differentiation in BCSCs, dietary interventions, dendritic cell vaccination, oncolytic viruses, nanotechnology, immunotherapy, and gene therapy. We particularly focused on studies reporting identification of BCSCs, their unique properties and the efficacy of various therapeutic modalities in targeting these cells. By dissecting these approaches, we aim to provide insights into the complex landscape of BC treatment and the potential pathways for improving patient outcomes through targeted BCSC therapies.

Impacts of immune microenvironment on musculoskeletal health

AbstractUnderstanding the interplay between immune cells and the bone marrow microenvironment is crucial for elucidating age‐related musculoskeletal changes. In this commentary manuscript, we summarized that studies have shown that proinflammatory immune cells in the bone marrow, such as macrophages and neutrophils, can inhibit bone formation by secreting grancalcin. Research on skeletal stem cells (SSCs) in aged mice reveals a shift towards pro‐inflammatory gene expression, affecting their osteogenic potential, while another study maps age‐related changes in cranial SSC niches, emphasizing the role of the CXCL12–CXCR4 axis in stem cell‐immune cell communication, Additionally, the immune system influences hematopoietic stem cells (HSCs); Niche ageing and accumulation of mutations with age lead to HSC exhaustion and a bias towards myeloid differentiation, with toll‐like receptors playing a key role in maintaining hematopoiesis and bone metabolism balance.

Abstract 3702: Dissecting circulating tumor cells phenotype in a cohort of locally advanced non-small cell lung cancer patients undergoing neoadjuvant chemotherapy

Abstract Background: Platinum-based neoadjuvant chemotherapy (pb-NACT) is the primary treatment approach for locally advanced non-small cell lung cancer (LA-NSCLC), despite offering only a limited overall survival (OS) gain. Cisplatin-induced cell damage has been shown to activate the SDF-1/CXCR4 axis in preclinical models of NSCLC, resulting in the recruitment of a cell subset co-expressing the stemness marker CD133 and CXCR4 (SDF-1 receptor) at distant sites. These cells, known as metastasis-initiating cells (MIC), exhibit prometastatic activity. By longitudinally monitoring circulating tumor cells (CTC) in the blood of LA-NSCLC patients (pts) undergoing pb-NACT, our objective is to assess the potential prognostic significance of CTC number and phenotype. Methods: Between February 2019 and August 2023, a total of 61 pts with LA-NSCLC undergoing pb-NACT, were included in the study. Blood samples were obtained at baseline (T1), post-pb-NACT (T2), and post-surgery (T3) for the characterization of CTC, including total CTC, CXCR4+CTCs, and CXCR4+CD133+ MIC. This characterization utilized a validated marker-independent strategy combining Parsortix and DEPArrayTM technologies. Logistic regression models were employed to examine the association between CTC subsets and the response to pb-NACT (Disease Control Rate [DCR], Overall Response Rate [ORR]), while Cox-regression models were used to assess their association with survival endpoints (event-free survival [EFS], OS, disease-free survival [DFS]). Results: Out of the 61 enrolled patients, 34 had available CTC data for analysis. In this population, DCR was 64.7%, and the ORR 29.4%. Surgery was performed in 14 pts (41.2%). At baseline (T1), elevated levels of CXCR4+CTCs were linked to a lower DCR (10 vs 3/mL, odds ratio [OR]=0.02, 95% confidence interval [CI]: 0-0.40, p=0.008), as well as shorter EFS (10 vs 3/mL, HR=4.20, 95% CI: 1.84-9.58, p=0.002) and OS (10 vs 3/mL, HR=3.14, 95% CI: 1.25-7.88, p=0.004). At the post- CT time point (T2), higher levels of MIC were associated with shorter EFS (3 vs 0/mL, HR=1.41, 95% CI: 1.00-1.99, p=0.047) and OS (3 vs 0/mL, HR=1.55, 95% CI: 1.03-2.34, p=0.035). Although the sample size was limited, a higher number of MIC at the post-surgery time point (T3) showed a trend toward reduced DFS. Discussion: CTC could serve as a novel biomarker for assessing the effectiveness of pb-NACT in LA-NSCLC. Elevated levels of CXCR4+CTC both at baseline and after pb-NACT, along with increased MIC post-pb-NACT and post-surgery, indicate unfavorable prognostic factors for survival. Specifically, a higher baseline of CXCR4+CTCs may be linked to a suboptimal response to treatment, suggesting the potential need for treatment intensification in patients exhibiting elevated CXCR4+CTC baseline levels. Citation Format: Rita Leporati, Teresa Beninato, Sara Iadecola, Chiara Andreon, Fabio Murianni, Melissa Balsamo, Antonia Martinetti, Benedetta Lombardi Stocchetti, Laura Mazzeo, Sara Manglaviti, Mario Occhipinti, Marta Brambilla, Arsela Prelaj, Claudia Proto, Rosalba Miceli, Luca Roz, Gabriella Sozzi, Filippo De Braud, Giuseppe Lo Russo, Giulia Bertolini. Dissecting circulating tumor cells phenotype in a cohort of locally advanced non-small cell lung cancer patients undergoing neoadjuvant chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3702.

