C-X-C Chemokine Receptor Type 4 Expression Research Articles

An in vivo tumour organoid model based on the chick embryonic chorioallantoic membrane mimics key characteristics of the patient tissue: a proof-of-concept study

BackgroundPatient-derived tumour organoids (PDOs) are highly advanced in vitro models for disease modelling, yet they lack vascularisation. To overcome this shortcoming, organoids can be inoculated onto the chorioallantoic membrane (CAM); the highly vascularised, not innervated extraembryonic membrane of fertilised chicken eggs. Therefore, we aimed to (1) establish a CAM patient-derived xenograft (PDX) model based on PDOs generated from the liver metastasis of a colorectal cancer (CRC) patient and (2) to evaluate the translational pipeline (patient – in vitro PDOs – in vivo CAM-PDX) regarding morphology, histopathology, expression of C-X-C chemokine receptor type 4 (CXCR4), and radiotracer uptake patterns.ResultsThe main liver metastasis of the CRC patient exhibited high 2-[18F]FDG uptake and moderate and focal [68Ga]Ga-Pentixafor accumulation in the peripheral part of the metastasis. Inoculation of PDOs derived from this region onto the CAM resulted in large, highly viable, and extensively vascularised xenografts, as demonstrated immunohistochemically and confirmed by high 2-[18F]FDG uptake. The xenografts showed striking histomorphological similarity to the patient’s liver metastasis. The moderate expression of CXCR4 was maintained in ovo and was concordant with the expression levels of the patient’s sample and in vitro PDOs. Following in vitro re-culturing of CAM-PDXs, growth, and [68Ga]Ga-Pentixafor uptake were unaltered compared to PDOs before transplantation onto the CAM. Although [68Ga]Ga-Pentixafor was taken up into CAM-PDXs, the uptake in the baseline and blocking group were comparable and there was only a trend towards blocking.ConclusionsWe successfully established an in vivo CAM-PDX model based on CRC PDOs. The histomorphological features and target protein expression of the original patient’s tissue were mirrored in the in vitro PDOs, and particularly in the in vivo CAM-PDXs. The [68Ga]Ga-Pentixafor uptake patterns were comparable between in vitro, in ovo and clinical data and 2-[18F]FDG was avidly taken up in the patient’s liver metastasis and CAM-PDXs. We thus propose the CAM-PDX model as an alternative in vivo model with promising translational value for CRC patients.Graphical

Airway Epithelium-derived CXCL14 Promotes Eosinophil Accumulation in Allergic Airway Inflammation.

C-X-C motif chemokine ligand 14 (CXCL14) is expressed in the airway epithelial cells of patients with asthma. However, the mechanisms of CXCL14 secretion and its effects on asthma pathogenesis remain unclear. Here, we investigated the role of CXCL14 in allergic airway inflammation and its effects on eosinophil infiltration. Our findings showed that Alternaria alternata, a major environmental allergen, stimulated CXCL14 secretion from airway epithelial cells via reactive oxygen species (ROS) generated in mitochondrial oxidative phosphorylation (OXPHOS) complexes, especially in OXPHOS complex II. In vivo, in a mouse model of allergic airway inflammation, intranasal administration of anti-CXCL14 antibody suppressed eosinophil and dendritic cell infiltration into the airways and goblet cell hyperplasia. In vitro, in human eosinophil-like cells, CXCL14 promoted cell migration through C-X-C chemokine receptor type 4 (CXCR4) binding. Eosinophil CXCR4 expression was upregulated by Alternaria stimulation via ROS production. These findings suggest that the crosstalk between Alternaria-stimulated airway epithelial CXCL14 secretion and eosinophil CXCR4 upregulation plays an important role in eosinophil infiltration into the lungs during allergic airway inflammation. In summary, this study demonstrates that CXCL14 could be a therapeutic target for allergic airway inflammation.

CXCR4 promotes migration, invasion, and epithelial-mesenchymal transition of papillary thyroid carcinoma by activating STAT3 signaling pathway.

Papillary thyroid cancer (PTC) is a serious threat to human health worldwide, while metastasis in the early phase limits therapeutic success and leads to poor survival outcomes. The CXC chemokine receptor type 4 (CXCR4) plays an important role in many cellular movements such as transcriptional modulation, cell skeleton rearrangement, and cell migration, and the change in CXCR4 levels are crucial in various diseases including cancer. In this study, we explored the role of CXCR4 in the migration and invasion of PTC and investigated the potential mechanisms underlying its effects. We analyzed the expression levels of CXCR4 in PTC tissues and cell lines. Would healing migration, Transwell invasion assay in vitro, and tail-vein lung metastasis assay In vivo were performed to evaluated the migration and invasion abilities of PTC cells with stable CXCR4 knockdown or overexpression. Signal transducers and activators of transcription (STAT3) signaling pathway-related protein expressions were examined by Western blotting assays. The results showed that CXCR4 was highly expressed in PTC cell lines and PTC tissues. CXCR4 knockdown in PTC cells dampened the migration, invasion, and epithelial-mesenchymal transition (EMT), whereas CXCR4 overexpression enhanced these properties. In vivo, we also found that CXCR4 promoted the metastasis of PTC. Mechanistic studies showed that CXCR4 played these vital roles through the STAT3 signaling pathway. Furthermore, PTC patients with high CXCR4 or p-STAT3 expression correlated with aggressive clinical characteristics such as extrathyroidal extension (ETE), and lymph node metastasis (LNM). We provided evidence that CXCR4 might activate the STAT3 signaling pathway and further promote PTC development. Thus, CXCR4 might be a novel therapeutic target for PTC.

