Contribution of Heparan Sulphate Binding in CCL21-Mediated Migration of Breast Cancer Cells

Chemokine receptor CCR7 is implicated in the metastasis of breast cancer to the lymph nodes. Chemokine function is dependent upon their binding to both cell-surface heparan sulphate (HS) and to their specific receptors; thus, the role of HS in CCR7-mediated lymph node metastasis was investigated by creating a non-HS binding chemokine CCL21 (mut-CCL21). Mut-CCL21 (Δ103–134) induced leukocyte chemotaxis in diffusion gradients but did not stimulate trans-endothelial migration of PBMCs (p < 0.001) and 4T1-Luc cells (p < 0.01). Furthermore, the effect of heparin and HS on the chemotactic properties of wild-type (WT) and mut-CCL21 was examined. Interestingly, heparin and HS completely inhibit the chemotaxis mediated by WT-CCL21 at 250 and 500 µg/mL, whereas minimal effect was seen with mut-CCL21. This difference could potentially be attributed to reduced HS binding, as surface plasmon resonance spectroscopy showed that mut-CCL21 did not significantly bind HS compared to WT-CCL21. A murine model was used to assess the potential of mut-CCL21 to prevent lymph node metastasis in vivo. Mice were injected with 4T1-Luc cells in the mammary fat pad and treated daily for a week with 20 µg mut-CCL21. Mice were imaged weekly with IVIS and sacrificed on day 18. Luciferase expression was significantly reduced in lymph nodes from mice that had been treated with mut-CCL21 compared to the control (p = 0.0148), suggesting the potential to target chemokine binding to HS as a therapeutic option

a The role of CXCR4, CXCR7 and CCR7 in breast cancer metastatis

PhD ThesisBreast cancer (BrCa) metastasis is a process mediated by the expression of chemokine receptors by BrCa cells, which migrate towards their chemokine ligands presented by glycosaminoglycans (GAGs) in distant organs. Although this metastatic spread is the cause of most BrCa deaths, there are no effective strategies to target this process. This study was designed to investigate whether BrCa cell migration can be controlled by cross-desensitisation of chemokine receptors or by use of non-GAG binding chemokines. Initial experiments showed that the chemokine receptors CXCR4, CXCR7 and CCR7 were upregulated in primary BrCa and that CXCR4 and CXCR7 could form heterodimers in transfected CHO cells. This co-expression modified CXCR4’s potential to activate Akt but did not affect ERK phosphorylation. To assess this signalling disparity, receptor internalisation was assessed. It was found that CXCR7 was recycled to the surface whilst CXCR4 was degraded, a process that could be partially inhibited with a proteasome inhibitor. Internalisation was also assessed using the CXCR7 agonist VUF11207, which caused both CXCR4 and CXCR7 to be degraded after internalization, highlighting its potential as a dual targeting drug. It was also found that only CXCR4 played a role in metastasis by promoting calcium flux, and CXCR7 co-expression did not significantly reduce CXCR4-mediated cellular migration. The role of GAGs in CCR7-mediated lymph node metastasis was investigated by creating a non-GAG binding CCL21 (mutCCL21). In vitro, mutCCL21 bound CCR7 and triggered diffusion gradient chemotaxis, but failed to induce transendothelial cell migration. This was recapitulated in a murine model of BrCa, where daily injections of mutCCL21 significantly reduced lymph node metastases, highlighting the therapy potential of disrupting GAG-chemokine interactions In summary, these findings suggest that each chemokine receptor has a different but vital role in BrCa metastasis, and should be considered as targets for future therapies.Woman’s Cancer Detection Society and the NHS Trust from the Queen Elizabeth hospital in Gateshea

Breast Cancer: An Examination of the Potential of ACKR3 to Modify the Response of CXCR4 to CXCL12

