anti-CXCR4 Antibody Research Articles

Disulfide bond replacement with non-reducible side chain to tail macrolactamization for the development of potent and selective CXCR4 peptide antagonists endowed with flanking binding sites

The present study describes a small library of peptides derived from a potent and selective CXCR4 antagonist (3), wherein the native disulfide bond is replaced using a side-chain to tail macrolactamization technique to vary ring size and amino acid composition. The peptides were preliminary assessed for their ability to interfere with the interaction between the receptor and anti-CXCR4 PE-conjugated antibody clone 12G5. Two promising candidates (13 and 17) were identified and further evaluated in a125I-CXCL12 competition binding assay, exhibiting IC50 in the low-nanomolar range. Furthermore, both candidates displayed high selectivity towards CXCR4 with respect to the cognate receptor CXCR7, ability to block CXCL12-dependent cancer cell migration, and receptor internalization, albeit at a higher concentration compared to 3. Molecular modeling studies on 13 and 17 produced a theoretical model that may serve as a guide for future modifications, aiding in the development of analogs with improved affinity. Finally, the study provides valuable insights into developing therapeutic agents targeting CXCR4-mediated processes, demonstrating the adaptability of our lead peptide 3 to alternative cyclization approaches and offering prospects for comprehensive investigations into the receptor region's interaction with its C-terminal region.

Expression of Concern: Human Anti-CXCR4 Antibodies Undergo VH Replacement, Exhibit Functional V-Region Sulfation, and Define CXCR4 Antigenic Heterogeneity.

Expression of Concern: Human Anti-CXCR4 Antibodies Undergo VH Replacement, Exhibit Functional V-Region Sulfation, and Define CXCR4 Antigenic Heterogeneity.

A HPRR-based diatomic catalyst electrochemical biosensor for detecting cancer-related extracellular vesicles.

Cancer-associated extracellular vesicles (EVs) are crucial biomarkers for cancer diagnosis as they contain abundant tumor cell information. To efficiently and accurately detect cancer-associated EVs, an electrochemical hydrogen peroxide reduction reaction (HPRR)-based biosensor was developed, utilizing enzyme-linked immunosorbent and diatomic catalyst strategies for catalytic HPRR current amplification for specific identification and highly sensitive detection. The anti-CXCR4 antibody was immobilized on a Au-plated electrode to selectively capture EVs from the sample. Subsequently, Fe/Cu diatomic catalysts, modified with an anti-CD63 antibody, were bound to the CD63 on the EVs. Quantitative detection of EVs was achieved by measuring the electrical signals from the HPRR catalyzed by the labeled Fe/Cu diatomic catalysts. Under optimized conditions, the electrochemical signals exhibited a linear relationship with EV concentration in the range of 500 to 1 × 107 particles mL-1, with a detection limit of 117 particles mL-1, maintaining accuracy even in FBS. With its affordability, high sensitivity, and ease of use, this sensor holds significant potential for medication guidance and postoperative evaluation.

Cx4Mab-1: A Novel Anti-Mouse CXCR4 Monoclonal Antibody for Flow Cytometry.

The CXC chemokine receptor 4 (CXCR4, CD184) is a member of the G protein-coupled receptor family that is expressed in most leukocytes. Overexpression of CXCR4 is associated with poor prognosis in not only hematopoietic malignancy but also solid tumors. Because CXCR4 is an attractive target for tumor therapy, reliable preclinical murine models using anti-CXCR4 monoclonal antibodies (mAbs) have been warranted. This study established a novel anti-mouse CXCR4 (mCXCR4) mAb using the Cell-Based Immunization and Screening method. Flow cytometric analysis showed that an anti-mCXCR4 mAb, Cx4Mab-1 (rat IgG2a, kappa), recognized mCXCR4-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR4) cells and endogenously mCXCR4-expressing mouse myeloma P3X63Ag8U.1 (P3U1) cells. Furthermore, Cx4Mab-1 did not recognize mCXCR4-knockout P3U1 cells. The dissociation constants of Cx4Mab-1 for CHO/mCXCR4 and P3U1 were determined as 6.4 × 10-9 M and 2.3 × 10-9 M, respectively, indicating that Cx4Mab-1 possesses a high affinity to both endogenous and exogenous mCXCR4-expressing cells. These results indicate that Cx4Mab-1 could be a useful tool for preclinical mouse models.

