anti-CCL2 Antibody Research Articles

Regulatory T cells require peripheral CCL2-CCR2 signaling to facilitate the resolution of medication overuse headache-related behavioral sensitization

Abstract Background Medication overuse headache (MOH) is the most common secondary headache disorder, resulting from chronic and excessive use of medication to treat headaches, for example, sumatriptan. In a recent study, we have shown that the peripheral C-C motif ligand 2 (CCL2), C-C motif chemokine receptor 2 (CCR2) and calcitonin-gene-related peptide (CGRP) signaling pathways interact with each other and play critical roles in the development of chronic migraine-related behavioral and cellular sensitization. In the present study, we investigated whether CCL2-CCR2 and CGRP signaling pathways play a role in the development of sumatriptan overuse-induced sensitization, and whether they are involved in its resolution by the low-dose interleukin-2 (LD-IL-2) treatment. Methods Mice received daily sumatriptan administration for 12 days. MOH-related behavioral sensitization was assessed by measuring changes of periorbital mechanical thresholds for 3 weeks. CCL2-CCR2 and CGRP signaling pathways were inhibited by targeted gene deletion or with an anti-CCL2 antibody. Ca2+-imaging was used to examine whether repetitive sumatriptan treatment enhances CGRP and pituitary adenylate cyclase–activating polypeptide (PACAP) signaling in trigeminal ganglion (TG) neurons. LD-IL-2 treatment was initiated after the establishment of sumatriptan-induced sensitization. Immunohistochemistry and flow cytometry analyses were used to examine whether CCL2-CCR2 signaling controls regulatory T (Treg) cell proliferation and/or trafficking. Results CCL2, CCR2 and CGRPα global KO mice exhibited robust sumatriptan-induced behavioral sensitization comparable to wild-type controls. Antibody neutralization of peripheral CCL2 did not affect sumatriptan-induced behaviors either. Repeated sumatriptan administration did not enhance the strength of CGRP or PACAP signaling in TG neurons. Nevertheless, LD-IL-2 treatment, which facilitated the resolution of sumatriptan-induced sensitization in wild-type and CGRPα KO mice, was completely ineffective in mice with compromised CCL2-CCR2 signaling. In CCL2 KO mice, we observed normal LD-IL-2-induced Treg expansion in peripheral blood, but the increase of Treg cells in dura and TG tissues was significantly reduced in LD-IL-2-treated CCL2 KO mice relative to wild-type controls. Conclusions These results indicate that the endogenous CCL2-CCR2 and CGRP signaling pathways are not involved in sumatriptan-induced behavioral sensitization, suggesting that distinct molecular mechanisms underlie chronic migraine and MOH. On the other hand, peripheral CCL2-CCR2 signaling is required for LD-IL-2 to reverse chronic headache-related sensitization. Graphical abstract

CCL2 and Lactate from Chemotherapeutics-Treated Fibroblasts Drive Malignant Traits by Metabolic Rewiring in Low-Migrating Breast Cancer Cell Lines.

While cytostatic chemotherapy targeting DNA is known to induce genotoxicity, leading to cell cycle arrest and cytokine secretion, the impact of these drugs on fibroblast-epithelial cancer cell communication and metabolism remains understudied. Our research focused on human breast fibroblast RMF-621 exposed to nonlethal concentrations of cisplatin and doxorubicin, revealing reduced proliferation, diminished basal and maximal mitochondrial respirations, heightened mitochondrial ROS and lactate production, and elevated MCT4 protein levels. Interestingly, RMF-621 cells enhanced glucose uptake, promoting lactate export. Breast cancer cells MCF-7 exposed to conditioned media (CM) from drug-treated stromal RMF-621 cells increased MCT1 protein levels, lactate-driven mitochondrial respiration, and a significantly high mitochondrial spare capacity for lactate. These changes occurred alongside altered mitochondrial respiration, mitochondrial membrane potential, and superoxide levels. Furthermore, CM with doxorubicin and cisplatin increased migratory capacity in MCF-7 cells, which was inhibited by MCT1 (BAY-8002), glutamate dehydrogenase (EGCG), mitochondrial pyruvate carrier (UK5099), and complex I (rotenone) inhibitors. A similar behavior was observed in T47-D and ZR-75-1 breast cancer cells. This suggests that CM induces metabolic rewiring involving elevated lactate uptake to sustain mitochondrial bioenergetics during migration. Treatment with the mitochondrial-targeting antioxidant mitoTEMPO in RMF-621 and the addition of an anti-CCL2 antibody in the CM prevented the promigratory MCF-7 phenotype. Similar effects were observed in THP1 monocyte cells, where CM increased monocyte recruitment. We propose that nonlethal concentrations of DNA-damaging drugs induce changes in the cellular environment favoring a promalignant state dependent on mitochondrial bioenergetics.

