Chemokine Receptor Inhibitors Research Articles

SATB1 prevents immune cell infiltration by regulating chromatin organization and gene expression of a chemokine gene cluster in T cells

SATB1, a key regulator of T cell development, governs lineage-specific transcriptional programs upon T cell activation. The absence of SATB1 has been linked to the initiation and progression of autoimmunity. However, its precise roles in this process remain incompletely understood. Here we show that conditional knockout of Satb1 in CD4+ T cells in mice led to T cell hyperactivation and inflammatory cell infiltration across multiple organs. Transcriptional profiling on activated T cells revealed that the loss of SATB1 led to aberrant upregulation of CC chemokines. Treating Satb1 conditional knockout mice with CC chemokine receptor inhibitor alleviated inflammatory cell infiltration. Intriguingly, SATB1’s transcriptional regulation of chemokine genes could not be attributed to its direct binding to chemokine promoters. Instead, SATB1 exerted its regulatory effects by controlling higher-order chromatin organization at a CC chemokine locus. The loss of SATB1 led to the emergence of a new chromatin domain encompassing the Ccl3, Ccl4, Ccl5, Ccl6, and Ccl9 genes and a distal enhancer, resulting in increased contacts between the enhancer and all five chemokine genes, thus inducing their upregulation. Collectively, these results demonstrate that SATB1 protects organs from immune cell infiltration by regulating chemokine expression, providing valuable insights into the development of autoimmunity-related phenotypes.

New Emerging Chemokine Receptors: CCR5 or CXCR5 on Tumor Is Associated with Poor Response to Chemotherapy and Poor Prognosis in Locally Advanced Triple-Negative Breast Cancer.

We aim to investigate any possible associations between chemokine receptor expression and responses to neoadjuvant chemotherapy (NAC) along with outcomes in patients with triple-negative breast cancer (TNBC) with locally advanced disease. Expressions of chemokine receptors were examined immunohistochemically after staining archival tissue of surgical specimens (n = 63) using specific antibodies for CCR5, CCR7, CXCR4, and CXCR5. Patients with high CCR5, CCR7, CXCR4, and CXCR5 expression on tumors and high CXCR4 expression on tumor-infiltrating lymphocytes (TILs) were less likely to have a pathological complete response (pCR) or Class 0-I RCB-Index compared to others. Patients with residual lymph node metastases (ypN-positive), high CCR5TM(tumor), and high CXCR4TM expressions had an increased hazard ratio (HR) compared to others (DFS: HR = 2.655 [1.029-6.852]; DSS: HR = 2.763 [1.008-7.574]), (DFS: HR = 2.036 [0.805-5.148]; DSS: HR = 2.689 [1.020-7.090]), and (DFS: HR = 2.908 [1.080-7.829]; DSS: HR = 2.132 (0.778-5.846)), respectively. However, patients without CXCR5TIL expression had an increased HR compared to those with CXCR5TIL (DFS: 2.838 [1.266-6.362]; DSS: 4.211 [1.770-10.016]). High expression of CXCR4TM and CCR5TM was found to be associated with poor prognosis, and CXCR5TM was associated with poor chemotherapy response in the present cohort with locally advanced TNBC. Our results suggest that patients with TNBC could benefit from a chemokine receptor inhibitor therapy containing neoadjuvant chemotherapy protocols.

Effect of AFPGC immune-microenvironment and resistance to immunotherapy.