IL-4/IL-4 Ab complex enhances the accumulation of both antigen-specific and bystander CD8 T cells in mouse lungs infected with influenza A virus

BackgroundUnlike conventional T cells, innate and virtual-memory CD8 T cells in naïve mice acquire their memory phenotypes and functions in the absence of antigenic encounters in a cytokine-dependent manner. The relevant cytokines include interleukin-4 (IL-4), type I interferon, and interleukin-15 (IL-15). Moreover, exogenous IL-4 can also induce de novo generation and/or expansion of the virtual-memory CD8 T cell population. In this study, we investigated whether exogenous IL-4 could enhance the immune response to a viral infection.ResultsIn vivo administration of IL-4 and an anti-IL-4 antibody complex (IL-4C) increased CXCR3 expression in both memory and naïve phenotype CD8 T cells in the absence of antigenic stimulation, and protected mice from lethal influenza infection. Flow cytometric analysis of lung-infiltrating immune cells on day 5 after virus infection revealed higher numbers of antigen-specific and bystander CD8 T cells in IL-4C-treated mice than in control mice. In particular, the bystander CD8 T cells were a naïve or evident memory phenotypes. Crucially, an anti-CXCR3 blocking antibody abrogated this IL-4C effect, reflecting that the increased accumulation of CD8 T cells in the lungs after IL-4C treatment is dependent on CXCR3.ConclusionsThese data demonstrate that exogenous IL-4C plays a protective role by enhancing CXCR3-dependent migration of CD8 T cells into influenza-infected lungs.

Chemokine Receptor CXCR4 Radioligand Targeted Therapy Using 177Lutetium-pentixather for Pulmonary Neuroendocrine Cancers.

Intermediate to high-grade lung neuroendocrine tumors (NETs; i.e., atypical carcinoid tumors) and neuroendocrine carcinomas (NECs) are currently difficult to cure. These tumors were found to express the CXCR4 G-protein coupled receptor that can be targeted with radioligands. PCR and flow cytometric analysis of lung NET and NEC cell lines using an anti-CXCR4 antibody demonstrated that all cell lines tested expressed CXCR4. PET/CT imaging with 68Galium-pentixafor in mouse xenografts of NETs and NECs verified tumor targeting that was blocked by a CXCR4 agonist. Clonogenic survival analysis demonstrated a more than additive enhancement of killing when 1 μM auranofin (a thioredoxin reductase inhibitor) was used as a radiosensitizer in combination with 177Lu-pentixather (10 μCi). DMS273 small cell lung cancer xenografts in female nude mice treated with 25 μCi/g 177Lu-pentixather induced inhibition of tumor growth and resulted in an increase in overall survival without causing unacceptable normal tissue toxicities. Immunohistochemical staining of 95 retrospective human samples (containing 90 small cell lung carcinomas) demonstrated 84% CXCR4 positivity. In a multivariable analysis of this cohort that included age, gender, stage, primary site, SSTR2 status, and CXCR4 status, Cox regression models determined that only distant metastasis at presentation (P < 0.01) and a CXCR4 H-score >30 (P = 0.04) were significantly associated with reduced survival. Prospective clinical testing of patient tumors identified CXCR4-positivity in 76% of 21 NECs, 67% of 15 lung NETs (including 8 of 10 atypical carcinoids), and 0% of 25 non-lung NETs (including 5 NETS G3s). These data support the hypothesis that CXCR4-targeted theranostics can be utilized effectively for select NETs and NECs.

Abstract 16577: Unraveling T-cell and Macrophage Chemokine Crosstalk in Immunotherapy-Induced Myocarditis

Introduction: Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that activate immunity against tumor cells but can cause immune related adverse events such as myocarditis, a rare but serious side effect with up to 40% mortality. Hypothesis: Macrophages contribute to recruitment of cytotoxic T-cell subsets to the heart in the setting of ICI, causing myocarditis. Methods: To study the role of macrophage depletion on ICI myocarditis, we treated MRL/Pdcd1-/- mice with liposomal clodronate. To identify timing of immune infiltration, we performed serial single cell T-cell receptor and RNA sequencing of CD45+ cells from the hearts/blood of MRL/Pdcd1-/- myocarditis mice at 1, 2, and 4 weeks of age. We treated MRL mice with anti-PD1/CTLA4 to induce myocarditis and assessed if anti-CXCR3 could decrease effector CD8+ T-cell cardiac infiltration and mitigate myocarditis. We studied the effects of in vitro CXCR3 blockade on CXCR3+ T-cell migration in a transwell assay. Results: Serial single-cell TCR sequencing/scRNA-seq of immune cells from MRL/ Pdcd1-/- vs. MRL control hearts showed infiltration of CXCL9/10+ macrophages ( Fig. 1, A ) along with progressive clonal expansion of CXCR3+ effector CD8+ T-cells in the heart starting at 2 weeks of age, prior to clinical onset of myocarditis. Macrophage depletion with liposomal clodronate reduced myocarditis and improved survival in MRL/ Pdcd1-/- mice ( Fig. 1, B-C) . CXCR3 blockade with anti-CXCR3 antibody reduced myocarditis, decreased CXCR3+ CD8+ T-cell infiltration, and improved survival in mice treated with anti-CTLA4/anti-PD1 antibodies (Fig. 1, D-E) . CXCR3 blockade in transwell assay also reduced migration of CD8+CXCR3+ T-cells towards the macrophage compartment (Fig. 1, F) . Conclusions: CXCL9/10+ macrophages likely play a critical role in development of ICI myocarditis through the recruitment of CXCR3+ effector T-cells into the heart, making this pathway an attractive therapeutic target.