PIEZO1 Promotes the Migration of Endothelial Cells via Enhancing CXCR4 Expression under Simulated Microgravity.

Exposure to microgravity during spaceflight induces the alterations in endothelial cell function associated with post-flight cardiovascular deconditioning. PIEZO1 is a major mechanosensitive ion channel that regulates endothelial cell function. In this study, we used a two-dimensional clinostat to investigate the expression of PIEZO1 and its regulatory mechanism on human umbilical vein endothelial cells (HUVECs) under simulated microgravity. Utilizing quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis, we observed that PIEZO1 expression was significantly increased in response to simulated microgravity. Moreover, we found microgravity promoted endothelial cells migration by increasing expression of PIEZO1. Proteomics analysis highlighted the importance of C-X-C chemokine receptor type 4(CXCR4) as a main target molecule of PIEZO1 in HUVECs. CXCR4 protein level was increased with simulated microgravity and decreased with PIEZO1 knock down. The mechanistic study showed that PIEZO1 enhances CXCR4 expression via Ca2+ influx. In addition, CXCR4 could promote endothelial cell migration under simulated microgravity. Taken together, these results suggest that the upregulation of PIEZO1 in response to simulated microgravity regulates endothelial cell migration due to enhancing CXCR4 expression via Ca2+ influx.

Abstract 3341: Targeting CXCR4 via the small molecule inhibitor NMX1 as a therapeutic strategy to treat high-risk malignancies

Abstract Introduction: Cytokines are secreted by various cells of the immune system, endothelium, and stroma that play a critical role in cell survival. The activation of C-X-C chemokine receptor type 4 (CXCR4) by its ligand SDF-1 leads to signaling by the AKT and mTOR pathways. CXCR4 is highly expressed in several types of cancer and contributes to tumor growth, metastasis, and resistance to therapy. Interleukin 11 (IL-11) is a cytokine that often drives disease progression, as well as contributing to tumor immune evasion. NMX1 (NovoMedix) is a novel oral small molecule that inhibits mTOR signaling, CXCR4 expression, IL-11 secretion, and activates AMPK. In animal models of breast cancer, NMX1 effectively prevented tumor growth and metastasis, while also protecting from doxorubicin-induced cardiotoxicity. In this study, we investigated the anticancer activity of NMX1 in preclinical models of high-risk pediatric and adult malignancies. Methods: Cell viability assays using alamar blue were performed in a panel of pediatric and adult cancer cells, including leukemia, brain, breast, lung, neuroblastoma, and sarcoma. Cells were treated with increasing concentrations of NMX1 ranging from 250 nM to 16 µM for 96 hours, followed by determination of half maximal inhibitory concentrations (IC50). To validate target modulation, the level of IL-11 and CXCR4 was determined by ELISA and immunoblot, respectively. Protein kinase activity was assessed by phosphorylation levels. Induction of apoptosis was confirmed by caspase-mediated PARP cleavage. To identify effective drug combinations, NMX1-treated cells were screened with a comprehensive drug library. Synergy was evaluated in the top candidates by dose-response matrices and SynergyFinder. Safety and tolerability was tested in animal models. Results: NMX1 treatment induced cytotoxicity at micromolar concentrations in diverse types of cancer, with IC50 values ranging from 0.2-8.7 µM and 0.4-4.4 µM for leukemia and solid tumor cells, respectively. NMX1 induced cancer cell death by PARP cleavage and apoptosis. Mechanistically, CXCR4 expression, mTOR signaling, and IL-11 secretion were decreased upon treatment with NMX1, validating effective target modulation. The drug combination screen identified several candidates in a tumor type-dependent manner. Synergistic effects were observed using NMX1 combined with clinically relevant concentrations of rucaparib, linsitinib, and crizotinib, which target PARP, IGFR, and c-MET, respectively. Conclusions: Our study shows antitumor activity by NMX1 through inhibition of CXCR4, IL-11, and mTOR signaling, providing an effective therapeutic strategy for high-risk and refractory malignancies. Preclinical data on feasible drug combinations and biological correlates of activity support development of early phase clinical studies involving NMX1. Citation Format: Patrick Sipila, Son Tran, Chunfen Zhang, Laura G. Corral, Kyle W. Chan, Cathy A. Swindlehurst, Aru Narendran. Targeting CXCR4 via the small molecule inhibitor NMX1 as a therapeutic strategy to treat high-risk malignancies [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 3341.