e16041 Background: Alpha-fetoprotein producing gastric cancer (AFPGC) is a rare subtype GC with high with high morbidity and mortality rates. Even though immunotherapy has become the cornerstone of first-line treatment, our previous results suggest that AFPGC was resistant to immunotherapy, standard regimen remains unclear. It is warranted to explore precision immunotherapy strategies for AFPGC. Methods: A retrospective cohort of 98 patients with gastric cancer receiving mono-immunotherapy was included. Kaplan-Meier survival plots for PFS and OS were generated based on the AFP levels. Log-rank tests were used to compare the survival curves. Chi-square test was used to analyze the differences in the efficacy of immunotherapy between patients with different levels of AFP. AFPGC in vivo models were constructed, and after in vivo administration, RNA-seq, flow cytometry, and Western blot were used to explore and validate the features of the AFPGC microenvironment and explore immunotherapy combination strategies. Results: Retrospectively analysis demonstrated that the response of mono-immunotherapy in AFPGC was inferior to that in non-AFPGC (ORR: 16.7% vs 29.3%; DCR: 33.0% vs 55.4%). The response of the subcutaneous transplant tumor model with AFP overexpression to PD-1 antibody was significantly reduced. Meanwhile, RNA-seq results from the secondary PD-1 antibody resistant model showed that the mRNA level of AFP was significantly upregulated in the resistant model. RNA-seq results from transplant tumors of AFPGC compared to nAFPGC revealed that the differentially expressed genes were significantly enriched in pathways related to neutrophil chemotaxis and angiogenesis. Flow cytometry confirmed that the proportion of neutrophils in AFPGC was significantly higher than that in nAFPGC. SB265610 (neutrophil chemokine receptor inhibitor) combined with PD-1 antibody and apatinib (anti-angiogenesis) plus PD-1 antibody were administrated in animal models. Both of the regimens could release the primary resistance to PD-1 antibody. Based on these preclinical findings, we designed and conducted a clinical study (NCT04609176) to explore the reponse of apatinib combined with PD-1 antibody in AFPGC. Primary endpoints were achieved and will be announced in the near future. Typical case is depicted as follows: A 79 years old male patients diagnosed with low-differentiated adenocarcinoma of GC, AFP: 135220 ng/ml. After fail in chemotherapy, patients were treated with third-line apatinib 250mg po d1-d21, PD-1 monoclonal antibody 200mg d1, q21d for 6 cycles. Patient was evaluated as partial response (PR) for 4 months and AFP sharply decreased to 945 ng/ml. Conclusions: AFPGC is resistant to PD-1 monoclonal antibody. Combined treatment with SB265610 or apatinib can alleviate or reverse the resistance, making it the most promising treatment regimen for AFPGC.

Platelet-derived growth factor signaling in pericytes promotes hypothalamic inflammation and obesity.

Pericytes are a vital component of the blood-brain barrier, and their involvement in acute inflammation was recently suggested. However, it remains unclear whether pericytes contribute to hypothalamic chronic inflammation and energy metabolism in obesity. The present study investigated the impact of pericytes on the pathophysiology of obesity by focusing on platelet-derived growth factor (PDGF) signaling, which regulates pericyte functions. Tamoxifen-inducible systemic conditional PDGF receptor β knockout mice (Pdgfrb∆SYS-KO) and Calcium/calmodulin-dependent protein kinase type IIa (CaMKIIa)-positive neuron-specific PDGF receptor β knockout mice (Pdgfrb∆CaMKII-KO) were fed a high-fat diet, and metabolic phenotypes before and 3 to 4weeks after dietary loading were examined. Intracellular energy metabolism and relevant signal transduction in lipopolysaccharide- and/or platelet-derived growth factor-BB (PDGF-BB)-stimulated human brain pericytes (HBPCs) were assessed by the Seahorse XFe24 Analyzer and Western blotting. The pericyte secretome in conditioned medium from HBPCs was studied using cytokine array kit, and its impact on polarization was examined in bone marrow-derived macrophages (BMDMs), which are microglia-like cells. Energy consumption increased and body weight gain decreased after high-fat diet loading in Pdgfrb∆SYS-KO mice. Cellular oncogene fos (cFos) expression increased in proopiomelanocortin (POMC) neurons, whereas microglial numbers and inflammatory gene expression decreased in the hypothalamus of Pdgfrb∆SYS-KO mice. No significant changes were observed in Pdgfrb∆CaMKII-KO mice. In HBPCs, a co-stimulation with lipopolysaccharide and PDGF-BB shifted intracellular metabolism towards glycolysis, activated mitogen-activated protein kinase (MAPK), and modulated the secretome to the inflammatory phenotype. Consequently, the secretome showed an increase in various proinflammatory chemokines and growth factors including Epithelial-derived neutrophil-activating peptide 78 (C-X-C motif chemokine ligand (CXCL)5), Thymus and activation-regulated chemokine (C-C motif chemokine (CCL)17), Monocyte chemoattractant protein 1 (CCL2), and Growth-regulated oncogene α (CXCL1). Furthermore, conditioned medium from HBPCs stimulated the inflammatory priming of BMDMs, and this change was abolished by the C-X-C motif chemokine receptor (CXCR) inhibitor. Consistently, mRNA expression of CXCL5 was elevated by lipopolysaccharide and PDGF-BB treatment in HBPCs, and the expression was significantly lower in the hypothalamus of Pdgfrb∆SYS-KO mice than in control Pdgfrbflox/flox mice (FL) following 4 weeks of HFD feeding. PDGF receptor β signaling in hypothalamic pericytes promotes polarization of macrophages by changing their secretome and contributes to the progression of obesity.