Prolonged CXCR4 Receptor Occupancy By Motixafortide Following a Single Subcutaneous Injection Is Associated with Extended Mobilization of CD34+ Cells in Peripheral Blood for > 24 Hours

Background: Interaction between CXCL12 (SDF-1) and its receptor, CXCR4, plays a critical role in the retention of CD34+ hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Motixafortide (motix) is a novel selective CXCR4 antagonist with high affinity for CXCR4 (IC 50 0.42 - 4.5nM), slow dissociation (Kd = 3.38e-5 s-1), and high plasma protein binding (&amp;gt;99%). In the Phase 3 GENESIS trial (NCT03246529), motix added to G-CSF (G) resulted in significantly higher mobilization of HSPCs in patients with multiple myeloma (MM) prior to autologous stem cell transplantation, with 88.8% of patients collecting ≥6x10 6 CD34+ cells/kg (local lab) in 1 leukapheresis procedure (LP) 10-14 hours following a single dose of motix (1.25mg/kg). Of note, GENESIS trial patients requiring additional LPs to meet HSPC mobilization goals underwent a second LP at 34-38 hours post-motix, with a total of 92.5% of patients meeting their collection goal. Here, we report in-vitro receptor occupancy, as well as clinical pharmacokinetics (PK) and pharmacodynamics (PD) of peripheral blood (PB) mobilization of CD34+ cells/ μL in healthy volunteers (HV) and patients with MM following motix administration. Methods: CXCR4 receptor occupancy and dissociation rate were assessed by incubating increasing doses of motix (3-500nM) with Jurkat cells in vitro followed by washing and incubation 0-72 hours later with either an anti-CXCR4 12G5 fluorophore-conjugated monoclonal antibody (mAb) (competes with motix for binding to CXCR4) or an anti-CXCR4 1D9 fluorophore-conjugated mAb (binds to CXCR4 but does not compete with motix) followed by flow cytometry. In HV (NCT05293171), PB samples were collected following a single subcutaneous (SC) administration of motix (1.25mg/kg). PK timepoints were pre-dose and at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 24 hours post-motix; and PD timepoints were pre-dose and at 0.75, 2, 4, 8, 12, 16, and 24 hours post-motix. In patients with MM, PB samples were collected following SC motix (1.25mg/kg) added to G. PK timepoints were pre-motix (post-G) and 0.25, 0.5, 1, 2, 4, 8, and 12 hours post- motix; and PD timepoints were pre-motix (post-G) and 12 hours post-motix. Results: In vitro studies demonstrated complete CXCR4 receptor occupancy at 3 nM (6.48 ng/mL) of motix, with increasing concentrations resulting in longer receptor occupancy of &amp;gt;72 hours. Due to high protein binding, a minimum total plasma concentration of 800 ng/mL is required to achieve a free fraction of 3nM. This level was achieved following administration with doses ≥ 1.0 mg/kg (Fig 1A). PK analysis in HVs and in patients with MM dosed with 1.25 mg/kg demonstrated that motix was rapidly absorbed (maximum concentration within 0.25-1 hours). Mean maximal plasma concentrations were 2245 ± 791 ng/mL (1040nM) in HVs and 1800 ± 695 ng/mL (834nM) in patients with MM. Notably, the half-life of motix was 1-3 hours. However, despite the short half-life, the high CXCR4 receptor affinity and slow dissociation rate of motix led to an extended PD effect. In HVs who received motix as monotherapy, median PB CD34+ counts increased from baseline of 1.6 CD34+ cells/µl to 17 CD34+ cells/ μL within 4 hours post-motix, reached maximal levels of 43 CD34+ cells/μL at 16 hours post-motix and remained elevated at 36 CD34+ cells/μL at the last observation time point 24 hours post-motix (Fig 1B). In MM patients who received motixafortide after 4 doses of G-CSF, there was a 6.6 fold increase in median PB CD34+ cell counts, from 15.8 cells/μL (post-G-CSF, pre-motix) to 116.0 CD34+ cells/μL at 12 hours post-motix (Fig 1B). Conclusions: In vitro studies demonstrated complete receptor occupancy by motix starting at a concentration of 3nM. Increasing concentrations resulted in longer duration of receptor occupancy. Mobilization of CD34+ cells to PB was observed within 2 hours after motix injection (1.25mg/kg), peaked at 12-16 hours, and remained elevated (&amp;gt;20 CD34+ cells/μL) for &amp;gt;24 hours post-motix. These PK/PD characteristics of motix result in rapid and robust PB CD34+ HSPC mobilization (median 116.0 CD34+ cells/μL); enabling 88.8% of patients to collect ≥6x10 6 CD34+ cells/kg in 1 LP with 1 dose of motix added to G. Furthermore, the extended PD effect of motix supports a flexible administration window that allows for LP to be performed &amp;gt; 24 hours post-motix administration.

IL8 derived from macrophages inhibits CD8+ T-cell function by downregulating TIM3 expression through IL8-CXCR2 axis in patients with advanced colorectal cancer.