Anti-CCL2 antibody combined with etoposide prolongs survival in a minimal residual disease mouse model of neuroblastoma

C–C motif chemokine ligand 2 (CCL2) is a monocyte chemoattractant that promotes metastatic disease and portends a poor prognosis in many cancers. To determine the potential of anti-CCL2 inhibition as a therapy for recurrent metastatic disease in neuroblastoma, a mouse model of minimal residual disease was utilized in which residual disease was treated with anti-CCL2 monoclonal antibody with etoposide. The effect of anti-CCL2 antibody on neuroblastoma cells was determined in vitro with cell proliferation, transwell migration, and 2-dimensional chemotaxis migration assays. The in vivo efficacy of anti-CCL2 antibody and etoposide against neuroblastoma was assessed following resection of primary tumors formed by two cell lines or a patient-derived xenograft (PDX) in immunodeficient NOD-scid gamma mice. In vitro, anti-CCL2 antibody did not affect cell proliferation but significantly inhibited neuroblastoma cell and monocyte migration towards an increasing CCL2 concentration gradient. Treatment of mice with anti-CCL2 antibody combined with etoposide significantly increased survival of mice after resection of primary tumors, compared to untreated mice.

Targeting conditioned media dependencies and FLT-3 in chronic lymphocytic leukemia

AbstractThe importance of the stromal microenvironment in chronic lymphocytic leukemia (CLL) pathogenesis and drug resistance is well established. Despite recent advances in CLL therapy, identifying novel ways to disrupt interactions between CLL and its microenvironment may identify new combination partners for the drugs currently in use. To understand the role of microenvironmental factors on primary CLL cells, we took advantage of an observation that conditioned media (CM) collected from stroma was protective of CLL cells from spontaneous cell death ex vivo. The cytokine in the CM-dependent cells that most supports CLL survival in short-term ex vivo culture was CCL2. Pretreatment of CLL cells with anti-CCL2 antibody enhanced venetoclax-mediated killing. Surprisingly, we found a group of CLL samples (9/23 cases) that are less likely to undergo cell death in the absence of CM support. Functional studies revealed that CM-independent (CMI) CLL cells are less sensitive to apoptosis than conventional stroma-dependent CLL. In addition, a majority of the CMI CLL samples (80%) harbored unmutated immunoglobulin heavy-chain variable (IGHV) region. Bulk-RNA sequence analysis revealed upregulation of the focal adhesion and RAS signaling pathways in this group, along with expression of fms-like tyrosine kinase 3 (FLT3) and CD135. Treatment with FLT3 inhibitors caused a significant reduction in cell viability among CMI samples. In summary, we were able to discriminate and target 2 biologically distinct subgroups of CLL based on CM dependence with distinct microenvironmental vulnerabilities.

Suppression of androgen receptor signaling induces prostate cancer migration via activation of the CCL20-CCR6 axis.

The suppression of androgen receptor (AR) expression exacerbates the migration potential of prostate cancer. This study identified a previously unrecognized regulation of the AR-controlled pathway that promotes migration potential in prostate cancer cells. Prostate cancer cells that pass through a transwell membrane (mig cells) have a higher migration potential with a decreased AR expression than parental cells. In this study, we aimed to elucidate the mechanism of migration enhancement associated with the suppression of AR signaling. Expression of C-C motif ligand 20 (CCL20) is upregulated in mig cells, unlike in the parental cells. Knockdown of AR with small interfering RNA (siAR) in LNCaP and C4-2B cells increased CCL20 secretion and enhanced the migration of cancer cells. Mig cells, CCL20-treated cells, and siAR cells promoted cell migration with an enhancement of AKT phosphorylation and Snail expression, while the addition of a C-C chemokine receptor 6 (CCR6, the specific receptor of CCL20) inhibitor, anti-CCL20 antibody, and AKT inhibitor suppressed the activation of AKT and Snail. With 59 samples of prostate cancer tissue, CCL20 secretion was profuse in metastatic cases despite low AR expression levels. Snail expression was associated with the expression of CCL20 and CCR6. A xenograft study showed that the anti-CCL20 antibody significantly inhibited Snail expression, thereby suggesting a new therapeutic approach for castration-resistant prostate cancer with the inhibition of the axis between CCL20 and CCR6.

CCL2: a Chemokine Potentially Promoting Early Seeding of the Latent HIV Reservoir.

ABSTRACTHIV infects long-lived CD4 memory T cells, establishing a latent viral reservoir that necessitates lifelong antiretroviral therapy (ART). How this reservoir is formed so quickly after infection remains unclear. We now show the innate inflammatory response to HIV infection results in CCL2 chemokine release, leading to recruitment of cells expressing the CCR2 receptor, including a subset of central memory CD4 T cells. Supporting a role for the CCL2/CCR2 axis in rapid reservoir formation, we find (i) treatment of humanized mice with anti-CCL2 antibodies during early HIV infection decreases reservoir seeding and preserves CCR2/5+ cells and (ii) CCR2/5+ cells from the blood of HIV-infected individuals on long-term ART contain significantly more integrated provirus than CCR2/5-negative memory or naive cells. Together, these studies support a model where the host’s innate inflammatory response to HIV infection, including CCL2 production, leads to the recruitment of CCR2/5+ central memory CD4 T cells to zones of virus-associated inflammation, likely contributing to rapid formation of the latent HIV reservoir.