Chemokine receptors in COVID-19 infection.

Chemokine receptors play diverse roles in the immune response against pathogens by recruiting innate and adaptive immune cells to sites of infection. However, their involvement could also be detrimental, causing tissue damage and exacerbating respiratory diseases by triggering histological alterations such as fibrosis and remodeling. This chapter reviews the role of chemokine receptors in the immune defense against SARS-CoV-2 infection. In COVID-19, CXCR3 is expressed mainly in T cells, and its upregulation is related to an increase in SARS-CoV-2-specific antibodies but also to COVID-19 severity. CCR5 is a key player in T-cell recruitment, and its suppression leads to reduced inflammation and viremia levels. Conversely, CXCR6 is implicated in the aberrant migration of memory T cells within airways. On the other hand, increased CCR4+ cells in the blood and decreased CCR4+ cells in lung cells are associated with severe COVID-19. Additionally, CCR2 is associated with an increase in macrophage recruitment to lung tissues. Elevated levels of CXCR1 and CXCR2, which are predominantly expressed in neutrophils, are associated with the severity of the disease, and finally, the expression of CX3CR1 in cytotoxic T lymphocytes affects the retention of these cells in lung tissues, thereby impacting the severity of COVID-19. Despite the efforts of many clinical trials to find effective therapies for COVID-19 using chemokine receptor inhibitors, no conclusive results have been found due to the small number of patients, redundancy, and co-expression of chemokine receptors by immune cells, which explains the difficulty in finding a single therapeutic target or effective treatment.

The Role of TAMs in the Regulation of Tumor Cell Resistance to Chemotherapy.

Tumor-associated macrophages (TAMs) are the predominant cell infiltrate in the immunosuppressive tumor microenvironment (TME). TAMs are central to fostering pro-inflammatory conditions, tumor growth, metastasis, and inhibiting therapy responses. Many cancer patients are innately refractory to chemotherapy and or develop resistance following initial treatments. There is a clinical correlation between the level of TAMs in the TME and chemoresistance. Hence, the pivotal role of TAMs in contributing to chemoresistance has garnered significant attention toward targeting TAMs to reverse this resistance. A prerequisite for such an approach requires a thorough understanding of the various underlying mechanisms by which TAMs inhibit response to chemotherapeutic drugs. Such mechanisms include enhancing drug efflux, regulating drug metabolism and detoxification, supporting cancer stem cell (CSCs) resistance, promoting epithelial-mesenchymal transition (EMT), inhibiting drug penetration and its metabolism, stimulating angiogenesis, impacting inhibitory STAT3/NF-κB survival pathways, and releasing specific inhibitory cytokines including TGF-β and IL-10. Accordingly, several strategies have been developed to overcome TAM-modulated chemoresistance. These include novel therapies that aim to deplete TAMs, repolarize them toward the anti-tumor M1-like phenotype, or block recruitment of monocytes into the TME. Current results from TAM-targeted treatments have been unimpressive; however, the use of TAM-targeted therapies in combination appears promising These include targeting TAMs with radiotherapy, chemotherapy, chemokine receptor inhibitors, immunotherapy, and loaded nanoparticles. The clinical limitations of these strategies are discussed.

Development of tolerance to chemokine receptor antagonists: current paradigms and the need for further investigation.