T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is a vital immune checkpoint that regulates the immune response. However, the specific role of TIM3 in patients with colorectal cancer (CRC) have rarely been studied. In this study, we investigated the effect of TIM3 on CD8+ T cells in CRC and explored the mechanism of TIM3 regulation in tumor microenvironment (TME). Peripheral blood and tumor tissues of patients with CRC were collected to evaluate TIM3 expression using flow cytometry. Cytokines in the serum of healthy donors and patients with early- and advanced-stage CRC were screened using a multiplex assay. The effects of interleukin-8 (IL8) on TIM3 expression on CD8+ T cells were analyzed using cell incubation experiments in vitro. The correlation between TIM3 or IL8 and prognosis was verified using bioinformatics analysis. TIM3 expression on CD8+ T cells was obviously reduced in patients with advanced-stage CRC, whereas a lower TIM3 expression level was associated with poorer prognosis. Macrophage-derived IL8, which could inhibit TIM3 expression on CD8+ T cells, was significantly increased in the serum of patients with advanced CRC. In addition, the function and proliferation of CD8+ and TIM3+CD8+ T cells were inhibited by IL8, which was partly depending on TIM3 expression. The inhibitory effects of IL8 were reversed by anti-IL8 and anti-CXCR2 antibodies. In summary, macrophages-derived IL8 suppresses TIM3 expression on CD8+ T cells through CXCR2. Targeting the IL8/CXCR2 axis may be an effective strategy for treating patients with advanced CRC.

Dual Stromal Targeting Sensitizes Pancreatic Adenocarcinoma for Anti-Programmed Cell Death Protein 1 Therapy

The stroma in pancreatic ductal adenocarcinoma (PDAC) contributes to its immunosuppressive nature and therapeutic resistance. Herein we sought to modify signaling and enhance immunotherapy efficacy by targeting multiple stromal components through both intracellular and extracellular mechanisms. A murine liver metastasis syngeneic model of PDAC was treated with focal adhesion kinase inhibitor (FAKi), anti-programmed cell death protein 1 (PD-1) antibody, and stromal hyaluronan (HA) degradation by PEGylated recombinant human hyaluronidase (PEGPH20) to assess immune and stromal modulating effects of these agents and their combinations. The results showed that HA degradation by PEGPH20 and reduction in phosphorylated FAK expression by FAKi leads to improved survival in PDAC-bearing mice treated with anti-PD-1 antibody. HA degradation in combination with FAKi and anti-PD-1 antibody increases T-cell infiltration and alters T-cell phenotype toward effector memory T cells. FAKi alters the expression of T-cell modulating cytokines and leads to changes in T-cell metabolism and increases in effector T-cell signatures. HA degradation in combination with anti-PD-1 antibody and FAKi treatments reduces granulocytes, including granulocytic- myeloid-derived suppressor cells and decreases C-X-C chemokine receptor type 4 (CXCR4)-expressing myeloid cells, particularly the CXCR4-expressing granulocytes. Anti-CXCR4 antibody combined with FAKi and anti-PD-1 antibody significantly decreases metastatic rates in the PDAC liver metastasis model. This represents the first preclinical study to identify synergistic effects of targeting both intracellular and extracellular components within the PDAC stroma and supports testing anti-CXCR4 antibody in combination with FAKi as a PDAC treatment strategy.

CXCR4 mediates leukemic cell migration and survival in the testicular microenvironment.

The testis is the second most frequent extramedullary site of relapse in pediatric acute lymphoblastic leukemia (ALL). The mechanism for B-cell (B) ALL cell migration towards and survival within the testis remains elusive. Here, we identified CXCL12-CXCR4 as the leading signaling axis for B-ALL cell migration and survival in the testicular leukemic niche. We combined analysis of primary human ALL with a novel patient-derived xenograft (PDX)-ALL mouse model with testicular involvement. Prerequisites for leukemic cell infiltration in the testis were prepubertal age of the recipient mice, high surface expression of CXCR4 on PDX-ALL cells, and CXCL12 secretion from the testicular stroma. Analysis of primary pediatric patient samples revealed that CXCR4 was the only chemokine receptor being robustly expressed on B-ALL cells both at the time of diagnosis and relapse. In affected patient testes, leukemic cells localized within the interstitial space in close proximity to testicular macrophages. Mouse macrophages isolated from affected testes, in the PDX model, revealed a macrophage polarization towards a M2-like phenotype in the presence of ALL cells. Therapeutically, blockade of CXCR4-mediated functions using an anti-CXCR4 antibody treatment completely abolished testicular infiltration of PDX-ALL cells and strongly impaired the overall development of leukemia. Collectively, we identified a prepubertal condition together with high CXCR4 expression as factors affecting the leukemia permissive testicular microenvironment. We propose CXCR4 as a promising target for therapeutic prevention of testicular relapses in childhood B-ALL. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract 670: CXCR4 expression in tumor associated circulating cells of patients with pancreatic cancer is prognostic for progression and overall survival