Abstract 2710: Combination CXCR4 and PD-1 blockade increases T cell infiltration and effector function in fibrolamellar carcinoma (FLC)

Abstract Introduction: Fibrolamellar carcinoma (FLC), a rare liver cancer affecting young patients without cirrhosis, has a poor prognosis and few systemic therapy options. Current evidence implicates an immunosuppressive tumor microenvironment (TME), including our preliminary data suggesting preferential localization of T cells in stromal bands. We hypothesized that dual blockade of C-X-C chemokine receptor type 4 (CXCR4) and programmed cell death protein (PD-1) would enhance T cell infiltration and activation, respectively, and thereby increase tumor killing. Methods: Flow cytometry and single-nuclear RNA sequencing (snRNA-seq) were used to analyze immune cells and evaluate gene expression, respectively, in patient-derived FLC and non-tumor liver (NTL). Immunohistochemistry (IHC) was used to assess CD3+ T cell localization in relation to CXCL12, the chemokine ligand of CXCR4. Tumor slice cultures (TSCs) from fresh FLC resection specimens (n=11) were treated with control (IgG1), a small molecule CXCR4 inhibitor (AMD3100), anti-PD-1 blocking antibody (αPD-1), or combination CXCR4 and PD-1 blockade (AMD3100/αPD-1). IHC, RNA expression (Nanostring), and live microscopy were used to evaluate T cell mobilization and function. Results: Flow cytometry analysis demonstrated fewer CD45+ cells (16% vs 54% of live cells, p=0.01) and heterogenous PD-1 expression on T cells in FLC compared with NTL. SnRNA-seq analysis showed that CXCR4 expression is upregulated in FLC lymphocyte and macrophage populations relative to NTL (15% vs 10% and 20% vs 5% expressed), and CXCL12 is expressed primarily in stellate cells. CD3+ T cells colocalized with CXCL12 in the FLC stroma on IHC. Multiplex IHC of treated TSCs revealed more T cells in the carcinoma (CK7+) than the stromal (SMA+) compartment after AMD3100 compared with IgG1 (63% vs 34% T cells in CK7+ compartment, p=0.05). Nanostring indicated upregulation of effector pathways (apoptosis, cytotoxicity, lymphocyte activation) after AMD3100/αPD-1 compared with IgG1. Time-lapse live microscopy revealed a greater percentage of apoptotic carcinoma cells (SR-FLICA+CK7+) colocalized with CD3+ T cells (37% vs 18%, p=0.005) and longer CD3+ T cell track length (27 vs 22 μm, p=0.03) after AMD3100/αPD-1 than before treatment. Cleaved caspase-3 (CC3) IHC demonstrated significantly increased tumor apoptosis after AMD3100/αPD-1 compared with IgG1 and monotherapy (53% vs 32% (IgG1), 39% (AMD3100), and 42% (αPD-1) CC3+ cells, p=0.002, p=0.03, p=0.04). Conclusion: The CXCR4/CXCL12 axis mediates T cell exclusion in the FLC TME. Combination CXCR4 and PD-1 blockade overcomes immunosuppression in FLC by mobilizing T cells into the carcinoma compartment and activating their effector function, with resultant tumor apoptosis. These findings support consideration of clinical trials testing drugs targeting CXCR4 and PD-1 in FLC. Citation Format: Lindsay K. Dickerson, Renske van den Bijgaart, Xiuyun Jiang, Sara K. Daniel, Jason A. Carter, Alaa Farghli, Kevin M. Sullivan, Yongjun Liu, Heidi L. Kenerson, Raymond S. Yeung, Teresa S. Kim, Ian N. Crispe, Praveen Sethupathy, Kevin C. Barry, Venu G. Pillarisetty. Combination CXCR4 and PD-1 blockade increases T cell infiltration and effector function in fibrolamellar carcinoma (FLC) [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 2710.

Abstract 6802: CXCR4 transgene improves in vivo migration and efficacy of engineered iPSC-derived natural killer cells

Abstract Chimeric antigen receptor (CAR)-engineered induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells have shown a favorable safety profile and early evidence of clinical efficacy in the treatment of patients with certain hematologic malignancies. CAR-iNK cells retain phenotypic and functional similarities to primary natural killer cells, with additional capabilities for recognition of tumors. For the treatment of cancers that are located in the bone marrow, it is critical that iNK cells are able to migrate efficiently to relevant sites of disease. CXCR4 (C-X-C chemokine receptor type 4) is a chemokine receptor specific for stromal-derived-factor-1 (SDF-1), and mediates the migration of hematopoietic cells to the bone marrow upon binding SDF-1. Here we describe novel engineering of iPSCs resulting in efficient migration and efficacy of iNK cells. iPSC were sequentially engineered at defined loci with Crispr/MAD7/HDR to incorporate Allo-EvasionTM edits, a CAR targeting antigens expressed on different hematological malignancies, constitutive interleukin-15 (IL-15) cytokine support, with and without wild-type CXCR4. iPSCs were then differentiated into natural killer cells using a proprietary differentiation process. Cytolysis of a panel of target cells by iNK was evaluated using an Incucyte-based killing assay. Anti-tumor efficacy was tested in disseminated xenograft models of hematological malignancies following intravenous injection of engineered iNK cells. Additionally, in vivo migration was evaluated using immunohistochemistry (IHC) and flow cytometry (FACS) of blood, bone marrow, and other organs. Engineering with CXCR4 resulted in a bulk population of iNK with a heterogenous expression of CXCR4. No significant impact of CXCR4 engineering was detected on iPSC differentiation, phenotype, or iNK yield. iNKCXCR4+ and iNKCXCR4− cells had equivalent target tumor cell cytolysis in vitro, indicating the CXCR4 transgene does not affect killing potential. In vivo, iNKCXCR4+ cells migrated to the bone marrow of both tumor-bearing and non-tumor-bearing animals, and the presence of these cells was enhanced in tumor-bearing mice, indicating CXCR4-mediated migration and possibly CAR-mediated expansion and/or retention of iNK. Migration to the bone marrow was not observed with iNKCXCR4− cells. As a result, iNKCXCR4+ cells significantly impacted anti-tumor efficacy, effectively eliminating bone disease. We have demonstrated the CAR-specific killing and enhanced iNK migration and subsequent tumor killing in the bone marrow, with the addition of a CXCR4 transgene in iPSC-derived NK cells. The addition of the CXCR4 transgene in iPSC-derived effector cells may allow for a higher therapeutic response in axial-skeletal located tumors. Citation Format: Hillary J. Millar, David Walker, Liam R. Campion, Ciara Budd, Aarti Kolluri, Harsh Sharma, Buddha Gurung, Jill M. Carton, Daniel J. Perry, Nicholas Alexander, Hyam Levitsky, Michael Naso, Barry A. Morse. CXCR4 transgene improves in vivo migration and efficacy of engineered iPSC-derived natural killer cells [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 6802.