Chemokine G-protein coupled receptors are validated drug targets for many diseases, including cancer, neurological, and inflammatory disorders. Despite much time and effort spent on therapeutic development, very few chemokine receptor antagonists are approved for clinical use. Among potential reasons for the slow progress in developing chemokine receptor inhibitors, antagonist tolerance, a progressive reduction in drug efficacy after repeated administration, is likely to play a key role. The mechanisms leading to antagonist tolerance remain poorly understood. In many cases, antagonist tolerance is accompanied by increased receptor concentration on the cell surface after prolonged exposure to chemokine receptor antagonists. This points to a possible role of altered receptor internalization and presentation on the cell surface, as has been shown for agonist (primarily opioid) tolerance. In addition, examples of antagonist tolerance in the context of other G-protein coupled receptors suggest the involvement of noncanonical signal transduction in opposing the effects of the antagonists. In this review, we summarize the available progress and challenges in therapeutic development of chemokine receptor antagonists, describe the available knowledge about antagonist tolerance, and propose new avenues for future investigation of this important phenomenon. Furthermore, we highlight the modern methodologies that have the potential to reveal novel mechanisms leading to antagonist tolerance and to propel the field forward by advancing the development of potent "tolerance-free" antagonists of chemokine receptors.

Abstract 3618: The role of endothelial ACKR1 in triple-negative breast cancer metastasis

Abstract Metastasis is the most common cause of breast cancer mortality. Of the breast cancer subtypes, triple-negative breast cancer (TNBC) is the deadliest due to its increased likelihood to metastasize. Tumor cell extravasation is a critical step of metastasis and allows circulating tumor cells to exit the vasculature and seed distant tissues. Clear understanding of the major regulators of tumor cell extravasation will provide insights into the progression of TNBC metastasis. One potential regulator of TNBC cell extravasation is ACKR1. In many contexts, ACKR1 expression is required in endothelial cells (EC) for leukocyte extravasation. Endothelial ACKR1 binds CXCL2, a promigratory chemokine, and localizes it to EC junctions to guide neutrophils through leukocyte extravasation. CXCL2 expression in TNBC cells is also necessary for tumor cell extravasation from lung microvasculature and for tumor metastasis. Our preliminary data show that ACKR1 is required in at least one stromal cell type for TNBC metastasis from the primary tumor to the lung. These data suggest that endothelial ACKR1-CXCL2 interactions may mediate tumor cell extravasation in TNBC metastasis. Therefore, we hypothesize that endothelial ACKR1 promotes metastasis via retention of CXCL2 at EC junctions, increasing tumor cell chemotaxis and extravasation. To address this hypothesis, we will examine the in vivo significance of endothelial ACKR1 expression using our validated ACKR1 endothelial cell-specific knockout mouse model. We will test the requirement for endothelial ACKR1 for metastasis of orthotopically implanted TNBC tumors to distant sites in the lung and for extravasation of circulating tumor cells into lung tissue. We will determine which steps of extravasation require ACKR1 by evaluating ACKR1-low and ACKR1-overexpressing ECs using an Ibidi flow co-culture system that recapitulates the shear stress conditions of pulmonary microvasculature. We will examine whether these steps are dependent on CXCL2 by introducing CXCL2-neutralizing antibodies to the Ibidi flow system and observing their effects on each extravasation step. Our proposed studies will establish the role of endothelial ACKR1 in TNBC metastatic progression and determine the specific steps of tumor cell extravasation in which endothelial ACKR1 and CXCL2 function. Understanding these processes may guide development of ACKR1 as a prognostic marker for metastasis and can provide mechanistic insight into candidate chemokine and chemokine receptor inhibitors under evaluation for treatment of breast cancer. Citation Format: Samuel Tanner Roach, Chinwe Ewenighi-Amankwah, J Dufraine, L.A. Naiche, Jan K. Kitajewski. The role of endothelial ACKR1 in triple-negative breast cancer metastasis. [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 3618.

Allosteric inhibition of CXCR1/2 by an orally bioavailable chemokine receptor inhibitor attenuates allergic lung inflammation in mice

CXCR1/2 chemokine receptors are expressed on the surface of inflammatory cells. We previously reported that parenterally-administered reparixin, a noncompetitive allosteric inhibitor of CXCR1 and CXCR2, inhibits allergic airway inflammation. Ladarixin is a second generation orally bioavailable dual CXCR1/2 inhibitor that binds an allosteric pocket of the trans-membrane region of both receptors with a 100-fold higher affinity than first generation CXCR1/2 inhibitors. Here we investigated whether oral ladarixin also inhibits allergic airway inflammation.