Abstract Background: CXCR4 is a chemotaxis receptor implicated in the progression of Pancreatic Cancer (PC) via the activation and invasion of tumor cells. To better elucidate its role in the PC metastasis, we examined the role of CXCR4 expression on a variety of tumor associated circulating cells (TACs) [i.e. circulating tumor cells (CTCs), epithelial to mesenchymal cells (EMTs), and cancer associated macrophage-like cells (CAMLs)] as it relates to patient outcomes. A PC cell line PANC-1 was used to model the activation and expression pathway of CXCR4 in cells. Then blood samples were procured from 30 PC patients prior to the start of therapeutic intent to evaluate CXCR4 expression in TACs. CAMLs, EMTs, and CTCs were found in 93%, 63%, and 30% of PC patients respectively, with high CXCR4 expression found to significantly relate with higher TAC populations in circulation. Further, in a 2-year univariate analysis it was found that high expression of CXCR4 in CAMLs or EMTs was significantly related to both progression free survival (PFS) and overall survival (OS). These data suggest that CXCR4 expression in circulating cells in PC is strongly related to the progression and metastatic spread of the disease. Methods: PANC-1 cells were used to examine the expression and upregulation of CXCR4 by staining with an anti-CXCR4 antibody after stimulation with Isoproterenol. A prospective pilot study was initiated using anonymized peripheral blood samples from (n=30) PC patients, obtained in accordance with local IRB regulations with informed consent. TACs were isolated from the blood using a low-flow CellSieve Microfiltration system and stained for cytokeratin, CXCR4, and CD45. Wilcoxon ranked sum test and univariate analysis were used to analyze the association of CXCR4 expression on all TACs and to evaluate 24-month PFS/OS. Results: Within the PANC-1 cell line, we observed a median expression of CXCR4 intensity of roughly 210 after activation and upregulation with Isoproterenol. Within TACs in the circulation, higher numbers of CAMLs (p=0.017), EMTs (p=0.001), and CTCs (p&amp;lt;0.001) were all significantly related to cells with ≥210 CXCR4 expression. Further, higher expression of CXCR4 on CAMLs correlated with worse PFS (HR=4.0, 95%CI 1.5-10.5, p=0.012) and worse OS (HR=4.8, 95%CI 1.7-13.1, p=0.006), with higher CXCR4 expression in EMTs also correlated with worse PFS (HR=4.6, 95%CI 1.2-15.4, p=0.033) and worse OS (HR=5.4, 95%CI 1.5-19.2, p=0.021). Expression of CXCR4 in CTCs was not significant for PFS or OS. Conclusion: Expression of chemotaxis related receptor CXCR4 appears to have a strong relationship to higher numbers of TACs in circulation. This higher CXCR4 expression in CAMLs and EMTs also appears to correlate with rapid progression and death, indicating a relationship CXCR4 in the spread of PC and its potential use as a target for therapy. Citation Format: Kirby P. Gardner, Susan Tsai, Mohammed Aldakkak, Stephen Gironda, Cha-Mei Tang, Daniel L. Adams. CXCR4 expression in tumor associated circulating cells of patients with pancreatic cancer is prognostic for progression and overall survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 670.

Extracellular DEK Regulates Hematopoiesis through CXCR2-Mediated Intracellular Signaling That Does Not Require Its Translocation into the Nucleus or Binding to DNA