Extracellular Hsp70 Is Involved in the CXCL12/CXCR4 Pathway in Primary Human Nasal Epithelial Cells: A Preliminary Study

Background and Objectives: To date, no studies have been conducted on the interaction between extracellular heat shock protein 70 (Hsp70) and C-X-C chemokine receptor type 4 (CXCR4) in the upper airway. We aimed to evaluate the relationship between extracellular Hsp70 and CXCR4 and their role in the primary human nasal epithelium.Methods: We cultured primary human nasal epithelial (HNE) cells in an air–liquid interface. Macrogen performed single-cell quantitative polymerase chain reaction and sequencing. We conducted western blot analysis for the CXCR4 and mitogen-activated protein kinase (MAPK) pathways.Results: Extracellular Hsp70 treatment significantly increased the genetic expression and protein levels of CXCR4 in primary HNE cells. Phospho-ERK expression was increased by cotreatment with Hsp70 and CXCL12, but inhibited by pretreatment with AMD3100, a CXCR4 inhibitor. Pretreatment with an anti-Hsp70 antibody reduced phospho-ERK expression upregulation induced by cotreatment with Hsp70 and CXCL12.Conclusion: Extracellular Hsp70 participates in the activation of the CXCR4-dependent downstream signaling pathway in HNE cells. Further studies should evaluate the extracellular Hsp70-CXCL12/CXCR4 axis and the role of its components in the development of inflammatory diseases.

CXCR4-BTK axis mediate pyroptosis and lipid peroxidation in early brain injury after subarachnoid hemorrhage via NLRP3 inflammasome and NF-κB pathway

C-X-C chemokine receptor type 4 (CXCR4) is critical for homeostasis of the adaptive and innate immune system in some CNS diseases. Bruton's tyrosine kinase (BTK) is an essential kinase that regulates inflammation in immune cells through multiple signaling pathways. This study aims to explore the effect of CXCR4 and BTK on neuroinflammation in the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Our results showed that the expression of CXCR4 and p-BTK increased significantly at 24 h after SAH in vivo and in vitro. Ibrutinib improved neurological impairment, BBB disruption, cerebral edema, lipid peroxidation, neuroinflammation and neuronal death at 24 h after SAH. Inhibition of BTK phosphorylation promoted the in vitro transition of hemin-treated proinflammatory microglia to the anti-inflammatory state, inhibited the p-P65 expression and microglial pyroptosis. NLRP3 deficiency can significantly reduce pyroptosis in SAH mice. Moreover, CXCR4 inhibition can suppress NLRP3-mediated pyroptosis, NF-κB activation and NOX2 expression in vitro, and ibrutinib can abolish CXCR4-aggravated BBB damage and pyroptosis in EBI after SAH. The levels of CXCR4 in CSF of SAH patients is significantly increased, and it is positively correlated with GSDMD and IL-1β levels, and have a moderate diagnostic value for outcome at 6-month follow-up. Our findings revealed the effect of CXCR4 and P-BTK on NLRP3-mediated pyroptosis and lipid peroxidation after SAH in vivo and in vitro, and the potential diagnostic role of CXCR4 in CSF of SAH patients. Inhibition of CXCR4-BTK axis can significantly attenuate NLRP3-mediated pyroptosis and lipid peroxidation by regulating NF-κB activation in EBI after SAH.

