Chemokine Receptor Expression Research Articles

Synthesis, Screening, and Evaluation of Theranostic Molecular CPCR4-Based Probe Targeting CXCR4.

Chemokines and chemokine receptors are indispensable to play a key role in the development of malignant tumors. As one of the most widely expressed chemokine receptors, chemokine (C-X-C motif) receptor 4 (CXCR4) has been a popular research focus. In most tumors, CXCR4 expression is significantly upregulated. Moreover, integrated nuclide diagnosis and therapy targeting CXCR4 show great potential. [68Ga]Ga-pentixafor, a radioligand targeting CXCR4, exhibits a strong affinity for CXCR4 both in vivo and in vitro. However, [177Lu]Lu-pentixather, the therapeutic companion of [68Ga]Ga-pentixafor, requires significant refinement to mitigate its pronounced hepatic biodistribution. The objective of this study was to synthesize theranostic molecular tracers with superior CXCR4 targeting functions. The Daudi cell line, which highly expressed CXCR4, and the MM.1S cell line, which weakly expressed CXCR4, were used in this study. Based on the pharmacophore cyclo (-d-Tyr-n-me-d-Orn-l-Arg-L-2-NAL-Gly-) (CPCR4) of pentixafor, six tracers were synthesized: [124I]I-1 ([124I]I-CPCR4), [99mTc]Tc-2 ([99mTc]Tc-HYNIC-CPCR4), [124I]I-3 ([124I]I-pentixafor), [18F]AlF-4 ([18F]AlF-NETA-CPCR4), [99mTc]Tc-5 ([99mTc]Tc-MAG3-CPCR4) and [124I]I-6 ([124I]I-pentixafor-Ga) and their radiochemical purities were all higher than 95%. After positron emission tomography (PET)/single-photon emission computed tomography (SPECT) imaging, the [124I]I-6 group exhibited the best target-nontarget ratio. At the same time, comparing the [68Ga]Ga-pentixafor group with the [124I]I-6 group, we found that the [124I]I-6 group had a better target-nontarget ratio and lower uptake in nontarget organs. Therefore, compound 6 was selected for therapeutic radionuclide (131I) labeling, and the tumor-bearing animal models were treated with [131I]I-6. The volume of the tumor site was significantly reduced in the treatment group compared with the control group, and no significant side effects were found. [124I]I-6 and [131I]I-6 showed excellent affinity for targeting CXCR4, and they showed great potential for the integrated diagnosis and treatment of tumors with high CXCR4 expression.

AESIS-1, a Rheumatoid Arthritis Therapeutic Peptide, Accelerates Wound Healing by Promoting Fibroblast Migration in a CXCR2-Dependent Manner.

In patients with autoimmune disorders such as rheumatoid arthritis (RA), delayed wound healing is often observed. Timely and effective wound healing is a crucial determinant of a patient's quality of life, and novel materials for skin wound repair, such as bioactive peptides, are continuously being studied and developed. One such bioactive peptide, AESIS-1, has been studied for its well-established anti-rheumatoid arthritis properties. In this study, we attempted to use the anti-RA material AESIS-1 as a therapeutic wound-healing agent based on disease-modifying antirheumatic drugs (DMARDs), which can help restore prompt wound healing. The efficacy of AESIS-1 in wound healing was assessed using a full-thickness excision model in diabetic mice; this is a well-established model for studying chronic wound repair. Initial observations revealed that mice treated with AESIS-1 exhibited significantly advanced wound repair compared with the control group. In vitro studies revealed that AESIS-1 increased the migration activity of human dermal fibroblasts (HDFs) without affecting proliferative activity. Moreover, increased HDF cell migration is mediated by upregulating chemokine receptor expression, such as that of CXC chemokine receptor 2 (CXCR2). The upregulation of CXCR2 through AESIS-1 treatment enhanced the chemotactic reactivity to CXCR2 ligands, including CXC motif ligand 8 (CXCL8). AESIS-1 directly activates the ERK and p38 mitogen-activated protein kinase (MAPK) signaling cascades, which regulate the migration and expression of CXCR2 in fibroblasts. Our results suggest that the AESIS-1 peptide is a strong wound-healing substance that increases the movement of fibroblasts and the expression of CXCR2 by turning on the ERK and p38 MAPK signaling cascades.