DEK is a highly conserved, ubiquitously expressed and structurally unique DNA-binding nuclear protein involved in transcription, DNA replication, and DNA repair implicating a role for DEK in gene regulation. Interestingly, hematopoietic cells, following stimulation with inflammatory mediators, can actively secrete DEK, which can then be taken up by cells via binding to heparan sulfate proteoglycans (HSPGs), translocating to the nucleus, and mediating its normal function of gene regulation. Previous work from our group indicates that both endogenous, intracellular DEK and secreted, extracellular DEK regulates normal hematopoiesis by decreasing numbers of hematopoietic progenitor cells (HPCs) and increasing numbers of hematopoietic stem cells (HSCs) suggesting that DEK plays a role in regulating HSC/HPC function and/or cell fate. Extracellular DEK-mediated regulation of hematopoiesis occurs in a HSPG-, CXCR2-, Gai protein-dependent manner. Neutralizing these pathways inhibits extracellular DEK regulation of HSCs and HPCs. However, what was not known is whether the HSPG-dependent internalization of DEK is required for the extracellular DEK-mediated regulation of hematopoiesis as HSPGs are also required for maximum binding efficiency of ELR-CXC chemokines to CXCR2. To address this unknown, two mutant forms of recombinant (r)DEK were utilized: DEK that lacks its nuclear translocation sequence (NTS) and DEK that lacks its DNA-binding motif (DBM). First we examined whether NTS- or DMB-rDEK could alter HPC or HSC numbers in vitro / ex vivo . We had previously shown that C57Bl/6 bone marrow (BM) cells when in culture with rDEK inhibited HPC colony formation in a dose-dependent manner, with the minimum dose of WT rDEK required for inhibition being 12.5 nM (p=0.002). Both NTS- and DMB-rDEK also inhibited HPC colony formation in a dose-dependent manner starting at 12.5 nM (p=0.03 and p=0.02 respectively) suggesting that neither the ability of DEK to get into the nucleus or bind to DNA is required to regulate HPC numbers in BM. To examine whether NTS- or DMB-rDEK can still enhance HSC numbers, ex vivo HSC expansion assays were performed. C57Bl/6 lineage negative BM cells were cultured in SCF, TPO, and Flt3L with vehicle control, WT-rDEK, NTS-rDEK, or DMB-rDEK then examined for HSC expansion on days 4 and 7. On both days, WT-, NTS-rDEK and DMB-rDEK significantly expanded long-term HSC numbers when compared to vehicle control, suggesting that like DEK-mediated suppression of HPCs, DEK-mediated expansion of HSCs does not require DEK to translocate to the nucleus or bind to DNA. To examine whether extracellular NTS- and DMB-rDEK can still mediate one of DEK's known nuclear functions, heterochromatin stability, DEK-/- lineage negative BM cells were incubated for 12 hours with WT rDEK, NTS-rDEK or DMB-rDEK then examined for H3K9 trimethylation in HSCs and HPCs by ImageStream flow cytometry. Neither NTS- or DMB-rDEK were able to enhance the trimethylation of H3K9 in DEK-/- HSCs or HPCs. However, incubating DEK-/- lineage negative BM with WT rDEK, increased H3K9me3 in the nucleus of the cells by ~26%. When utilizing WT BM, incubating cells for 12 hours with WT, NTS- or DMB-rDEK did not result in changes in H3K9Me3 that was detectable by ImageStream technology suggesting that extracellular DEK-mediated changes in chromatin remodeling in HSCs and HPCs only occurs when the cells lack endogenous DEK. Co-culturing C57Bl/6 BM cells with WT rDEK mediated AKT and ERK1/2 phosphorylation in HSCs and ERK1/2 in HPCs which is blocked when BM cells are pretreated with a neutralizing antibody against CXCR2 or when preteated with heparin neutralizing HSPGs. Both NTS- and DMB-rDEK were able to mediate similar activation of AKT and ERK1/2 in HSCs and HPCs as WT rDEK but not when the BM was pretreated with anti-CXCR2 antibody or heparin. Taken together, these data suggest that extracellular DEK functions to regulate HSC and HPC numbers/function by binding to CXCR2 to mediate intracellular signaling events in C57Bl/6 BM cells. The fact that HSPGs are required for extracellular DEK's effect, but DEK does not have to get into the nucleus and bind DNA, also suggests that internalization of DEK is not required to regulate hematopoiesis in cells with normal levels of endogenous DEK. Therefore, like the ELR-CXC chemokines, DEK requires HSPGs for its CXCR2-mediated signaling processes in both HSCs and HPCs. DisclosuresNo relevant conflicts of interest to declare.

Plasmonic-doped melanin-mimic for CXCR4-targeted NIR-II photoacoustic computed tomography-guided photothermal ablation of orthotopic hepatocellular carcinoma

Effective and noninvasive diagnosis and prompt treatment of early-stage hepatocellular carcinoma (HCC) are urgently needed to reduce its mortality rate. Herein, the integration of high-resolution diagnostic second near-infrared (NIR-II) photoacoustic computed tomography (PACT) and imaging-guided targeted photothermal ablation of orthotopic small HCC (SHCC) is presented for the first time, which was enabled by a plasmonic platinum (Pt)-doped polydopamine melanin-mimic nanoagent. As designed, an antibody-modified nanoagent (designated Pt@PDA-c) with a plasmonic blackbody-like NIR absorption and superior photothermal conversion efficiency (71.3%) selectively targeted and killed CXCR4-overexpressing HCC (HepG2) cells, which was validated in in vitro experiments. The targeted accumulation properties of Pt@PDA-c in vivo were previously recognized by demonstrating effective NIR-II PA imaging and photothermal ablation in a subcutaneous HCC mouse model. Subsequently, with real-time quantitative guidance by PACT for the accurate diagnosis of intraabdominal SHCC (approximately 4 mm depth), the effective and noninvasive photothermal ablation of SHCCs was successfully demonstrated in an orthotopic tumor-bearing mouse model without damaging adjacent liver tissues. These results show a great potential of NIR-II PACT-guided noninvasive photothermal therapy as an innovative phototheranostic approach and expand the biomedical applications of melanin-mimic materials. Statement of significanceIn this paper, we report the first diagnostic NIR-II photoacoustic computed tomography (PACT)-guided noninvasive photothermal ablation of small hepatocellular carcinoma (SHCC) located in deep tissues in orthotopic tumor-bearing mice; this process is empowered by a polydopamine-based melanin-mimic tumor-targeting nanoagent doped with plasmonic platinum that provides superior NIR-II (1064 nm) absorption and photothermal conversion efficiency of 71.3%. Following surface modification with anti-CXCR4 antibodies, the nanoagent (namely Pt@PDA-c) can selectively target CXCR4-overexpressed HepG2 carcinoma cells and tumor lesions, and serve as the theranostic agent for both NIR-II PACT-based diagnosis of orthotopic SHCC (diameter less than 5 mm) and efficient NIR-II PTT in vivo. This study may also extend the potential of melanin-derived blackbody materials for optical-biomedical and water distillation applications.