Check update pattern of tumorigenic vasculature signature based on MMP9 and CXCR4 expression in locally advanced breast cancer

Context: Locally advanced breast cancers (LABC) are the most common women malignant tumors. Appropriate vasculature is required for tumor growth support, formed by involving protein signaling, including matrix metalloprotein 9 (MMP9) and C-X-C chemokine receptor type 4 (CXCR4). Neoadjuvant chemotherapy (NAC) administration to inoperable LABC commonly exhibits a positive response, although recurrences may be encountered in a few cases. Aims: To evaluate the MMP9 and CXCR4 expression shifting after the NAC procedure to establish evidence of the anti-angiogenic effect of NAC, which encourages knowledge of tumor size reduction pathways in LABC. Methods: Observational designs were conducted in this study. Tissue specimens before and after NAC were collected from 45 LABC-enrolled subjects. The targeted protein expression was analyzed by immunohistochemistry, and stained sections were classified according to the percentage of nuclear-stained tumor cells. Clinicopathological features of LABC were recorded. Tumor size was measured by Vernier caliper before and after NAC. Results: The results showed the nuclear expression of MMP9 and CXCR4 protein were observed in all tissue specimens. The expression of MMP9 and CXCR4 tended to decrease after the NAC but was not statistically significant for MMP9. There was a significant correlation between expression levels of CXCR4 and tumor size reduction (p<0.001) but not for MMP9. Conclusions: The results of this study demonstrate the anti-angiogenic effect of NAC by inhibiting MMP9 and CXCR4, which may be integrated with tumor size reduction in LABC. Further studies are required to highlight the possibility of recurrence following inhibition of MMP9 and CXCR4 by NAC.

SAT381 Effect of Obesity on Androgen-induced Splenic Dysregulation in Polycystic Ovary Syndrome

Abstract Disclosure: S. Rezq: None. J. Basnet: None. A.M. Huffman: None. L.L. Yanes Cardozo: None. D.G. Romero: None. Background: Polycystic ovary syndrome (PCOS) is characterized by ovarian dysfunction and hyperandrogenism. Women with PCOS exhibit low-grade systemic inflammation, obesity, and immune dysregulation. The spleen plays an important role in immune function and physiological inflammation regulation. Androgens and obesity negatively impact splenic structure and function. However, the effect of androgens on spleen architecture and function and its modulation by obesogenic diet is unknown in PCOS. Using a well-established PCOS model of hyperandrogenemia we aim to test the hypothesis that androgens cause splenic dysregulations that are exacerbated by obesity. Methods: Three-week-old female mice were treated with the androgen dihydrotestosterone (DHT, 8 mg, s.c) or vehicle. Animals were fed a high-fat diet (HFD; 60% kcal fat) or a low-fat diet (LFD; 10% kcal fat) for 90 days (n=6-8/group). Body composition (EchoMRI) and spleen weight were assessed. Splenic mRNA or protein expression of multiple immune cells/regulatory markers such as chemokines ligands Cxcl12 and Lymphotoxin B (Ltb), and their corresponding receptors C-X-C chemokine receptor type 4 (CXCR4) and Ltb receptor (LTBR), as well as the chemokine monocyte chemoattractant protein-1 (MCP-1), proliferation markers Csf1, Csf2, and Csf3, the adhesion molecule Vcam1, and extracellular matrix (ECM) remodeling marker Coch were assessed by RT-qPCR or Western blot. Additionally, splenic androgen receptor (AR) protein expression was measured by Western blot, while the inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) were detected by ELISA. Results: DHT significantly (p<0.05) increased fat mass (6.2 ± 0.5 vs. 3.4 ± 0.3 g) and spleen weight (11.9 ± 1.6 vs. 4.9 ± 0.22 g) in LFD-fed mice, which was exacerbated by HFD. DHT-mediated splenomegaly was associated with a significant (p<0.05) decrease in the expression of AR (90%), the chemokine Cxcl12 (70-80%), the proliferation markers Csf1 and Csf2 (65-66%), and the ECM marker Coch (70%) in mice that were fed on both diets. PCOS mice had significantly (p<0.05) higher CXCR4 (4.4-4.7-fold) and LTBR (5.7-7.6-fold) expression, independent of diet. Interestingly, only the HFD-fed PCOS mice had significantly (p<0.05) lower splenic Vcam1 (76%), IL-6 (25%), and TNF-α (4.6 ± 0.2 vs. 5.6 ± 0.2 pg/μg protein) levels, while they also had higher MCP-1 protein levels (2-fold, p<0.05). Conclusion and significance: Our data show that androgens cause splenic structural and molecular alterations that are modulated by obesity. These findings could explain PCOS-associated immune dysregulation that is more predominant in obese women with PCOS. The study highlights the spleen as a novel key target organ of androgen-mediated pathology in PCOS. Furthermore, our study suggests that weight loss could potentially attenuate androgen-associated splenic derangements in PCOS. Presentation Date: Saturday, June 17, 2023

The CXCR4-AT1 axis plays a vital role in glomerular injury via mediating the crosstalk between podocyte and mesangial cell