Abstract A39: Targeting tumor-microenvironmental dependencies as a therapeutic approach in Chronic Lymphocytic Leukemia (CLL)

Abstract Interaction between CLL and its microenvironment is a major factor in drug resistance. Bone marrow derived stroma cells provide a protective and supportive niche to allow CLL cells to survive drug-induced apoptosis. Therefore, a current challenge is to develop new strategies to target the microenvironment. To understand the role of tumor microenvironment in CLL survival, we studied conditioned media (CM) derived from NKTert cells. Human NKTert stroma cells provide a survival advantage to primary CLL cells by protecting them from spontaneous apoptosis in ex-vivo culture. We identified three cytokines (IL6, IL8 and CCL2) from CM as candidates for mediating survival. We exposed CLL cells to neutralizing antibodies against each cytokine and found significant decrease in CLL viability in presence of anti-CCL2 suggesting its role in CLL survival. BH3 profiling revealed an increase in apoptotic priming with anti-CCL2 treatment as well. Using dynamic BH3 profiling (DBP), we found that anti-CCL2 increased dependence on BCL-2 as well as BCL-xL in primary CLL cells suggesting a potential role of CCL2 on CLL survival. To our surprise, we also identified a CLL group (CM independent, CMI) that were resilient to cell death in the absence of stroma support in ex-vivo culture. In addition to this, anti-CCL2 antibody showed no effect on their survival. Next, we exposed CMI CLL cells to venetoclax and found that these CLL cells were less sensitive to apoptosis compared to the CLL samples that are stroma dependent (CM dependent, CMD). BH3 profiling showed that CMI CLL cells are less primed for apoptosis. Consistent with our findings, DBP with anti-CCL2, venetoclax or the combination didn’t increase any drug induced apoptotic priming in CMI CLL samples. In our study, 80% of CMI harbor unmutated IgHV. To provide additional insight to the molecular mechanism, we performed western blot analyses for BCL-2 family proteins and found decreased BAX ad BCL-2 expression in CMI samples. This result suggests altered BCL-2 family proteins expression might be a mechanism for differential priming. Bulk RNA seq analysis revealed upregulation of focal adhesion genes and Ras signaling pathway in CMI samples. In summary, our study suggest that in the CMD subset of CLL, anti-CCL2 therapy enhances apoptotic priming and venetoclax efficacy. Furthermore, we have also functionally defined two biologically distinct CLL subgroups based on stroma dependence. Citation Format: Salma Parvin, Aditi Aryal, Shanye Yin, Matthew S. Davids, Geoffrey G. Fell, Catherine J. Wu, Anthony Letai. Targeting tumor-microenvironmental dependencies as a therapeutic approach in Chronic Lymphocytic Leukemia (CLL) [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A39.

Development of an Electrochemical CCL5 Chemokine Immunoplatform for Rapid Diagnosis of Multiple Sclerosis

Serum level of CCL5 chemokine is considered an emerging biomarker for multiple sclerosis (MS). Due to the lack of specific assays for this disease, the development of a point-of-care test for rapid detection of MS could lead to avoiding diagnostics delays. In this paper, we report the first electrochemical immunoplatform for quantification of the CCL5 biomarker at the clinically required levels, able to discriminate between patients diagnosed with MS and healthy individuals. The immunosensing device involves protein capture from biological samples by complexation with biotinylated specific antibodies immobilized onto neutravidin-functionalized microparticles and sandwich assay with anti-CCL5 antibody and IgG labelled with horseradish peroxidase (HRP) for the enzyme-catalyzed amperometric detection of H2O2 using hydroquinone (HQ) as the redox mediator. The method shows excellent analytical performance for clinical application with a wide linear range of concentrations (0.1–300 ng·mL−1 CCL5, R2 = 0.998) and a low detection limit (40 pg·mL−1 CCL5). The biosensing platform was applied to the determination of the CCL5 endogenous content in 100-fold diluted sera both from healthy individuals and patients diagnosed with MS, with no further sample treatment in just two hours. The results were successfully compared with those obtained by the ELISA methodology.

Abstract 2043: The role of CCL20 in response to radiation in head and neck squamous cell carcinomas