Acute Kidney Injury Instigates Malignant Renal Cell Carcinoma via CXCR2 in Mice with Inactivated Trp53 and Pten in Proximal Tubular Kidney Epithelial Cells.

Renal cell carcinoma (RCC) is one of the most common urologic malignancies with the highest mortality rates worldwide. However, relevant mouse models that recapitulated the genetic alterations found in RCC have been lacking. In this study, we crossed Trp53 and Pten conditional knockout mice with Ggt1-Cre mice to generate a Ggt1-Cre; Trp53LoxP/LoxP ; PtenLoxP/LoxP ; YFPLoxP/LoxP (GPPY) mouse model, which resulted in the formation of dysplastic lesions involving kidney tubular epithelial cells (TEC), with only approximately 25% of mice developing RCC at an advanced age. Combining CRISPR/Cas9-mediated Vhl knockout in these mice increased the frequency of dysplasia, but failed to increase the incidence of RCC. Assessments of whether ischemic injury of TECs in the GPPY kidney without Vhl knockout influences the emergence of RCC revealed that advanced RCC predominantly emerged in the contralateral, noninjured kidney with 100% penetrance at a younger age, but rarely in the injured kidney due to severely damaged ischemic TEC. Injured TEC released CXCL1 into the microenvironment that traveled systemically to activate fibroblasts and recruit neutrophils to enable emergence of RCC in the contralateral kidney. Fibroblasts responded to CXCL1 via CXCR2 and recruited tumor-associated neutrophils, which in turn mediated tumor-promoting inflammation and angiogenesis. Treatment with anti-CXCR2 antibodies abolished the emergence of malignant RCC. Collectively, these results demonstrate a defining functional role of systemic inflammation and microenvironment in the emergence of malignant cancer from preestablished dysplastic precursor lesions. SIGNIFICANCE: These results identify a role for CXCL1/CXCR2 and the tumor microenvironment in the development of RCC. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/10/2690/F1.large.jpg.See related commentary by Kusmartsev, p. 2584.

A critical role of STING-triggered tumor-migrating neutrophils for anti-tumor effect of intratumoral cGAMP treatment.

Stimulator of interferon genes (STING) contributes to anti-tumor immunity by activating antigen-presenting cells and inducing mobilization of tumor-specific T cells. A role for tumor-migrating neutrophils in the anti-tumor effect of STING-activating therapy has not been defined. We used mouse tumor transplantation models for assessing neutrophil migration into the tumor triggered by intratumoral treatment with STING agonist, 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Intratumoral STING activation with cGAMP enhanced neutrophil migration into the tumor in an NF-κB/CXCL1/2-dependent manner. Blocking the neutrophil migration by anti-CXCR2 monoclonal antibody impaired T cell activation in tumor-draining lymph nodes (dLNs) and efficacy of intratumoral cGAMP treatment. Moreover, the intratumoral cGAMP treatment did not show any anti-tumor effect in type I interferon (IFN) signal-impaired mice in spite of enhanced neutrophil accumulation in the tumor. These results suggest that both neutrophil migration and type I interferon (IFN) induction by intratumoral cGAMP treatment were critical for T-cell activation of dLNs and the anti-tumor effect. In addition, we also performed in vitro analysis showing enhanced cytotoxicity of neutrophils by IFN-β1. Extrinsic STING activation triggers anti-tumor immune responses by recruiting and activating neutrophils in the tumor via two signaling pathways, CXCL1/2 and type I IFNs.

Autoantibodies against C5aR1, C3aR1, CXCR3, and CXCR4 are decreased in primary Sjogren’s syndrome

BackgroundNetworks formed of numerous autoantibodies (aabs) directed against G-protein coupled receptors (GPCR) have been suggested to play important role in autoimmune disorders. In present study, we aimed to evaluate the association between anti-GPCR antibodies and primary Sjogren’s syndrome (pSS) to determine the potential pathogenic factors. MethodsBy applying a cell membrane-based ELISA technique, which is capable of detecting aabs against conformational epitopes within GPCR, serum levels of fourteen GPCR were determined in well-characterized patients with pSS (n = 52) and gender-matched healthy controls (n = 54). Comparisons between groups were analyzed by two-tailed Mann-Whitney U test, Bonferroni correction was applied for multiple comparisons. Spearman`s rank correlation coefficients were calculated between variables and visualized by heat map. ResultsCompared to healthy subjects, sera of patients with pSS showed significantly higher binding to β2AR and ETAR, but lower binding to C5aR1, C3aR1, CXCR3, and CXCR4. Autoantibodies against C5aR1, C3aR1, CXCR3, and CXCR4 were also decreased in patients with rheumatoid arthritis. In pSS patients, levels of anti-CXCR3 and anti-CXCR4 antibodies were negatively correlated with circulating lymphocyte counts. Furthermore, correlation signatures of anti-GPCR antibodies changed dramatically in the patients with pulmonary involvement. ConclusionsThis study demonstrates an association between pSS and autoantibodies recognizing GPCR, especially those functionally involved in immune cell migration and exocrine glandular secretion.