Abstract 6125: Differential expression of chemokine genes by race and Breast Cancer Consensus Subtypes in hormone receptor positive breast cancer

Abstract Hormone receptor positive (HR+) breast cancer is a heterogeneous subtype and African American women (AAW) are twice as likely to have HR+ tumors with lower levels of HR staining (1-10%) compared to European American women (EAW). These tumors are associated with substantially worse survival compared to strongly HR+ tumors. We further hypothesized that HR+ tumors from AAW would be more likely to have molecular features typically associated with HR-negative breast cancer and poor prognosis. To test this, we used the Breast Cancer Consensus Subtyping (BCCS) method and evaluated expression of chemokine receptors known to be differentially expressed by race, PAM50 subtypes, and survival. We downloaded The Cancer Genome Atlas (TCGA) expression data for 394 female EA and AA HR+/HER2- breast cancer patients using the cBio Cancer Genomics Portal. Breast Cancer Consensus Subtypes (BCCS) were determined using the R program BCCSclassifier. BCCS classification results in five subtypes: ER-negative non-basal-like (BCS1), ER-negative basal-like (BCS2), ER+ highly proliferative with poor prognosis (BCS3), ER+ stromal infiltration with good prognosis (BCS4), and ER+ hormone response genes highly expressed with good prognosis (BCS5). High and low expression of chemokines (ACKR1, ACKR4, CCR3, CCR6, CCRL2, CXCR1, CXCR2, CXCR4, CXCR6, CX3CR1) was dichotomized based on median expression of each gene within our study cohort. Distribution of BCCS by race was assessed with multinomial logistic regression. Associations between chemokine gene expression, BCC subtype and race were evaluated using logistic regression. An alpha value of 0.05 was set to determine statistical significance. From the TCGA cohort, AAW were more likely to have the BCS2 ER-negative basal-like phenotype (OR=7.17, p<0.001) as well as the poor prognosis highly proliferative ER+ BCS3 phenotype (OR=2.83, p=0.036) versus the high hormone expression BCS5 subtype. In multivariable models evaluating both race and BCCS subtype, AAW were 6.17 times more likely to have lower than median expression of CX3CR1 (p<0.001) and CXCR2 (OR=2.23, p=0.0096) compared to EAW. Notably, all chemokine receptors consistently showed significantly lower expression in BCS1-4 subtypes compared to BCS5. For CCRL2, the effect estimates for BCS4 vs. BCS5 significantly differed by race (p=0.039). In AAW, BCS4 was associated with higher than median expression (OR=1.99, p=0.49), although not statistically significant. In EAW, BCS4 was associated with lower than median expression (0.23, p<0.001). Our findings show that AAW with HR+ breast cancer have tumors with more inherently aggressive molecular subtypes associated with poor prognosis compared to EAW. These results also further support the premise that HR+/HER2- tumors are highly heterogeneous and strongly suggest that chemokine gene expression is associated with breast cancer subtype. Citation Format: Abigail M. Fielder, Manohar Ratnam, Gregory Dyson, Kristen Purrington. Differential expression of chemokine genes by race and Breast Cancer Consensus Subtypes in hormone receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6125.

Phenotype and function of MAIT cells in patients with alveolar echinococcosis.

Mucosal-associated invariant T (MAIT) cells are a subpopulation of unconventional T cells widely involved in chronic liver diseases. However, the potential role and regulating factors of MAIT cells in alveolar echinococcosis (AE), a zoonotic parasitic disease by Echinococcus multilocularis (E. multilocularis) larvae chronically parasitizing liver organs, has not yet been studied. Blood samples (n=29) and liver specimens (n=10) from AE patients were enrolled. The frequency, phenotype, and function of MAIT cells in peripheral blood and liver tissues of AE patients were detected by flow cytometry. The morphology and fibrosis of liver tissue were examined by histopathology and immunohistochemistry. The correlation between peripheral MAIT cell frequency and serologic markers was assessed by collecting clinicopathologic characteristics of AE patients. And the effect of in vitro stimulation with E. multilocularis antigen (Emp) on MAIT cells. In this study, MAIT cells are decreased in peripheral blood and increased in the close-to-lesion liver tissues, especially in areas of fibrosis. Circulating MAIT exhibited activation and exhaustion phenotypes, and intrahepatic MAIT cells showed increased activation phenotypes with increased IFN-γ and IL-17A, and high expression of CXCR5 chemokine receptor. Furthermore, the frequency of circulating MAIT cells was correlated with the size of the lesions and liver function in patients with AE. After excision of the lesion site, circulating MAIT cells returned to normal levels, and the serum cytokines IL-8, IL-12, and IL-18, associated with MAIT cell activation and apoptosis, were altered. Our results demonstrate the status of MAIT cell distribution, functional phenotype, and migration in peripheral blood and tissues of AE patients, highlighting their potential as biomarkers and therapeutic targets.

In-vitro assessment of cutaneous immune responses to aedes mosquito salivary gland extract and dengue virus in Cambodian individuals.