Abstract Background: Radioresistance is responsible for most cases of tumor progression and recurrence in head and neck squamous cell carcinomas (HNSCC). Combined radiotherapy (RT) and immunotherapy (IT) treatment has improved survival for a minority of patients with inflamed tumors or tumors susceptible to RT-induced inflammation. To enhance the response of radioresistant tumors to radiation, an understanding of the factors that suppress anti-tumor immunity is necessary. In this regard, regulatory T cells (Tregs) are critical mediators of immune suppression in HNSCCs and their depletion improves tumor response to RT. In this study, we investigated how radiation modulates Treg infiltration through the chemokine CCL20. We hypothesized that radiation induces CCL20 secretion resulting in Treg infiltration and suppression of anti-tumor immunity and that blocking CCL20 could lead to improved response to RT. Methods: Human (Cal27, SCC9) and mouse (MOC1, MOC2) cell lines with different immune phenotypes were irradiated at doses of 2Gy or 10Gy. Conditioned media (CM), RNA and protein were collected for the assessment of CCL20. qPCR was used to determine CCL20 gene expression. In vivo, MOC2 cell were were implanted into the right buccal cavity of mice and the effect of neutralizing CCL20 antibody was determined alone and in combination with RT. Blood samples were collected before and after RT for the analysis of serum levels of CCL20. Tumor samples were analyzed by flow cytometry to determine immune infiltrates, including CD8 T cells and Tregs. Results: Cal27 and MOC2 tumors had a gene signature associated with immunosuppression and Treg infiltration whereas SCC9 and MOC1 tumors displayed a gene signature associated with an inflamed microenvironment. In vitro, high-dose radiation significantly induced CCL20 in Cal27 and MOC2 cells relative to control. Multiplex analysis of chemokines from conditioned media samples showed that induction of CCL20 was associated with increased secretion of GM-CSF by MOC2 and Cal27 cells. In vivo, inhibition of CCL20 alone resulted in a significant decrease in tumor growth compared to control in MOC2 tumors. The combination of RT and anti-CCL20 showed a significant decrease in tumor volume compared to RT alone. Conclusion: Our data suggests that high-dose radiation promotes the induction of CCL20 secretion by tumor cells which is responsible for attraction of Tregs. Inhibition of CCL20 secretion using anti-CCL20 antibodies can slow down tumor growth and enhance the response to radiation. Citation Format: Cleopatra Rutihinda, Ayman J. Oweida, Patrick Delage, Léanie Moreau, Safia Chelighem, Nour Elhouda Saidi. The role of CCL20 in response to radiation in head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2043.

Endometriotic Peritoneal Fluid Stimulates Recruitment of CD4+CD25highFOXP3+ Treg Cells.

Endometriosis is a common gynecological disorder characterized by the presence of endometrial-like tissue outside the uterus. The disease is associated with disturbed local and systemic immunity. It has been reported that the proportion of CD4+CD25highFOXP3+ Treg cells may be significantly increased in the peritoneal fluid of patients with endometriosis. Therefore, the aim of our study was to investigate whether the proportions of Treg cells in the peritoneal cavity of patients with endometriosis are related to the chemotactic and stimulatory activity of the local peritoneal milieu. The peritoneal fluid was collected from 13 women with ovarian endometriosis and 12 control women without the disease. T cell populations were analyzed by flow cytometry, cytokines and chemokines were evaluated using the cytometric bead kit, and cell chemotaxis was studied by cell migration assay. We confirmed that the proportions of Treg cells are increased in the peritoneal fluid of women with endometriosis as compared to the control women. Endometriosis was also associated with elevated concentrations of IL-6, IL-10, and TGF-β1/2 as well as CCL20, CXCL8, CXCL9, and CXCL10. We did not reveal any changes in the proportion of peritoneal Th17 cells and concentrations of IL-17A. Peritoneal Treg cells positively correlated with concentrations of TGF-β, IL-10, and CCL20. Endometriotic peritoneal fluid stimulated chemotaxis of both CD4+ and Treg cells. This chemotactic activity positively correlated with concentrations of CCL20. CCL20 stimulated the migration of Treg cells, and the chemotactic activity of the endometriotic peritoneal fluid was inhibited by neutralizing anti-CCL20 antibodies. These results imply that increased proportions of the peritoneal Treg cells in women with endometriosis may result from attraction and activation by local chemokines and cytokines, especially CCL20 and TGF-β. Since Treg cells contribute to the immunopathogenesis of endometriosis, their chemotaxis and activation may be considered as a target for therapeutic intervention.

Tongue Cancer Cell-Derived CCL20 Induced by Interaction With Macrophages Promotes CD163 Expression on Macrophages.

BackgroundCD163-positive macrophages contribute to the aggressiveness of oral squamous cell carcinoma. We showed in a previous report that CD163-positive macrophages infiltrated not only to the cancer nest but also to its surrounding epithelium, depending on the presence of stromal invasion in tongue carcinogenesis. However, the role of intraepithelial macrophages in tongue carcinogenesis remains unclear. In this study, we assessed the biological behavior of intraepithelial macrophages on their interaction with cancer cells.Materials and MethodsWe established the indirect coculture system (intraepithelial neoplasia model) and direct coculture system (invasive cancer model) of human monocytic leukemia cell line THP-1-derived CD163-positive macrophages with SCC25, a tongue squamous cell carcinoma (TSCC) cell line. Conditioned media (CM) harvested from these systems were analyzed using cytokine array and enzyme-linked immunosorbent assay and extracted a specific upregulated cytokine in CM from the direct coculture system (direct CM). The correlation of both this cytokine and its receptor with various clinicopathological factors were evaluated based on immunohistochemistry using clinical samples from 59 patients with TSCC. Moreover, the effect of this cytokine in direct CM on the phenotypic alterations of THP-1 was confirmed by real-time polymerase chain reaction, western blotting, immunofluorescence, and transwell migration assay.ResultsIt was shown that CCL20 was induced in the direct CM specifically. Interestingly, CCL20 was produced primarily in SCC25. The expression level of CCR6, which is a sole receptor of CCL20, was higher than the expression level of SCC25. Our immunohistochemical investigation showed that CCL20 and CCR6 expression was associated with lymphatic vessel invasion and the number of CD163-positive macrophages. Recombinant human CCL20 induced the CD163 expression and promoted migration of THP-1. We also confirmed that a neutralizing anti-CCL20 antibody blocked the induction of CD163 expression by direct CM in THP-1. Moreover, ERK1/2 phosphorylation was associated with the CCL20-driven induction of CD163 expression in THP-1.ConclusionsTongue cancer cell-derived CCL20 that was induced by interaction with macrophages promotes CD163 expression on macrophages.