Cenicriviroc for the treatment of COVID-19: first interim results of a randomised, placebo-controlled, investigator-initiated, double-blind phase II trial

ObjectivesC-C-chemokine receptors (CCRs) are expressed on a variety of immune cells and play an important role in many immune processes, particularly leukocyte migration. Comprehensive preclinical research demonstrated CCR2/CCR5-dependent pathways as pivotal for the pathophysiology of severe COVID-19. Here we report human data on use of a chemokine receptor inhibitor in patients with COVID-19. MethodsInterim results of a 2:1 randomised, placebo-controlled, investigator-initiated trial on the CCR2/CCR5-inhibitor Cenicriviroc (CVC) 150 mg BID orally for 28 d in hospitalised patients with moderate to severe COVID-19 are reported. The primary endpoint is the subject's responder status defined by achieving grade 1 or 2 on the 7-point ordinal scale of clinical improvement on day 15. ResultsOf the 30 patients randomised, 18 were assigned to receive CVC and 12 to placebo. Efficient CCR2- and CCR5 inhibition was demonstrated through CCL2 and CCL4 elevation in CVC-treated patients (485% and 80% increase on day 3 compared to the baseline, respectively). In the modified intention-to-treat population, 82.4% of patients (14/17) in the CVC group met the primary endpoint, as did 91.7% (11/12) in the placebo group (OR = 0.5, 95% CI = 0.04–3.41). One patient treated with CVC died of progressive acute respiratory distress syndrome, and the remaining had a favourable outcome. Overall, treatment with CVC was well tolerated, with most adverse events being grade I or II and resolving spontaneously. ConclusionsOur interim analysis provides proof-of-concept data on CVC for COVID-19 patients as an intervention to inhibit CCR2/CCR5. Further studies are warranted to assess its clinical efficacy.

CCR1 and CCR5 mediate cancer-induced myelopoiesis and differentiation of myeloid cells in the tumor

BackgroundCancer-induced ‘emergency’ myelopoiesis plays a key role in tumor progression by inducing the accumulation of myeloid cells with a suppressive phenotype peripherally and in the tumor. Chemokine receptors (CCRs) and,...

Prognostic significance of immunohistochemical expression of chemokine receptor (CXCR4) and RAF kinase inhibitor protein (RKIP) in gastric carcinoma and premalignant lesions of the stomach

Background: CXCR4 and RKIP have been implicated in initiation and progression in many cancers, but their role in gastric adenocarcinoma remains vague.Aim: The aim of this work is to assess the possible significance of both markers in gastric adenocarcinoma and premalignant lesions of the stomach.Methods: This retrospective study was carried upon 50 cases of gastric adenocarcinoma and 20 of premalignant lesions. Immunohistochemistry was performed to examine the expression of CXCR4 and RKIP in both gastric adenocarcinoma and premalignant lesions of the stomach.Results: CXCR4 was found to be highly expressed in gastric adenocarcinoma compared to premalignant lesions (P value=0.027). It was significantly correlated with tumor grade (P value=0.027), depth of tumor invasion (P value=0.019), lymph node metastasis (P value=0.025), distant metastasis (P value=0.044) and TNM stage (P value=0.002). No statistically significant correlation between CXCR4 expression and histopathological types (P value=0.8). RKIP expression has no significant correlation with progression from premalignant gastric lesions to adenocarcinoma (P value=0.078). RKIP was negatively associated with advanced tumor grade (P value=0.001), depth of invasion (P value=0.018), distant metastasis (P value=0.029) and TNM stage (P value=0.007). No statistically significant correlation between RKIP expression and histopathological types (P value=0.17) and lymph node status (P value=0.053). No significant correlation between CXCR4 and RKIP expression in studied cases was detected (P value=0.178).Conclusions: These results suggested that CXCR4 might be involved in gastric carcinogenesis.Keyword: Gastric adenocarcinoma, premalignant, CXCR4, RKIP.

Enhancing tumor infiltration

Targeting a chemokine receptor inhibitor enhances the trafficking of antitumor T cells into tumors.