Dengue virus (DENV) poses a global health threat, affecting millions individuals annually with no specific therapy and limited vaccines. Mosquitoes, mainly Aedes aegypti and Aedes albopictus worldwide, transmit DENV through their saliva during blood meals. In this study, we aimed to understand how Aedes mosquito saliva modulate skin immune responses during DENV infection in individuals living in mosquito-endemic regions. To accomplish this, we dissociated skin cells from Cambodian volunteers and incubated them with salivary gland extract (SGE) from three different mosquito strains: Ae. aegypti USDA strain, Ae. aegypti and Ae. albopictus wild type (WT) in the presence/absence of DENV. We observed notable alterations in skin immune cell phenotypes subsequent to exposure to Aedes salivary gland extract (SGE). Specifically, exposure lead to an increase in the frequency of macrophages expressing chemokine receptor CCR2, and neutrophils expressing CD69. Additionally, we noted a substantial increase in the percentage of macrophages that became infected with DENV in the presence of Aedes SGE. Differences in cellular responses were observed when Aedes SGE of three distinct mosquito strains were compared. Our findings deepen the understanding of mosquito saliva's role in DENV infection and skin immune responses in individuals regularly exposed to mosquito bites. This study provides insights into skin immune cell dynamics that could guide strategies to mitigate DENV transmission and other arbovirus diseases.

Expression of monocyte chemokine receptors in diabetes after non-surgical periodontal treatment: A pilot study

The aim of this study was to evaluate the effect of non-surgical periodontal treatment in the expression of chemokine receptors, in individuals with Periodontitis, associated or not with Diabetes. Pilot study, which included patients (n = 45) with Periodontitis, associated (n = 25) or not (n = 20) with Diabetes, submitted to the non-surgical periodontal treatment for one month. The expression of chemokine receptors CCR2, CCR5, and CX3CR1 at the mRNA level was evaluated in the peripheral mononuclear cells, as well as the expression of these receptors at the protein level was verified in monocyte subtypes (classical, intermediate, and non-classical monocytes). There was higher expression of CCR2 and CCR5 receptors at the initial visit in the group with Diabetes, with no differences for CX3CR1 (p = 0.002; p = 0.018, and p = 0.896, respectively), without differences after treatment. There was higher expression of CCR2 and CCR5 proteins in the group with Diabetes at the initial visit for classical, intermediate, and nonclassical monocytes, with no differences for CX3CR1 (CCR2: p = 0.004; p = 0.026; p = 0.024; CCR5: 0.045; p = 0.045; p = 0.013; CX3CR1: p = 0.424; p = 0.944; p = 0.392, respectively), without differences after the end of treatment. Concerning each group separately, there were reductions in the expression of CCR2 as well as CCR5 in classical, intermediate, and nonclassical monocytes, and reduction of CX3CR1 in classical monocytes after treatment in the group with Diabetes (p = 0.003; p = 0.006; p = 0.039; p = 0.007; p = 0.006; p = 0.004; p = 0.019, respectively), without differences in the group without Diabetes. The expression of the chemokine receptors CCR2 and CCR5, in patients with Periodontitis associated with Diabetes, is favorably modified after the end of the non-surgical periodontal treatment.

A novel CXCR4-targeted peptide for SPECT/CT imaging in tumor

As the most commonly expressed chemokine receptor in tumor cells, CXCR4 is associated with the development and metastasis of a variety of tumors, which has always been an important target for tumor diagnosis and treatment. We present a series of peptides designed to target CXCR4 and determine their affinity values (Kd) for human CXCR4 proteins using surface ion resonance technology. Despite the significant variations in their affinities, our results demonstrate the feasibility of our peptide engineering strategy. Among them, YQ-X-9 (Gly-Gly-Pro-D-Tyr-Arg-D-Val-Gly-Arg-D-2Nal-Pro-NH2) had the highest affinity (Kd=530 nM) compared to the positive control AMD3100 (Kd=240 nM). Furthermore, YQ-X-9 was selected as the target and 99 mTc was functionalized by HYNIC to form a specific targeting probe (99 mTc-HYNIC-YQ-X-9). Additionally, we established a xenograft mouse model of B16 melanoma cells for in vivo SPECT/CT imaging, resulting in evident tumor uptake, excellent imaging efficacy, and high contrast. And in vivo distribution experiments confirmed that in B16 tumor-bearing mice, tumors had significant uptake, reaching 2.4% ID/g. Therefore, 99 mTc-HYNIC-YQ-X-9 may serve as a promising peptide-based probe for clinical imaging of CXCR4 overexpressed tumors.’