Chronic treatment with the (iso-)glutaminyl cyclase inhibitor PQ529 is a novel and effective approach for glomerulonephritis in chronic kidney disease

Glomeruli and renal tubule injury in chronic kidney disease (CKD) is reported to involve induction of macrophage activation through the CCL2/CCR2 axis. The effects of inhibitors of the CCL2/CCR2 axis, such as anti-CCL2 antibody and CCR2 antagonist, on kidney function in animal models or humans with kidney dysfunction have been demonstrated. The N-terminal glutamine on immature CCL2 is replaced with pyroglutamate (pE) by glutaminyl cyclase (QC) and isoQC. pE-CCL2 is stable and resistant to peptidases. We hypothesized that inhibiting QC/isoQC activity would lead to the degradation of CCL2, thereby ameliorating CKD and reducing kidney inflammation. To test this hypothesis, we investigated the renoprotective properties of the QC/isoQC inhibitor PQ529 in anti-glomerular basement membrane (GBM) antibody–induced glomerulonephritis Wistar Kyoto (WKY) rats. Three-week repeated administration of PQ529 (30 and 100 mg/kg, twice daily) significantly reduced the serum and urine CCL2 and urinary protein excretion in a dose-dependent manner. Correlations between the urinary protein level and serum or urinary CCL2 levels were confirmed in tested animals. Repeated administration of PQ529 significantly reduced the expression of CD68, a macrophage marker, in the kidney cortex and mononuclear infiltration into the tubulointerstitium. In addition, decreased levels of urinary KIM-1, β2 microglobulin, and clusterin were detected, suggesting the inhibition of inflammation in both the proximal and distal tubules. These results suggest that PQ529 suppresses the progression of inflammation-induced renal dysfunction by inhibiting the CCL2/CCR2 axis. Inhibition of QC/isoQC may thus be a viable alternative therapeutic approach for treating glomerulonephritis and CKD patients.

Structure and Functional Characterization of a Humanized Anti-CCL20 Antibody following Exposure to Serum Reveals the Formation of Immune Complex That Leads to Toxicity.

mAbs have revolutionized the treatment of autoimmune disorders. Even though mAbs have shown impressive efficacy in blocking T cell or B cell activation and/or recruitment to sites of inflammation, this group of biologicals are not devoid of adverse effects. The most serious adverse effects include infusion reactions, including the activation of the complement pathway. In this study, we present a detailed structure-function study of an anti-CCL20 humanized IgG1 mAb that neutralizes CCL20 chemokine and prevents the recruitment of Th17 cells to sites of inflammation. We demonstrate that the anti-CCL20 Ab changes significantly following administration to humans and monkeys and exposure to human serum. Analysis of the drug product revealed that the anti-CCL20 Ab has unexpectedly high C1q binding. This high binding was linked to immune complex formation in vivo but not during in vitro serum incubation. The immune complex contained multiple complement components. Anti-CCL20 Ab-mediated, complement-dependent cytotoxicity occurred when the Ab bound to CCL20 tethered to the cell membrane of target cells. Taken together, these results provide a likely cause for the animal toxicity observed. In addition, anti-CCL20 revealed progressive acidification because of N100 (located in CDR) deamidation over time, which did not directly impact Ag binding. Our study demonstrates that the safety profiling of mAbs should include the evaluation of effector functions in addition to typical stressed conditions.