Safety and Efficacy Trial of Escalation of Plerixafor for Mobilization of CD34+ Hematopoietic Progenitor Cells (HPCs) for Globin Gene Transfer in Patients with Sickle Cell Disease

▪There is presently no curative therapy for SCD other than allogeneic hematopoietic progenitor cell (HPC) transplantation. Incoming trials of autologous gene therapy approaches in patients with sickle cell disease (SCD) include the need of HPCs for gene transduction. However, the use of G-CSF for mobilization of HPCs is contraindicated in this patient population, having resulted in sickle crises and death in some patients.Plerixafor is an inhibitor of the CXCR4 chemokine receptor on HPCs and blocks binding of its ligand, stromal cell-derived factor-1α (SDF-1α). It is FDA-approved at doses of 240 μg/kg subcutaneously in combination with G-CSF for autologous HPC mobilization in non- Hodgkin lymphoma and multiple myeloma patients.We designed a multi center 3+3, phase 1 study of dose escalation of plerixafor for mobilization of HPC in patients with SCD. The primary goal of this trial is to evaluate the safety of, and the optimal dose for the mobilization of HPCs with plerixafor alone. The desired optimal peripheral blood CD34 concentration was set at > 30 cells/μl. Plerixafor was supplied by Sanofi following their approval of the protocol. Because of the ethical considerations, and the risks associated with the mobilization of patients with SCD, the protocol was submitted to, and approved by the FDA; IND 122657. It was registered with ClinicalTrials.gov; identifier: NCT02193191.Fourteen patients with SCD were entered on the study to date, and 13 patients were evaluable. Patients' age was 21-41 years (median 30 years). Patients had a history of SCD complications including vaso-occlusive crises, acute chest syndrome, osteomyelitis, cerebral aneurysm, retinal ischemia, and skin ulcers. Nine patients were on, and 4 patients were off hydroxyurea at the time of accrual on study.Six patients were treated at the first dose level of 80 μg/Kg, three patients at the second dose level of 160 μg/Kg, and to date, four patients at the third dose level of 240 μg/Kg. Two patients developed adverse events of vaso-occlusive crisis, 2 and 4 days following completion of plerixafor at the first and third dose level respectively, requiring increasing the first and third dose cohorts to 6 patients.White blood cell counts (WBC), absolute neutrophil counts (ANC), absolute lymphocyte counts (ALC) (x 10^9/L), CD34+ and CD34+CD38- cell counts were measured at the following time points: 0, 6-12 and 20-24 hours post-treatment with Plerixafor, which allowed to identify the optimal CD34 peak time in SCD patients.The results of peak WBC, ANC and ALC, and fold increase for these counts, as well as CD34+ concentrations (cells/μl), and number of patients achieving a CD34+count > 30 cells/μl are shown in Table 1 for patients at the 3 dose levels. There was a 1.8-2.8 fold increase in WBC and 1.0-2.2 fold increase in ANC at the 3 dose levels, and CD34 concentrations were: 7-132 cells/μl, 27-251 cells/μl and 19-95 cells/μl for the 3 respective dose levels. There were no significant variations in the levels of hemoglobin and platelets.In summary, this is the first trial on the use of plerixafor for HPC mobilization in patients with SCD. Our data in 13 patients support the safety of plerixafor administration at doses of 80, 160 μg/Kg, and 240 μg/Kg, which is especially significant, in view of the prior toxicity associated with G-CSF in this patient population. Plerixafor increased WBC, ANC and ALC to acceptable levels (1.8-2.8 fold), and allowed the mobilization of high numbers of CD34+ cells for 8 of 13 patients treated at the 3 dose levels. This study will allow us to define the optimal conditions for effective globin gene transfer after optimization of lentivirus-mediated globin gene transfer with the TNS9.3.55 vector in an upcoming trial. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Molecular docking studies of 3-substituted 4-phenylamino coumarin derivatives as chemokine receptor inhibitor

Molecular docking studies of 3-substituted 4-phenylamino coumarin derivatives as chemokine receptor inhibitor

Differential Analyses of Peripheral Blood Parameters in CCR5 Inhibition-treated Colorectal Cancer Patients Reveal Dynamic Changes Linked to Clinical Outcomes.