Glomeruli stand at the center of nephrons to accomplish filtration and albumin interception.Podocytes and mesangial cells are the major constituents in the glomeruli. However, their interdependency in glomerular injury has rarely been reported. Herein, we investigated the role of C-X-C chemokine receptor type 4 (CXCR4) in mediating the crosstalk between podocytes and mesangial cells. We found CXCR4 and angiotensin II (AngII) increased primarily in injured podocytes. However, type-1 receptor of angiotensin II (AT1) and stromal cell-derived factor 1α (SDF-1α), a ligand of CXCR4, were evidently upregulated in mesangial cells following the progression of podocyte injury. Ectopic expression of CXCR4 in 5/6 nephrectomy mice increased the decline of renal function and glomerular injury, accelerated podocyte injury and mesangial cell activation, and initiated CXCR4-AT1 axis signals. Additionally, treatment with losartan, an AT1 blocker, interrupted the cycle of podocyte injury and mesangial matrix deposition triggered by CXCR4. Podocyte-specific ablation of CXCR4 gene blocked podocyte injury and mesangial cell activation. In vitro, CXCR4 overexpression induced oxidative stress and renin angiotensin system (RAS) activation in podocytes, and triggered the communication between podocytes and mesangial cells. In cultured mesangial cells, AngII treatment induced the expression of SDF-1α, which was secreted into the supernatant to further promote oxidative stress and cell injury in podocytes. Collectively, these results demonstrate that the CXCR4-AT1 axis plays a vital role in glomerular injury via mediating pathologic crosstalk between podocytes and mesangial cells. Our findings uncover a novel pathogenic mechanism by which the CXCR4-AT1 axis promotes glomerular injury.

Utility of mutation-specific immunohistochemical markers for predicting post-induction remission among acute myeloid leukemia patients – A prognostic test accuracy study

Objectives: Various prognostically important genetic mutations are associated with acute myeloid leukemia (AML). Studies have found correlation between these mutations and expression of certain abnormal proteins in the tumor cells by immunohistochemistry (IHC). Common genetic mutations are nucleophosmin 1 (NPM1) and FMS like tyrosine kinase 3 (FLT3). This study aimed at studying the prognostic utility of surrogate IHC for these mutations-NPM1 IHC for NPM1 mutation, whereas C-X-C Chemokine Receptor type 4 (CXCR4) and Cluster of Differentiation 123 (CD123) IHC for FLT3 mutation in AML patients. Material and Methods: This was a prognostic test accuracy study done in a tertiary care centre over a period of two years (2018–2020) under two subgroups: who attained remission (remission group) and who failed to achieve remission (not in remission group) after induction therapy. Prognostic IHC markers were performed on the diagnostic bone marrow biopsy. Results: There were 70 cases in remission and 49 cases not in remission with median age of 32 and 31 years, respectively. Median total leucocyte count was significantly more in remission group (P = 0.02). AML subtype and cytogenetics wise, remission group, had significantly more M3 and M4 subtypes and translocations, while not in remission group had more M2 and M1 and more of normal and complex cytogenetics (P = 0.01 and 0.03, respectively). NPM1 and FLT3 mutation did not show significant association with remission status. IHC for NPM1, CXCR4, and CD123 was performed in the diagnostic bone marrow biopsy. Loss of nuclear localization of NPM1 and CXCR4 positivity by IHC was more in remission than not in remission (34.3% vs. 28.6% and 54.3% vs. 44.9%, respectively) which was not statistically significant. The expression of NPM1, CXCR4, and CD123 IHC had low sensitivity (34%, 54%, and 4.3%, respectively) to predict remission status. NPM1 IHC was highly significantly associated with NPM1 mutation and had high sensitivity (89%) and specificity (86%) to predict NPM1 mutation whereas CXCR4 and CD123 had low sensitivity, specificity to predict FLT3 mutation. Conclusion: NPM1 IHC can be used as a surrogate to predict NPM1 mutation whereas CXCR4 and CD123 are not effective surrogates to predict FLT3 mutation.

HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple‑negative breast cancer metastasis via the inhibition of CXCR4 expression.

Triple‑negative breast cancer (TNBC), a highly metastatic subtype of breast cancer, and it has the worst prognosis among all subtypes of breast cancer. However, no effective systematic therapy is currently available for TNBC metastasis. Therefore, novel therapies targeting the key molecular mechanisms involved in TNBC metastasis are required. The present study examined whether the expression levels of human epidermal growth factor receptor 3 (HER3) were associated with the metastatic phenotype of TNBC, and evaluated the potential of HER3 as a therapeutic target invitro and invivo. A new highly metastatic 4T1 TNBC cell line, termed 4T1‑L8, was established. The protein expression levels in 4T1‑L8 cells were measured using luminex magnetic bead assays and western blot analysis. The HER3 expression levels and distant metastasis‑free survival (DMFS) in TNBC were analyzed using Kaplan‑Meier Plotter. Transwell migration and invasion assays were performed to detect migration and invasion. The anti‑metastatic effects were determined in an experimental mouse model of metastasis. The results revealed that the increased expression of the HER3/Akt/mTOR pathway was associated with a greater level of cell migration, invasion and metastasis of TNBC cells. In addition, it was found that high expression levels of HER3 were associated with a poor DMFS. The inhibition of the HER3/Akt/mammalian target of rapamycin (mTOR) pathway decreased the migration, invasion and metastasis of TNBC cells by decreasing the expression of C‑X‑C chemokine receptor type 4 (CXCR4). Furthermore, treatment of metastatic TNBC cells with everolimus inhibited their migration, invasion and metastasis by decreasing CXCR4 expression. Thus, targeting the HER3/Akt/mTOR pathway opens up a new avenue for the development of therapeutics against TNBC metastasis; in addition, everolimus may prove to be an effective therapeutic agent for the suppression of TNBC metastasis.