Combination Therapy of Bone Marrow Mesenchymal Stem Cell Transplantation and Electroacupuncture for the Repair of Intrauterine Adhesions in Rats: Mechanisms and Functional Recovery.

Transplantation of bone marrow mesenchymal stem cells (BMSCs) has demonstrated promising clinical utility in the treatment of endometrial injury and the restoration of fertility. However, since the efficacy of BMSCs after transplantation is not stable, it is very important to find effective ways to enhance the utilisation of BMSCs. Electroacupuncture (EA) has some positive effects on the chemotaxis of stem cells and diseases related to uterine injury. In this study, we established the intrauterine adhesion (IUA) model of the Sprague-Dawley rat using lipopolysaccharide infection and mechanical scratching. Phosphate-buffered saline, BMSCs alone, and BMSCs combined with EA were randomly administered to the rats. Fluorescent cell labelling showed the migration of transplanted BMSCs. H&E staining, Masson staining, Western blot, immunohistochemistry, ELISA, and qRT-PCR were utilised to detect changes in endometrial morphology and expressions of endometrial receptivity-related factors, endometrial pro-inflammatory factors, and fibrosis factors. Finally, we conducted a fertility test to measure the recovery of uterine function. The results showed that EA promoted transplanted BMSCs to migrate into the injured uterus by activating the SDF-1/CXCR4 axis. Endometrial morphology showed the most significant improvement in the BMSC + EA group. The expressions of endometrial pro-inflammatory factors and fibrosis indexes in the BMSC + EA group were lower than those in the model and BMSC groups. Further studies revealed that the expression of endometrial receptivity-related factors and the number of embryos implanted on day 8 of gestation increased in the BMSC + EA group compared with the model group and the BMSC group.

SDF-1 involvement in orthodontic tooth movement after tooth extraction

The stromal cell-derived factor 1 (SDF-1)/chemokine receptor type 4 (CXCR4) axis plays a key role in alveolar bone metabolism during orthodontic tooth movement (OTM). Herein, the effects of the SDF-1/CXCR4 axis on the regional acceleratory phenomenon (RAP) in OTM velocity and on changes in the surrounding periodontium after adjacent tooth extraction in rats were investigated. Six-week-old male Wistar/ST rats underwent left maxillary first molar (M1) extraction and mesial OTM of the left maxillary second molar (M2) with a 10-g force closed-coil spring. Phosphate-buffered saline, immunoglobulin G (IgG) isotype control antibody, or anti-SDF-1 neutralizing monoclonal antibody were injected at the M1 and M2 interproximal areas (10 μg/0.1 mL) for the first three days. Analyses were performed after 1, 3, and 7 days (n = 7). The results demonstrated a significant increase in SDF-1 expression from day 1, which was effectively blocked via anti-SDF-1 neutralizing monoclonal antibody injection. On day 3, the M2 OTM distance and the number of positively stained osteoclasts significantly reduced alongside a reduction in inflammatory markers in the experimental group. Our results demonstrated that serial local injection of the anti-SDF-1 neutralizing monoclonal antibody reduces M2 OTM, osteoclast accumulation, and localized inflammatory responses in an OTM model with tooth extraction-induced RAP.