Upon binding with the chemokine CXCL12, the chemokine receptor CXCR4 has been shown to promote breast cancer progression. This process, however, can be affected by the expression of the atypical chemokine receptor ACKR3. Given ACKR3’s ability to form heterodimers with CXCR4, we investigated how dual expression of both receptors differed from their lone expression in terms of their signalling pathways. We created single and double CXCR4 and/or ACKR3 Chinese hamster ovary (CHO) cell transfectants. ERK and Akt phosphorylation after CXCL12 stimulation was assessed and correlated with receptor internalization. Functional consequences in cell migration and proliferation were determined through wound healing assays and calcium flux. Initial experiments showed that CXCR4 and ACKR3 were upregulated in primary breast cancer and that CXCR4 and ACKR3 could form heterodimers in transfected CHO cells. This co-expression modified CXCR4’s Akt activation after CXCL12’s stimulation but not ERK phosphorylation (p < 0.05). To assess this signalling disparity, receptor internalization was assessed and it was observed that ACKR3 was recycled to the surface whilst CXCR4 was degraded (p < 0.01), a process that could be partially inhibited with a proteasome inhibitor (p < 0.01). Internalization was also assessed with the ACKR3 agonist VUF11207, which caused both CXCR4 and ACKR3 to be degraded after internalization (p < 0.05 and p < 0.001), highlighting its potential as a dual targeting drug. Interestingly, we observed that CXCR4 but not ACKR3, activated calcium flux after CXCL12 stimulation (p < 0.05) and its co-expression could increase cellular migration (p < 0.01). These findings suggest that both receptors can signal through ERK and Akt pathways but co-expression can alter their kinetics and internalization pathways

Contribution of Heparan Sulphate Binding in CCL21-Mediated Migration of Breast Cancer Cells

SIMPLE SUMMARY: Breast cancer is a leading cause of cancer-related deaths worldwide, predominantly caused by metastasis. Chemokine receptor CCR7 and its ligand CCL21 are implicated in the metastasis of breast cancer to the lymph nodes. Chemokine function is dependent upon binding to their specific chemokine receptors and negatively charged molecules on the cell surface (heparan sulphate). The role of heparan sulphate in CCR7-mediated lymph node metastasis was investigated by creating a non-heparan sulphate binding mutant chemokine CCL21. Mutant-CCL21 was tested in vitro in a range of assays, including cell migration, calcium flux and surface plasmon resonance spectroscopy. Mutant-CCL21 induced leukocyte chemotaxis in diffusion gradients but did not stimulate trans-endothelial migration of breast cancer cells. A murine model was used to assess the potential of mutant-CCL21 to prevent lymph node metastasis in vivo. Lymph node metastasis was significantly reduced by the administration of mutant-CCL21 compared to the control. Targeting chemokine–heparan sulphate interactions may be a promising approach to inhibit chemokine activity and metastasis. ABSTRACT: Chemokine receptor CCR7 is implicated in the metastasis of breast cancer to the lymph nodes. Chemokine function is dependent upon their binding to both cell-surface heparan sulphate (HS) and to their specific receptors; thus, the role of HS in CCR7-mediated lymph node metastasis was investigated by creating a non-HS binding chemokine CCL21 (mut-CCL21). Mut-CCL21 (Δ103–134) induced leukocyte chemotaxis in diffusion gradients but did not stimulate trans-endothelial migration of PBMCs (p < 0.001) and 4T1-Luc cells (p < 0.01). Furthermore, the effect of heparin and HS on the chemotactic properties of wild-type (WT) and mut-CCL21 was examined. Interestingly, heparin and HS completely inhibit the chemotaxis mediated by WT-CCL21 at 250 and 500 µg/mL, whereas minimal effect was seen with mut-CCL21. This difference could potentially be attributed to reduced HS binding, as surface plasmon resonance spectroscopy showed that mut-CCL21 did not significantly bind HS compared to WT-CCL21. A murine model was used to assess the potential of mut-CCL21 to prevent lymph node metastasis in vivo. Mice were injected with 4T1-Luc cells in the mammary fat pad and treated daily for a week with 20 µg mut-CCL21. Mice were imaged weekly with IVIS and sacrificed on day 18. Luciferase expression was significantly reduced in lymph nodes from mice that had been treated with mut-CCL21 compared to the control (p = 0.0148), suggesting the potential to target chemokine binding to HS as a therapeutic option

Global patterns of antigen receptor repertoire disruption across adaptive immune compartments in COVID-19