Chemokine receptor inhibition is an immunotherapy that modulates the innate arm of the immune system. Previous work in microsatellite-stable metastatic colorectal cancer showed an exploitive loop that could be successfully targeted via C-C-motive-chemokine-receptor 5 (CCR5) specific blocking, resulting in a selective anti-tumoral activation of macrophages. In the respective trial (MARACON trial, NCT01736813) the peripheral blood laboratory markers and cytokine values were measured over time. Little is known on their role as biomarkers or stratification parameters in immunotherapy trials. Systematic analyses of key laboratory parameters are presented, highlighting specific dynamics of lymphocyte and monocyte percentages, lactate dehydrogenase as well as interleukin-6 and interleukin-8 levels as parameters of a systemic inflammatory readout. Specific dynamical changes of lymphocyte and monocyte compositions were noted between different patients, showing a stabilization (or increase) versus decreased numbers over time for monocytes. While lactate dehydrogenase, interleukin-6 and interleukin-8 showed almost uniformly rising levels over time, the systemic monocyte patterns prompted a further evaluation. Stabilized or increasing monocyte percentages were associated with improved overall survival (Kaplan Meier analysis, p=0.025) and with induced overt radiologic necrosis in patients. The observed association between monocyte dynamics and imaging findings as well as overall survival suggests that analyses of dynamical parameters in the peripheral blood should be implemented in immunotherapy trials.

A potential novel therapeutic target in diabetic retinopathy: a chemokine receptor (CCR2/CCR5) inhibitor reduces retinal vascular leakage in an animal model.

We have previously shown that the chemokine CCL2 plays an important role in monocyte trafficking into the retina and alteration of the BRB in an animal model of diabetic retinopathy. In this study, we examined the effect of pharmacologically targeting the chemokine pathway to reduce the increased retinal vascular permeability in this model. C57BL/6J mice were made diabetic using streptozotocin. After 4months of diabetes, mice (n = 10) were treated by intraperitoneal injections of TAK-779 (dual CCR2/CCR5 inhibitor) (30mg/kg) daily for 2weeks. Retinal vascular permeability and protein expression were done using western blot. The SDF-1 levels were measured by ELISA. Immune cell infiltration in the retinas was measured using flow cytometry. The dual inhibitor significantly decreased retinal vascular permeability in diabetic animals. There was a significant reduction in macrophage/microglia infiltration in the retinas of treated animals. Levels of SDF-1 and ICAM-1 were significantly reduced and the tight junction protein ZO-1 level was increased, and phospho-VE-Cad was significantly reduced with drug treatment. A chemokine receptor inhibitor (CCR2/CCR5) can reduce retinal vascular permeability in diabetic animals. Targeting the chemokine pathway pharmacologically may be used as a novel therapeutic strategy in management of diabetic macular edema.

Abstract A16: Natural killer cell therapy efficacy is enhanced through inhibition of myeloid-derived suppressor cell trafficking

Abstract Immunotherapies designed to activate or replace T-cell effector immunity, such as immune checkpoint blockade or adoptive transfer of T cells, are limited by the ability of subpopulations of tumor cells to escape T-cell immunity. This can occur through immunologic selection of tumor cells that present weak T-cell antigens or harbor genomic or epigenetic alterations that alter antigen processing or presentation or responses to interferon. Immunotherapy designed to activate or replace NK cell immunity may overcome these limitations through antigen and MHC class I-independent tumor cell recognition and killing. However, similar to suppression of T-cell function, local immunosuppression within the tumor microenvironment may limit the function of NK cells. We hypothesized that immature, immunosuppressive myeloid cells (myeloid derived suppressor cells, or MDSCs) may limit optimal function of NK cells and that NK cell control of tumors can be enhanced through abrogation of MDSC trafficking and tumor infiltration. To model new NK cell therapy products that are available for clinical investigation, we studied how MDSCs alter the ability of culturable murine NK (KIL) cell line to kill oral cancer cells. We used an oral cancer model, MOC2, that has few genomic alterations and forms highly aggressive, non-T cell-inflamed tumors in wild-type C57BL/6 mice. CXCR2+ neutrophilic-MDSCs (PMN-MDSCs) sorted from MOC2 tumors significantly suppressed the ability of KIL to kill MOC2 tumor cells in vitro as assessed by real-time impedance analysis. NK suppression mediated by PMN-MDSC appeared to be mediated by both cell surface TGFβ and arginase. In vivo, we aimed to block chemotaxis and MOC2 tumor infiltration of CXCR2+ PMN-MDSC with the use of SX-682, a clinical-stage small-molecule inhibitor of CXCR1/2. After validating that SX-682 blocks tumor PMN-MDSC trafficking into tumors, we treated mice bearing established MOC2 tumors with SX-682 and adoptive transfer of KIL, alone or in combination. While SX-682 alone produced no tumor growth inhibition (TGI) and KIL adoptive transfer alone produced modest TGI, the combination resulted in significant TGI and prolonged survival of mice. Immune correlative analysis revealed increased tumor infiltration of adoptively transferred KIL with inhibition of PMN-MDSC tumor infiltration. These data suggest that the efficacy of NK cell therapies can be enhanced with PMN-MDSC inhibition and support the clinical investigation of chemokine receptor inhibitors such as SX-682 in combination with NK cell therapy products. Citation Format: Sarah Greene, Yvette Robbins, Paul E. Clavijo, Dean Maeda, John Zebala, Jeffrey Schlom, Claudia Palena, Clint Allen. Natural killer cell therapy efficacy is enhanced through inhibition of myeloid-derived suppressor cell trafficking [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A16.