Apt‐19s‐Functionalized 3D‐Printed Mesoporous Bioactive Glass Scaffold Promotes BMSC Recruitment in Bone Regeneration via SDF‐1α/CXCR4 Axis and MAPK Signaling

AbstractDirectional migration and differentiation of stem cells in situ are two essential processes for tissue regeneration. However, transplanted scaffold itself lacks sufficient ability to induce stem cell recruitment. As an artificial synthesized nucleic acid, aptamer 19s (Apt‐19s), with high cost‐effectiveness and bioactivity, demonstrates a robust capacity for specific binding and recruitment of mesenchymal stem cells. In this study, a mesoporous bioactive glass scaffold fabricated via a seamless route of self‐assembly and 3D printing (3D‐MBG), functionalized with Apt‐19s is successfully prepared via physical adsorption and lyophilization with optimized release kinetics and bone marrow‐derived mesenchymal stem cells (BMSCs) recruiting efficiency in vitro. This scaffold significantly enhanced migration and osteogenic differentiation of BMSCs in vitro and in vivo. The mechanism underlying Apt‐19s‐induced BMSCs recruitment is elucidated for the first time, that Apt‐19s promoted BMSCs migration by upregulating stromal cell‐derived factor‐1α (SDF‐1α) gene expression, facilitating SDF‐1α protein translation and secretion, and further activating the SDF‐1α/CXCR4 signaling axis and MAPK pathway. In summary, the Apt‐19s‐functionalized 3D‐MBG scaffold offers an economical and efficient solution for customized bone regeneration in situ, and the elucidation of Apt‐19s‐induced BMSCs recruitment at molecular biological level may shed light on its broader clinical applications.

Mechanism of Bushen Zhuangjin Decoction to Promote BMSCs Homing and Protect Articular Cartilage in Mice by the SDF-1/CXCR4 Axis

Mechanism of Bushen Zhuangjin Decoction to Promote BMSCs Homing and Protect Articular Cartilage in Mice by the SDF-1/CXCR4 Axis

Abstract A031: Biotherapeutic strategies targeting the CXCR2 axis for depletion of myeloid-derived suppressor cells in pancreatic ductal adenocarcinoma

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) has a 5-year survival rate of only 10%, and limited treatment options exist. Immune checkpoint inhibitors are effective in a variety of cancer types; however, the complex immunosuppressive tumor microenvironment (TME) of PDAC is a significant barrier to immunotherapy. Myeloid-derived suppressor cells (MDSCs) are abundant in PDAC tumors from humans and mouse models. MDSCs suppress effector T cell function and promote the expansion of other immunosuppressive cell types. This led us to hypothesize that targeting tumor MDSCs would be an effective therapeutic strategy for PDAC and enhance the efficacy of T cell based immunotherapy. Our preliminary data and the work from others shows that CXCR2 ligands such as CXCL8/IL-8, CXCL5/ENA78, and CXCL1/GROα are the most abundant soluble factors secreted by PDAC cells. CXCR2 ligands are critical for the homing of myeloid cells to sites of injury in cutaneous wound healing, suggesting that dysregulated secretion of CXCR2 chemokines in PDAC may mimic a wound healing response that attracts myeloid cells and promotes the expansion of MDSCs and an immunosuppressive TME. To test this hypothesis, we have designed and produced multiple drug-conjugated CXCR2 ligand-based engineered proteins to target and eliminate MDSCs from PDAC tumors. We are referring to these novel biotherapeutic molecules as Ligand Drug Conjugates (LDCs). We have demonstrated binding, internalization, induction of signaling, and cytotoxicity of these LDCs to CXCR2 expressing myeloid cells. Our data suggests that our engineered proteins are superior binders and activators of CXCR2 axis signaling compared to WT CXCR2 ligands. Utilizing CXCR2 knockin models we demonstrated that the cytotoxicity of our LDCs is CXCR2 dependent. In vivo, we are utilizing the KPC (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre) mouse model of PDAC, which we've shown in preliminary studies to, like human PDAC, support an abundance of MDSCs in tumors as well as secondary lymphoid organs such as the spleen. We anticipate that this study will demonstrate the therapeutic potential and actionability of targeting MDSCs and will provide novel biotherapeutic drug candidates that will ultimately improve immunotherapy outcomes in patients with PDAC. Citation Format: Benjamin N. Christopher, Lety Reyes, Reeder Robinson, Lena Golick, Nathan Dolloff. Biotherapeutic strategies targeting the CXCR2 axis for depletion of myeloid-derived suppressor cells in 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 A031.