CCR2 Expression Signature Can Classify and Predict Outcome in a Subpopulation of Chronic Lymphocytic Leukemia (CLL) Patients

Despite the recent advances in chronic lymphocytic leukemia (CLL) treatment with the development of novel targeted agents such as Bruton's tyrosine kinase (BTK) inhibitor ibrutinib and the BCL2 antagonist Venetoclax, the disease remains incurable for most patients, and resistance mechanisms to these novel agents have already been identified. Therefore, identifying new therapeutic vulnerabilities to treat and prevent resistant tumors is of great importance. Stromal cells provide CLL cells a protective niche which increases resistance to drug-induced apoptosis. Therefore, a current challenge is to develop new strategies by targeting not only CLL cells, but also the microenvironment. We found that soluble factors can recapitulate much of the protective function of stromal cells. To understand the role of microenvironmental factors on CLL cells, we took advantage of the conditioned media (CM) collected from the stromal cell line NK-tert, which protects CLL cells from spontaneous cell death. First, we identified three major cytokines (IL6, IL8, CCL2) from the conditioned media using Proteome Profiler Human XL Cytokine Array. Next, we evaluated the effect of each cytokine on peripheral blood (PB) CLL survival (n=5, treatment naïve, unselected regarding genetic background). CLL cells were cultured ex-vivo in CM and treated with neutralizing antibody against either Il6, IL8 or CCL2 for 48hrs and found a significant decrease in cell viability in presence of anti-CCL2 antibody (median % decrease in cell viability over isotype control is 50%, p<0.0001). Similarly, we also found an increase in cell viability when CLL cells were cultured in RPMI in presence of recombinant-CCL2 protein than those are cultured in RPMI only, suggesting that CCL2 has an important role in CLL survival. BH3 profiling quantifies apoptotic signaling based on cytochrome c loss as a measure of mitochondrial outer membrane permeabilization (MOMP) by flow cytometry in response to BH3-only synthetic peptides that mimic pro-apoptotic BCL-2 family proteins, applied to digitonin permeabilized cells. We performed BH3 profile on PB derived -CLL samples (n=5, treatment naive) cultured in CM and exposed to either isotype control or anti-CCL2 antibody. Anti-CCL2 treated CLL samples showed an increase in MOMP in response to BIM, PUMA, MS1 and HRK peptides compared to CLL samples exposed to isotype control. This suggests that CCL2 suppresses apoptotic signaling in CLL cells. Furthermore, Dynamic BH3 profiling (DBP), a measure of drug-induced apoptotic signaling as previously described (Montero et al., Cell, 2015) showed that a combination of anti-CCL2 and venetoclax increased dependence on BCL-2 (BAD peptide)MCL-1(MS1 peptide) as well as BCL-xL (HRK peptide) in CLL cells. To our surprise, we also found a subgroup of treatment naive PB-CLL samples (CM independent) that undergo minimal spontaneous cell death in the absence of stroma support. In addition to this, IL-6, IL8, CCL2 neutralizing antibodies showed no effect on their survival. These CM independent CLL samples(n=5) are less sensitive to venetoclax treatment compared to the CLL samples that need stroma support (CM dependent). Moreover, baseline BH3 profile showed that CM independent CLL cells are less primed for apoptosis compared to CM dependent CLL cells, and exposure to venetoclax and anti-CCL2 antibody did not increase any drug-mediated apoptotic priming. Next, we checked mRNA expression level of the CCL2 receptor, CCR2 from CM dependent and CM independent CLL samples and found that CM dependent CLL cells have higher expression of CCR2, suggesting a distinct signature discriminating CM dependent and independent patients. In summary, our study shows that CCL2 may serve as a novel biomarker to target in CLL, especially in relapsed/refractory patients. Disclosures Davids: Verastem: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Surface Oncology: Research Funding; MEI Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Gilead Sciences: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Research Funding; Janssen: Consultancy; Genentech: Consultancy, Research Funding; Eli Lilly: Consultancy; Celgene: Consultancy; AstraZeneca: Consultancy, Research Funding; BeiGene: Consultancy; Ascentage Pharma: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; AbbVie: Consultancy; Zentalis: Consultancy; Sunesis: Consultancy; Syros Pharmaceuticals: Consultancy; Research to Practice: Honoraria. Letai:Chugai: Other: Lecture Fees; Novartis: Research Funding; AbbVie: Consultancy; AstraZeneca: Consultancy; Zentalis: Membership on an entity's Board of Directors or advisory committees; Flash Therapeutics: Membership on an entity's Board of Directors or advisory committees; Dialectic: Membership on an entity's Board of Directors or advisory committees.

Tp0136 targets fibronectin (RGD)/Integrin β1 interactions promoting human microvascular endothelial cell migration

Lesion healing without treatment is a unique clinical characteristic of the early stages of syphilis infection. Angiogenesis, which involves endothelial cell migration, is an important process in wound healing. Tp0136, an outer membrane protein of T. pallidum, has the ability to bind host fibronectin-producing cells, which plays a crucial role in the pathogenesis of syphilis. In this research, we purposed to analyze the role of Tp0136 in the migration of human microvascular endothelial (HMEC-1) cells and to explore the related mechanism. First, Tp0136 significantly promoted HMEC-1 cell migration. Furthermore, the levels of C–C motif ligand 2 (CCL2) mRNA and protein expression rose with the concentration and time increasing of Tp0136. The migration of HMEC-1 cells was significantly suppressed by an anti-CCL2 antibody and a CCR2 (the CCL2 receptor) inhibitor. Further study revealed that, in cells pretreated with anti-fibronectin antibody, anti-integrin β1 antibody or RGD (Arg-Gly-Asp), the expression levels of CCL2 induced by Tp0136 were notably decreased. Additionally, after pretreatment with an anti-fibronectin antibody, an anti-integrin β1 antibody or RGD, the migration of HMEC-1 cells treated with Tp0136 was obviously suppressed. These results show that Tp0136 promots the migration of HMEC-1 cells by inducing CCL2 expression via the interaction of the fibronectin RGD domain with integrin β1 and the CCL2/CCR2 signaling pathway, and these interactions may contribute to the mechanisms that increase the capacity for self-healing syphilis infection.