Anti-CXCR4 Single-Chain Variable Fragment Antibodies Have Anti-Tumor Activity.

Monoclonal antibodies (mAbs) are large and have limitations as cancer therapeutics. Human single-chain variable fragment (scFv) is a small antibody as a good alternative. It can easily enter cancer tissues, has no immunogenicity and can be produced in bacteria to decrease the cost. The chemokine receptor CXCR4 is overexpressed in different cancer cells. It plays an important role in tumor growth and metastasis. Its overexpression is associated with poor prognosis in cancer patients and is regarded as an attractive target for cancer treatment. In this study, a peptide on the CXCR4 extracellular loop 2 (ECL2) was used as an antigen for screening a human scFv antibody library by yeast two-hybrid method. Three anti-CXCR4 scFv antibodies were isolated. They could bind to CXCR4 protein and three cancer cell lines (DU145, PC3, and MDA-MB-231) and not to 293T and 3T3 cells as negative controls. These three scFvs could decrease the proliferation, migration, and invasion of these cancer cells and promote their apoptosis. The two scFvs were further examined in a mouse xenograft model, and they inhibited the tumor growth. Tumor immunohistochemistry also demonstrated that the two scFvs decreased cancer cell proliferation and tumor angiogenesis and increased their apoptosis. These results show that these anti-CXCR4 scFvs can decrease cancer cell proliferation and inhibit tumor growth in mice, and may provide therapy for various cancers.

CXCR3 chemokine receptor contributes to specific CD8+T cell activation by pDC during infection with intracellular pathogens.

Chemokine receptor type 3 (CXCR3) plays an important role in CD8+ T cells migration during intracellular infections, such as Trypanosoma cruzi. In addition to chemotaxis, CXCR3 receptor has been described as important to the interaction between antigen-presenting cells and effector cells. We hypothesized that CXCR3 is fundamental to T. cruzi-specific CD8+ T cell activation, migration and effector function. Anti-CXCR3 neutralizing antibody administration to acutely T. cruzi-infected mice decreased the number of specific CD8+ T cells in the spleen, and those cells had impaired in activation and cytokine production but unaltered proliferative response. In addition, anti-CXCR3-treated mice showed decreased frequency of CD8+ T cells in the heart and numbers of plasmacytoid dendritic cells in spleen and lymph node. As CD8+ T cells interacted with plasmacytoid dendritic cells during infection by T. cruzi, we suggest that anti-CXCR3 treatment lowers the quantity of plasmacytoid dendritic cells, which may contribute to impair the prime of CD8+ T cells. Understanding which molecules and mechanisms guide CD8+ T cell activation and migration might be a key to vaccine development against Chagas disease as those cells play an important role in T. cruzi infection control.

Myeloid-derived suppressor cells modulate aortic aneurysm formation via CXCR2 and IL-17 signaling

Abstract Aortic aneurysm formation is associated with vascular inflammation and remodeling mediated via CD4+ T cell-secreted IL-17A production. Our hypothesis is that innate myeloid-derived suppressor cells (MDSCs), which are a heterogeneous population of myeloid precursor and progenitor cells, exhibit an immunoregulatory role in the progression of aortic inflammation during abdominal aortic aneurysm (AAA) formation. The trafficking and migration of MDSCs to the aortic tissue and role of CXCR2 and programmed death-1 (PD-1) receptor signaling was deciphered using an in vivo elastase-treatment model of AAA as well as in vitro co-culture experiments. We used an established murine AAA model of topical elastase-treatment in the present study. Preliminary data showed that elastase-treatment of C57BL/6 (wild-type; WT) mice resulted in significant increase in infrarenal aortic diameter causing AAA formation on day 14 that was significantly inhibited by a combined treatment with anti-CXCR2 and PD-1 antibodies. A multi-fold increase in MDSCs (CD11b+Gr-1+) in aortic tissue of elastase-treated WT mice on days 7 and 14 occurred compared to controls. Co-culture of MDSCs with aortic endothelial cells in the presence of anti-CXCR2 antibody inhibited the transmigration of MDSCs through the endothelial barrier. Moreover, transient elastase treatment of co-cultures of anti-CD3/CD28 treated mononuclear cells with MDSCs significantly enhanced IL-17A secretion compared to cultures without MDSCs. Our results indicate that MDSCs may play a contributory role in aortic inflammation by trafficking in a CXCR2-dependent manner and promotion of IL-17A secretion, signifying the importance of MDSC-mediated immunoregulation of aortic aneurysms.