Tumor-associated macrophages promoting PD-L1 expression in infiltrating B cells through the CXCL12/ CXCR4 axis in human hepatocellular carcinoma

Tumor-associated macrophages promoting PD-L1 expression in infiltrating B cells through the CXCL12/ CXCR4 axis in human hepatocellular carcinoma

Dynamic Oscillation of Tetraspanin CD9 Orchestrates Engraftment and Mobilization of Human Hematopoietic Stem/Progenitor Cells

Background Trafficking of hematopoietic stem/progenitor cells (HSPCs) to and from their bone marrow (BM) niche is pivotal for engraftment and mobilization during clinical transplantation, where both processes are controlled by highly orchestrated molecular programs. We previously identified the tetraspanin CD9 as a downstream effector of the SDF-1/CXCR4 axis driving migration, adhesion and homing of human CD34+ HSPCs, but its consequential impact on stem cell engraftment and mobilization as well as the underlying mechanisms remains unknown. Methods Umbilical cord blood (UCB) CD34+ cells were exposed to a CD9 neutralizing antibody ALB6 prior to infusion into sublethally irradiated NOD/SCID mice, with overall engraftment and lineage output assessed by enumerating human CD45+ (pan-leukocytes), CD19+ (B-lymphoid), CD33+ (myeloid) and CD34+ (stem/progenitors) cells in the recipient BM at 8 weeks post-transplantation. CD9 expression on engrafted HSPCs was characterized by flow cytometry and compared to those before transplantation or after G-CSF mobilization. The oscillation pattern was then validated on HSPCs in pairwise BM, peripheral blood (PB) and G-CSF-mobilized peripheral blood (MPB) samples from healthy donors. Activity of integrin VLA-4 (an established binding partner of CD9) was evaluated on BM-residing and mobilized HSPCs by VCAM-1 binding assay. Results A short exposure of UCB CD34+ cells to the CD9 neutralizing antibody markedly inhibited their BM engraftment by 64.2% ( P=0.003), reflected by the general decline in human CD45+ cell population. There was no bias in lymphoid/myeloid lineage outputs, suggesting CD9-mediated engraftment is not restricted to specific subsets of hematopoietic precursors. CD9 expression was drastically elevated on engrafted CD34+ cells in the BM from 9.6% to 91.4% ( P<0.001) compared to pre-transplantation. In contrast, CD9 expression was significantly downregulated on BM CD34+ cells from 73.4% to 33.5% ( P<0.001) upon G-CSF mobilization, with the decline observed in both primitive stem cells (CD34+CD38-) and committed progenitors (CD34+CD38+), indicating that the oscillation of CD9 is highly associated with HSPC trafficking. Concordantly, pairwise analysis of steady-state HSPCs in BM and PB from healthy donors revealed higher CD9 level in BM-residing CD34+ cells than the PB-circulating portion (46.1% vs. 26.5%, P=0.004; n=13). In addition, a significantly higher CD9 expression was observed on BM CD34+ cells (47.7%; n=16) than those in MPB (32.3%, n=25; P=0.003). CD9 expression on MPB CD34+ cells was enhanced upon SDF-1 exposure ( P=0.006), revealing a dynamic regulation. In normal BM, CD9+ HSPCs exhibited elevated VLA-4 activities with preferential binding to soluble VCAM-1 compared to the CD9- counterpart (51.4% vs. 25.9%, P<0.001; n=10). Superiority in VCAM-1 binding capacity was also witnessed in BM-residing CD34+ cells compared to MPB (37.7% vs. 4.1%, P=0.014), implicating that the influence of CD9 on HSPC trafficking may due to the alteration of integrin activities and downstream adhesive properties. Conclusion This study describes the dynamic regulation of CD9 during entry and exit of HSPCs to and from their BM niche via modulation of integrin activities. The findings extend our understandings on HSPC trafficking that could potentially lead to new strategies to enhance clinical stem cell transplantation and mobilization.