Umbilical cord mesenchymal stem cells overexpressing CXCR7 facilitate treatment of ARDS-associated pulmonary fibrosis via inhibition of Notch/Jag1 mediated by the Wnt/β-catenin pathway

The therapeutic efficacy of umbilical cord mesenchymal stem cells (UCMSCs) in acute respiratory distress syndrome (ARDS) is mainly limited by the efficiency of homing of UCMSCs toward tissue damage. C-X-C chemokine receptor type 7 (CXCR7), which is involved in the mobilization of UCMSCs, is only expressed on the surface of a small proportion of UCMSCs. This study examined whether overexpression of CXCR7 in UCMSCs (UCMSCsOE-CXCR7) could improve their homing efficiency, and therefore, improve their effectiveness in fibrosis repair at the site of lung injury caused by ARDS. A lentiviral vector expressing CXCR7 was built and then transfect into UCMSCs. The impacts of CXCR7 expression of the proliferationand homing of UCMSCs were examined in a lipopolysaccharide-induced ARDS mouse model. The potential role and underlying mechanism of CXCR7 were examined by performing scratch assays, transwell assays, and immunoassays. The therapeutic dose and treatment time of UCMSCsOE-CXCR7 were directly proportional to their therapeutic effect on lung injury. In addition, overexpression of CXCR7 increased SDF-1-induced proliferation and migration of lung epithelial cells (Base-2b cells), and upregulation of CXCR7 inhibited α-SMA expression, suggesting that CXCR7 may have a role in alleviating pulmonary fibrosis caused by ARDS. Overexpression of CXCR7 in UCMSCs may improve their therapeutic effect of acute lung injury mouse, The mechanism of fibrosis repair by CXCR7 is inhibition of Jag1 via suppression of the Wnt/β-catenin pathway under the chemotaxis of SDF-1.

The N-terminus of CXCR4 splice variants determines expression and functional properties.

C-X-C motif chemokine ligand 12(CXCL12) is an essential chemokine for organ development and homeostasis in multiple tissues. Its receptor, C-X-C chemokine receptor type 4(CXCR4), is expressed on the surface of target cells. The chemokine and receptor are expressed almost ubiquitously in human tissues and cells throughout life, and abnormal expression of CXCL12 and CXCR4 is observed in pathological conditions, such as inflammation and cancer. CXCR4 is reportedly translated into five splicing variants of different lengths, which each have different amino acids in the N-terminus. As the N-terminus is the first recognition site for chemokines, CXCR4 variants may respond differently to CXCL12. Despite these differences, the molecular and functional properties of CXCR4 variants have not been thoroughly described or compared. Here, we explored the expression of CXCR4 variants in cell lines and analyzed their roles in cellular responses using biochemical approaches. RT-PCR revealed that most cell lines express more than one CXCR4 variant. When expressed in HEK293 cells, the CXCR4 variants differed in protein expression efficiency and cell surface localization. Although variant 2 demonstrated the strongest expression and cell surface localization, variants 1, 3, and 5 also mediated chemokine signaling and induced cellular responses. Our results demonstrate that the N-terminal sequences of each CXCR4 variant determine the expression of the receptor and affect ligand recognition. Functional analyses revealed that CXCR4 variants may also affect each other or interact during CXCL12-stimulated cellular responses. Altogether, our results suggest that CXCR4 variants may have distinct functional roles that warrant additional investigation and could contribute to future development of novel drug interventions.

Abstract 5854: Suppression of phosphorylated focal adhesion kinase+ (pFAK+) fibroblasts and C-X-C chemokine receptor type 4 (CXCR4) expressing granulocytes is associated with exceptional clinical outcomes in pancreatic ductal adenocarcinoma (PDAC)

Abstract Background: PDAC is an aggressive and treatment-refractory cancer due to its desmoplastic and immunosuppressive tumor microenvironment (TME). Overexpression of pFAK, a master regulator of the TME, and CXCR4, a receptor involved immunosuppression and tumor progression, are associated with poorer outcomes in PDAC. Exceptional responders, broadly defined by extended survival and durable treatment responses, represent a rare subset in PDAC. We hypothesize that the immune and stromal signatures of exceptional responders vary significantly from non-exceptional responders, specifically exhibiting decreased p-FAK and CXCR4 expression and decreased immunosuppressive myeloid phenotypes in the TME. Methods: We performed correlative quantitative analysis of multiplex immunohistochemistry (mIHC) employing 40 biomarkers on stage I/II PDAC, surgically resected formalin-fixed paraffin-embedded tissues from 9 non-exceptional responders and 11 exceptional responders. Exceptional responders were selected based on degree and duration of disease-free survival or overall survival more than double the historical matched medians. mIHC and image cytometry were used to distinguish and quantify total immune cell populations and colocalize biomarker expression across each distinct cell type. Cell populations and expression levels were compared using unpaired T-tests. Results: Exceptional responders showed significantly decreased overall pFAK expression (p=0.02) and overall CXCR4 expression (p=0.04). In exceptional responders, pFAK expression and CXCR4 expression were significantly decreased in immunosuppressive CD66b+ granulocytes (p=0.01, p=0.03, respectively) and in tumor associated macrophages (p=0.01, p=0.006, respectively). While not significant, exceptional responders demonstrated a marked decrease in immunosuppressive pFAK+ fibroblasts (p=0.12) when compared to non-exceptional responders. No change was seen in the CD8+ T cell infiltration between groups; however, a trend was observed towards an increased proportion of granzyme B+ CD3+ CD8+ cytotoxic lymphocytes (p=0.57) in exceptional responders. Conclusion: Exceptional responders selected in this preliminary study have significantly lower FAK and CXCR4 expression and markedly reduced populations of immunosuppressive FAK+ and CXCR4+ myeloid cells. Suppression of pFAK and CXCR4 in fibroblasts and granulocytes, respectively are associated with exceptional clinical outcomes. Future expanded research comparing the distinct TME and genetic signatures of exceptional responders to non-exceptional is warranted. Citation Format: John Davelaar, Anser A. Abbas, Angela Minasyan, Omer H. Elmadbouh, Natalie Moshayedi, Brent Larson, Nicholas Nissen, Simon Lo, Srinivas Gaddam, Kambiz Kosari, Jun Gong, Andrew Hendifar, Stephen Pandol, Arsen Osipov. Suppression of phosphorylated focal adhesion kinase+ (pFAK+) fibroblasts and C-X-C chemokine receptor type 4 (CXCR4) expressing granulocytes is associated with exceptional clinical outcomes in pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5854.