Combined G-CSF and Plerixafor enhance hematopoietic recovery of CD34+ cells from poor mobilizer patients in NSG mice

Transplantable CD34+ hematopoietic stem/progenitor cells (HSPCs) are currently isolated mainly from peripheral blood after mobilization with granulocyte colony-stimulating factor (G-CSF). These mobilized CD34+ cells have the potential to generate all blood cell types. For autologous transplantation, the minimal number of mobilized CD34+ cells is 2 × 106 CD34+ cells/kg body weight. However, up to 30% of patients fail to mobilize enough peripheral CD34+ cells after G-CSF treatment. To overcome this limitation, a combination of G-CSF and Plerixafor, a CXCR4 chemokine receptor inhibitor, is proposed to enhance CD34+ cell mobilization in poor mobilizer patients. However, only limited data are available on quantification of the functional quality of such patients' mobilized hematopoietic stem cells. Here, for six poor mobilizer patients, a head-to-head comparison of their CD34+ cells mobilized without versus with Plerixafor was performed to assess their properties with respect to the reconstitution of human hematopoiesis in vivo in immune-deficient mice. Our results indicate that mobilized CD34+ cells recovered after the G-CSF + Plerixafor mobilization protocol have an enhanced intrinsic hematopoietic reconstitution potential compared with CD34+ cells mobilized with G-CSF alone.

Per- and Polyfluoroalkyl Substances Differentially Inhibit Placental Trophoblast Migration and Invasion InVitro.

Per- and polyfluoroalkyl substances (PFAS) are used as industrial surfactants and chemical coatings for household goods such as Teflon. Despite regulatory efforts to phase out legacy PFAS, they remain detectable in drinking water throughout the United States. This is due to the stability of legacy PFAS and the continued use of replacement compounds. In humans, PFAS have been detected in placenta and cord blood and are associated with low birth weight and preeclampsia risk. Preeclampsia is a leading cause of maternal mortality and is driven by insufficient endometrial trophoblast invasion, resulting in poor placental blood flow. PFAS alter invasion of other cell types, but their impact on trophoblasts is not understood. We therefore assessed the effects of PFAS on trophoblast migration, invasion, and gene expression in vitro. Trophoblast migration and invasion were assessed using a modified scratch assay in the absence or presence of Matrigel, respectively. Treatment with perfluorooctanoic sulfate (PFOS), perfluorooctanoic acid (PFOA), and GenX (1000 ng/ml) each decreased trophoblast migration over 24 h. However, only GenX (1000 ng/ml) significantly inhibited trophoblast invasion. Treatment with PFOS, PFOA, and GenX also decreased trophoblast expression of chemokines (eg, CCL2), chemokine receptors (eg, CCR4), and inflammatory enzymes (eg, ALOX15) involved in migration. Inhibition of chemokine receptors with pertussis toxin (10 ng/ml), a G-protein inhibitor, inhibited trophoblast migration similar to the PFAS. Taken together, PFAS decrease trophoblast migration, invasion, and inflammatory signaling. By understanding the mechanisms involved, it may be possible to identify the biological and exposure factors that contribute to preeclampsia.