Identification of Competing Endogenous RNA Networks and Potential Therapeutic Drugs for Gastroesophageal Reflux Disease

Background: The diagnosis and cure rate of gastroesophageal reflux disease (GERD) is low, and its pathogenesis remains unclear. Aims: In this study, we aimed to construct the competing endogenous RNA (ceRNA) of GERD by bioinformatics and explore the potential therapeutic drugs for GERD. Methods: The differentially expressed genes (DEGs) were got by limma package and visualized by ggplot2 and pheatmap package in R version 4.1.3 from the GSE63401 gene microarray data. The pro-tein-protein interaction (PPI) network was constructed by the STRING database. The hub genes and subnetwork modules were obtained by Cytoscape software. The microRNA and lncRNA were pre-dicted by the miRTarBase and miRNet databases to construct the ceRNA network. Potential drugs tar-geting DEGs were screened by the CMAP database. Results: In this study, a total of 571 GEDs were obtained, including 462 up-regulated genes and 109 down-regulated genes, which mainly participated in the leukocyte migration process and the cytokine-cytokine receptor interaction pathway. Seventeen miRNAs and three key lncRNAs involved in regulat-ing hub genes were screened through miRTarBase and miRNet databases. Finally, nine ceRNA net-works were obtained, including TTN-AS1/NEAT1/XIST - miR-139-5p - CXCR4 axis, TTN-AS1/NEAT1/XIST - miR-185-5p - IL1B axis and TTN-AS1/NEAT1/XIST - miR-495-3p - LYN axis. In addition, five potential drugs (Pimavanserin, BMS-182874, CL-218872, Losartan, and Laropiprant) for GERD were obtained through the CMAP database. Conclusion: We constructed nine GERD-related ceRNA networks and screened five potential thera-peutic drugs for GERD, providing a theoretical basis for further research on the pathogenesis of GERD.

Implantable SDF-1α-loaded silk fibroin hyaluronic acid aerogel sponges as an instructive component of the glioblastoma ecosystem: Between chemoattraction and tumor shaping into resection cavities

In view of inevitable recurrences despite resection, glioblastoma (GB) is still an unmet clinical need. Dealing with the stromal-cell derived factor 1-alpha (SDF-1α)/CXCR4 axis as a hallmark of infiltrative GB tumors and with the resection cavity situation, the present study described the effects and relevance of a new engineered micro-nanostructured SF-HA-Hep aerogel sponges, made of silk fibroin (SF), hyaluronic acid (HA) and heparin (Hep) and loaded with SDF-1α, to interfere with the GB ecosystem and residual GB cells, attracting and confining them in a controlled area before elimination. 70 µm-pore sponges were designed as an implantable scaffold to trap GB cells. They presented shape memory and fit brain cavities. Histological results after implantation in brain immunocompetent Fischer rats revealed that SF-HA-Hep sponges are well tolerated for more than 3 months while moderately and reversibly colonized by immuno-inflammatory cells. The use of human U87MG GB cells overexpressing the CXCR4 receptor (U87MG-CXCR4+) and responding to SDF-1α allowed demonstrating directional GB cell attraction and colonization of the device in vitro and in vivo in orthotopic resection cavities in Nude rats. Not modifying global survival, aerogel sponge implantation strongly shaped U87MG-CXCR4+ tumors in cavities in contrast to random infiltrative growth in controls. Overall, those results support the interest of SF-HA-Hep sponges as modifiers of the GB ecosystem dynamics acting as “cell meeting rooms” and biocompatible niches whose properties deserve to be considered toward the development of new clinical procedures. Statement of SignificanceBrain tumor glioblastoma (GB) is one of the worst unmet clinical needs. To prevent the relapse in the resection cavity situation, new implantable biopolymer aerogel sponges loaded with a chemoattractant molecule were designed and preclinically tested as a prototype targeting the interaction between the initial tumor location and its attraction by the peritumoral environment. While not modifying global survival, biocompatible SDF1-loaded hyaluronic acid and silk fibroin sponges induce directional GB cell attraction and colonization in vitro and in rats in vivo. Interestingly, they strongly shaped GB tumors in contrast to random infiltrative growth in controls. These results provide original findings on application of exogenous engineered niches that shape tumors and serve as cell meeting rooms for further clinical developments.