CCL2/CCR2 signaling protects against bladder cancer growth in a T cell dependent manner

Abstract The chemokine CCL2 (C-C motif ligand 2) is an important signaling axis underlying recruitment of pro-tumorigenic myeloid cells and is associated with worse outcomes in several tumor models. Currently, anti-CCL2 antibodies are being tested in cancer trials for solid tumors. To test CCL2 signaling in bladder carcinogenesis, wild type (WT) mice and mice deficient in CCL2 (CCL2KO) were given the urothelial carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Surprisingly, tumor incidence was higher in CCL2KO mice demonstrating an unanticipated protective role for CCL2 signaling in bladder cancer (BC). We then orthotopically challenged WT, CCL2KO, and CCR2KO mice (lacking the major CCL2 receptor) with MB49 BC and confirmed the protective effect of CCL2/CCR2. WT mice had significantly more intratumoral T cells compared to CCL2KO mice suggesting that CCL2 is involved in recruiting T cells to the bladder. Further, depletion of T cells abolished this protective effect of CCL2. Adoptive transfer of CCR2+ T cells into CCR2KO mice restored protection against MB49. CCR2+ T cells were also more activated, functional and tumor specific compared to their CCR2− counterparts. We found that anti-CCL2 promotes BC growth highlighting a concern for use of anti-CCL2 in BC. Moreover, intravesical recombinant CCL2 (rCCL2) either alone or in combination with intravesical gemcitabine reduced bladder tumor and improved survival of mice with MB49 BC. We further developed a slow-release encapsulated nanoparticle formulation of rCCL2 which successfully treated MB49, C3H mice with MBT-2 BC and reduced urine hematuria in our first ever humanized model of orthotopic BC. Our results are rapidly translatable and identify a novel treatment strategy in BC.

Transcription factor Fli-1 impacts IL-17 expression and affects immune cell infiltration into the kidney in lupus-prone mice

Abstract Objectives Transcription factor Fli-1 affects lupus nephritis development through regulating several cytokines and chemokines. Th17 immune response is important to maintain inflammation in lupus nephritis. The purpose of this study is to elucidate whether Fli-1 affects renal IL-17 expression and influence immune cell infiltration into the kidney. Methods Sera were collected from Fli-1-heterozygous (Fli-1+/−) MRL/lpr mice and wild-type (WT) littermates, and the concentration of IL-17 was measured by enzyme-linked immunosorbent assay. Expression of IL-17 and related molecules were measured by real-time polymerase chain reaction (rt-PCR) analysis. Kidneys from Fli-1+/− and WT MRL/lpr mice were stained and evaluated the grade of inflammation. IL-17 expression was detected using anti-mouse IL-17 antibodies. Immunofluorescence staining was performed using anti-CD3, CD4, and anti-IL-17 antibodies to detect T cells related to Th17 responses. In addition, to detect CCL20 expression as a chemoattractant of Th17 cells, anti-CCL20 antibodies and anti-CD11b antibodies were used to detect monocytes expressed CCL20. Results Fli-1+/− MRL/lpr mice had tendency of decreased serum IL-17 levels. Expression of IL-17 was significantly decreased in Fli-1+/− MR/lpr mice kidney interstitium, accompanied by decreased IL-6, STAT3, and IL-1β expression by rt-PCR. CD3 or CD4/IL-17 double-positive immune cells were significantly decreased in Fli-1+/− MLR/lpr mice; similarly, CCL20 positive monocytes were significantly decreased. Conclusion Fli-1 impacts IL-17 expression into the kidney of MRL/lpr mice by reducing CD3 and CD4 positive lymphocytes expressing IL-17 as well as CD11b positive monocytes expressing CCL20.

FOXO1 promotes tumor progression by increased M2 macrophage infiltration in esophageal squamous cell carcinoma.