Enterovirus 71 induces pyroptosis of human neuroblastoma SH-SY5Y cells through miR-146a/ CXCR4 axis

Enterovirus 71 (EV71) is a predominant causative pathogen of hand-foot-and-mouth disease (HFMD) in children. Compared with other HFMD-associated viruses, EV71 tends to induce more severe neurological complications and even death. However, the detailed mechanism of EV71 causes nervous system disorder is still unclear. In this study, we found that EV71 induced the GSDMD/NLRP3-mediated pyroptosis of SH-SY5Y cells through up-regulated miR-146a. Through bioinformatic analysis, we identified C-X-C chemokine receptor type 4 (CXCR4) as the potential target of miR-146a. We noticed that the expression of CXCR4 was regulated by miR-146a during EV71 infection. Moreover, our results show that over-expression of CXCR4 attenuated EV71-induced pyroptosis of SY-SY5Y cells. These results reveal a previously unrecognized mechanism in which EV71 induces nervous system cells damage through regulating miR-146a/CXCR4 mediated pyroptosis.

KDM6B promotes gastric carcinogenesis and metastasis via upregulation of CXCR4 expression

KDM6B (Lysine-specific demethylase 6B) is a histone lysine demethyltransferase that plays a key role in many types of cancers. However, its potential role in gastric cancer (GC) remains unclear. Here, we focused on the clinical significance and potential role of KDM6B in GC. We found that the KDM6B expression is upregulated in GC tissues and that its high expression in patients is related to poor prognosis. KDM6B ectopic expression promotes GC cells’ proliferation and metastasis, while its inhibition has opposite effects in vitro and in vivo. Mechanistically, KDM6B promotes GC cells proliferation and metastasis through its enzymatic activity through the induction of H3K27me3 demethylation near the CXCR4 (C-X-C chemokine receptor type 4) promoter region, resulting in the upregulation of CXCR4 expression. Furthermore, H. pylori was found to induce KDM6B expression. In conclusion, our results suggest that KDM6B is aberrantly expressed in GC and plays a key role in gastric carcinogenesis and metastasis through CXCR4 upregulation. Our work also suggests that KDM6B may be a potential oncogenic factor and a therapeutic target for GC.

Comprehensive analysis of the immunological implication and prognostic value of CXCR4 in non-small cell lung cancer

CXCR4 (C-X-C chemokine receptor type 4) is the most commonly expressed of all chemokine receptors in malignant tumors. However, studies on CXCR4 in non-small cell lung cancer (NSCLC) tumor immune microenvironment, including those determining its immune efficacy and prognostic potential, are still scarce. Therefore, in this study, we determined the ability of CXCR4 to predict immunotherapy response and prognosis in NSCLC using immunohistochemical staining and RT-PCR, respectively, in two independent cohorts from the National Cancer Center of China. We analyzed transcriptome sequencing data and clinical information from multiple public databases to assess immune cell infiltration in NSCLC and constructed immune risk prognostic signatures based on CXCR4-related immunomodulators. We found that immune cell infiltration is significant differences in NSCLC tissues and is moderately correlated with CXCR4 expression. High CXCR4 expression was significantly associated with poor prognosis in NSCLC patients and a higher response rate to immunotherapy. The ROC curve showed that CXCR4 expression exhibited excellent performance in predicting the efficacy of immunotherapy in NSCLC. We identified 30 CXCR4-related immunomodulators in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) and constructed immune prognostic signatures based on CXCR4-related immunomodulators and CXCR4-related mutant genes. The signature-based prognostic risk score showed good performance in predicting patient prognosis in both LUAD and LUSC; high risk scores were significantly associated with poor prognosis (P < 0.0001) and was established as an independent prognostic factor by multivariate Cox regression. We postulate that CXCR4 is a potential predictive marker of immunotherapy efficacy in NSCLC and should be used in clinical settings. Moreover, the constructed signatures may be valuable in predicting patient prognosis in NSCLC.