Whereas pathogen-specific T and B cells are a primary focus of interest during infectious disease, we have used COVID-19 to ask whether their emergence comes at a cost of broader B cell and T cell repertoire disruption. We applied a genomic DNA-based approach to concurrently study the immunoglobulin-heavy (IGH) and T cell receptor (TCR) β and δ chain loci of 95 individuals. Our approach detected anticipated repertoire focusing for the IGH repertoire, including expansions of clusters of related sequences temporally aligned with SARS-CoV-2–specific seroconversion, and enrichment of some shared SARS-CoV-2–associated sequences. No significant age-related or disease severity–related deficiencies were noted for the IGH repertoire. By contrast, whereas focusing occurred at the TCRβ and TCRδ loci, including some TCRβ sequence–sharing, disruptive repertoire narrowing was almost entirely limited to many patients aged older than 50 y. By temporarily reducing T cell diversity and by risking expansions of nonbeneficial T cells, these traits may constitute an age-related risk factor for COVID-19, including a vulnerability to new variants for which T cells may provide key protection

Acute immune signatures and their legacies in severe acute respiratory syndrome coronavirus-2 infected cancer patients

Given the immune system's importance for cancer surveillance and treatment, we have investigated how it may be affected by SARS-CoV-2 infection of cancer patients. Across some heterogeneity in tumor type, stage, and treatment, virus-exposed solid cancer patients display a dominant impact of SARS-CoV-2, apparent from the resemblance of their immune signatures to those for COVID-19+ non-cancer patients. This is not the case for hematological malignancies, with virus-exposed patients collectively displaying heterogeneous humoral responses, an exhausted T cell phenotype and a high prevalence of prolonged virus shedding. Furthermore, while recovered solid cancer patients' immunophenotypes resemble those of non-virus-exposed cancer patients, recovered hematological cancer patients display distinct, lingering immunological legacies. Thus, while solid cancer patients, including those with advanced disease, seem no more at risk of SARS-CoV-2-associated immune dysregulation than the general population, hematological cancer patients show complex immunological consequences of SARS-CoV-2 exposure that might usefully inform their care

Pseudomonas aeruginosa antibiotic susceptibility profiles, genomic epidemiology and resistance mechanisms: a nation-wide five-year time lapse analysisResearch in context

Summary: Background: Pseudomonas aeruginosa healthcare-associated infections are one of the top antimicrobial resistance threats world-wide. In order to analyze the current trends, we performed a Spanish nation-wide high-resolution analysis of the susceptibility profiles, the genomic epidemiology and the resistome of P. aeruginosa over a five-year time lapse. Methods: A total of 3.180 nonduplicated P. aeruginosa clinical isolates from two Spanish nation-wide surveys performed in October 2017 and 2022 were analyzed. MICs of 13 antipseudomonals were determined by ISO-EUCAST. Multidrug resistance (MDR)/extensively drug resistance (XDR)/difficult to treat resistance (DTR)/pandrug resistance (PDR) profiles were defined following established criteria. All XDR/DTR isolates were subjected to whole genome sequencing (WGS). Findings: A decrease in resistance to all tested antibiotics, including older and newer antimicrobials, was observed in 2022 vs 2017. Likewise, a major reduction of XDR (15.2% vs 5.9%) and DTR (4.2 vs 2.1%) profiles was evidenced, and even more patent among ICU isolates [XDR (26.0% vs 6.0%) and DTR (8.9% vs 2.6%)] (p < 0.001). The prevalence of Extended-spectrum β-lactamase/carbapenemase production was slightly lower in 2022 (2.1%. vs 3.1%, p = 0.064). However, there was a significant increase in the proportion of carbapenemase production among carbapenem-resistant strains (29.4% vs 18.1%, p = 0.0246). While ST175 was still the most frequent clone among XDR, a slight reduction in its prevalence was noted (35.9% vs 45.5%, p = 0.106) as opposed to ST235 which increased significantly (24.3% vs 12.3%, p = 0.0062). Interpretation: While the generalized decrease in P. aeruginosa resistance, linked to a major reduction in the prevalence of XDR strains, is encouraging, the negative counterpart is the increase in the proportion of XDR strains producing carbapenemases, associated to the significant advance of the concerning world-wide disseminated hypervirulent high-risk clone ST235. Continued high-resolution surveillance, integrating phenotypic and genomic data, is necessary for understanding resistance trends and analyzing the impact of national plans on antimicrobial resistance. Funding: MSD and the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea—NextGenerationEU