Objective: The transcription factor forkhead box protein O1 (FOXO1) is critical for regulating cytokine and chemokine secretion. However, its function in the tumor microenvironment (TME) remains largely unexplored. In this study, we characterized the prognostic value of FOXO1 and the interaction between tumor-derived FOXO1 and M2 macrophages in esophageal squamous cell carcinoma (ESCC).Methods: FOXO1 expression and macrophage infiltration in clinical samples and mouse models were quantified using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry staining. Western blotting, qRT-PCR, and enzyme-linked immunosorbent assay were used to evaluate chemokine ligand 20 (CCL20) and colony stimulating factor 1 (CSF-1) expression in FOXO1(+) and FOXO1(-) tumor cells. Macrophage phenotypes were determined using qRT-PCR, flow cytometry, and RNA sequencing. Transcriptional activity was measured using chromatin immunoprecipitation (ChIP)-qPCR. Tumor viability was investigated using XTT proliferation and foci formation assays.Results: FOXO1 upregulation in tumor tissues was found to drive the polarization of M0 macrophages and infiltration of M2 macrophages into the TME, resulting in worse prognosis in ESCC patients. CSF-1, a vital factor inducing M0-to-M2 polarization, was upregulated via a FOXO1-mediated mechanism. RNA sequencing results corroborated that the FOXO1-induced macrophages exhibited similar molecular signatures to the IL4-stimulated M2 macrophages. The transwell assays showed that FOXO1 promoted the migration of M2 macrophages via CCL20 secretion, which could be inhibited using an anti-CCL20 antibody. FOXO1(+) tumor-induced M2 macrophages promoted tumor proliferation via the FAK-PI3K-AKT pathway and the PI3K inhibitor could effectively impede the oncogenical process.Conclusions: FOXO1 facilitated M0-to-M2 polarization and the recruitment of M2 macrophages in the TME via the transcriptional modulation of CCL20 and CSF-1. Our data deciphered the FOXO1-dependent mechanism in M2 macrophage infiltration in the TME of ESCC, which has implications for the development of novel prognostic and therapeutic targets to optimize the current treatment against ESCC.

Abstract LB-126: S100A14 promotes breast cancer metastasis via CCL2 dependent macrophage recruitment

Abstract S100A14 is a member of S100 calcium-binding protein family. Our previous studies have reported that S100A14 modulates a variety of tumorigenic processes by functioning both as intracellular and extracellular factors. Emerging evidence indicated that the biology of most S100 proteins is complex and multifactorial. Herein, we identify S100A14 is upregulated in Lymph Node (LN) metastasis breast cancer (BC) and associated with LN metastasis and prognosis. Through gain and loss of function approaches, we find that S100A14 promotes cell migratory and invasive capabilities in vitro and lung metastasis in vivo. More convincingly, S100A14 knockout also suppresses the lung metastases without affecting the primary tumor growth in MMTV-PyMT mouse models. Using a combination of iTRAQ based quantitative proteomics and transcriptomics, we identified CCL2 as a downstream target of S100A14. Importantly, systemic delivery of anti-CCL2 antibody significantly inhibited S100A14-enhanced cell migration and invasion, tumor-associated macrophages (TAMs) accumulation and lung metastasis. Finally, we indicated that the increased serum S100A14 levels could be used for the detection of BC and the prediction of LN metastases. These findings provide a plausible mechanism for S100A14-modulated tumor microenvironment in metastasis and show that S100A14-mediated CCL2 signaling plays a prominent role in breast cancer metastasis, which may have clinical implications for providing novel biomarkers and potential therapeutic targets of breast cancer in future. Note: This abstract was not presented at the meeting. Citation Format: Hongyan Chen, Xukun Li, Zhihua Liu. S100A14 promotes breast cancer metastasis via CCL2 dependent macrophage recruitment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-126.

CCL20-CCR6 axis modulated traumatic brain injury-induced visual pathologies

BackgroundTraumatic brain injury (TBI) is a major cause of death and disability in the USA and the world; it constitutes 30% of injury-related deaths (Taylor et al., MMWR Surveill Summ 66:1-16, 2017). Contact sports athletes often experience repetitive TBI (rTBI), which exerts a cumulative effect later in life. Visual impairment is a common after-effect of TBI. Previously, we have shown that C-C chemokine 20 (CCL20) plays a critical role in neurodegeneration and inflammation following TBI (Das et al., J Neuroinflammation 8:148, 2011). C-C chemokine receptor 6 (CCR6) is the only receptor that CCL20 interacts with. The objective of the present study was to investigate the role of CCL20-CCR6 axis in mediating rTBI-induced visual dysfunction (TVD).MethodsWild type (WT) or CCR6 knock out (CCR6−/−) mice were subjected to closed head rTBI. Pioglitazone (PG) is a peroxisome proliferator-activated receptor γ (PPARγ) agonist which downregulates CCL20 production. Subsets of WT mice were treated with PG following final rTBI. A subset of mice was also treated with anti-CCL20 antibody to neutralize the CCL20 produced after rTBI. Histopathological assessments were performed to show cerebral pathologies, retinal pathologies, and inflammatory changes induced by rTBI.ResultsrTBI induced cerebral neurodegeneration, retinal degeneration, microgliosis, astrogliosis, and CCL20 expression. CCR6−/− mice showed reduced retinal degeneration, microgliosis, and inflammation. Treatment with CCL20 neutralization antibody or PG showed reduced CCL20 expression along with reduced retinal degeneration and inflammation. rTBI-induced GFAP-positive glial activation in the optic nerve was not affected by knocking out CCR6.ConclusionThe present data indicate that rTBI-induced retinal pathology is mediated at least in part by CCL20 in a CCR6-dependent manner.