Main circulating CD8<sup>+</sup> T cell subsets in patients with systemic lupus erythematosus

Relevance. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by loss of immune tolerance and sustained production of autoantibodies.The aim of the study – to compare composition of peripheral blood cytotoxic CD8+ T lymphocytes (Tc) subsets and assess the clinical significance of them in systemic lupus erythematosus. Materials and methods. A total of 35 SLE patients and 49 healthy volunteers were included in the study. Phenotyping of peripheral blood T cell subpopulations was carried out by means of flow cytometry. T lymphocytes were determined using CD3+, CD4+, CD8+ antibodies. Tc were identified by using CD45RA and CD62L antibodies. Also the expression of chemokine receptors (CCR4, CCR6, CXCR3 and CXCR5) on Tc cells was assessed and the main Tc subpopulations were determined: Type 1 (Tc1), type 2 (Tc2), type 17 (Tc17), type 17/1 (Tc17.1), type 17/22 (Tc17.22) cytotoxic cells and T follicular cytotoxic cells (Tfc).Results. The absolute and relative number of Tc was significantly higher in the group of patients with SLE compared with the control group. Additionally, there was a significant decrease in the relative number of Tc1, Tc 17.1 and Tfc1 and a significant increase in the relative number of Tc2, Tfc 17 and Tfc17.1 within the SLE group when compared to the control group. There were significant positive correlationfor Tc1 and levels of C3 and C4 complement components (r=0.404, p<0.05).Conclusions. The absolute and relative number of peripheral blood Tc subsets is altered in SLE patients compared with the control group. It was found that patients with SLE contained increased number of Tc2 cells, which seems to be associated with markers of disease activity. These results demonstrate a prominent pathological role of Tc2 in SLE. While Tc1, Tc17, Tc17.1, Tfc subsets probably have regulatory functions

Directing the migration of serum-free, ex vivo-expanded Vγ9Vδ2 T cells.

Vγ9Vδ2 T cells represent a promising cancer therapy platform because the implementation of allogenic, off-the-shelf product candidates is possible. However, intravenous administration of human Vγ9Vδ2 T cells manufactured under good manufacturing practice (GMP)-compliant, serum-free conditions are not tested easily in most mouse models, mainly because they lack the ability to migrate from the blood to tissues or tumors. We demonstrate that these T cells do not migrate from the circulation to the mouse bone marrow (BM), the site of many malignancies. Thus, there is a need to better characterize human γδ T-cell migration in vivo and develop strategies to direct these cells to in vivo sites of therapeutic interest. To better understand the migration of these cells and possibly influence their migration, NSG mice were conditioned with agents to clear BM cellular compartments, i.e., busulfan or total body irradiation (TBI), or promote T-cell migration to inflamed BM, i.e., incomplete Freund's adjuvant (IFA), prior to administering γδ T cells. Conditioning with TBI, unlike busulfan or IFA, increases the percentage and number of γδ T cells accumulating in the mouse BM, and cells in the peripheral blood (PB) and BM display identical surface protein profiles. To better understand the mechanism by which cells migrate to the BM, mice were conditioned with TBI and administered γδ T cells or tracker-stained red blood cells. The mechanism by which γδ T cells enter the BM after radiation is passive migration from the circulation, not homing. We tested if these ex vivo-expanded cells can migrate based on chemokine expression patterns and showed that it is possible to initiate homing by utilizing highly expressed chemokine receptors on the expanded γδ T cells. γδ T cells highly express CCR2, which provides chemokine attraction to C-C motif chemokine ligand 2 (CCL2)-expressing cells. IFNγ-primed mesenchymal stromal cells (MSCs) (γMSCs) express CCL2, and we developed in vitro and in vivo models to test γδ T-cell homing to CCL2-expressing cells. Using an established neuroblastoma NSG mouse model, we show that intratumorally-injected γMSCs increase the homing of γδ T cells to this tumor. These studies provide insight into the migration of serum-free, ex vivo-expanded Vγ9Vδ2 T cells in NSG mice, which is critical to understanding the fundamental properties of these cells.

Exploring the Interplay of RUNX2 and CXCR4 in Melanoma Progression.

Overexpression of the Runt-related transcription factor 2 (RUNX2) has been reported in several cancer types, and the C-X-C motif chemokine receptor 4 (CXCR4) has an important role in tumour progression. However, the interplay between CXCR4 and RUNX2 in melanoma cells remains poorly understood. In the present study, we used melanoma cells and a RUNX2 knockout (RUNX2-KO) in vitro model to assess the influence of RUNX2 on CXCR4 protein levels along with its effects on markers associated with cell invasion and autophagy. Osteotropism was assessed using a 3D microfluidic model. Moreover, we assessed the impact of CXCR4 on the cellular levels of key cellular signalling proteins involved in autophagy. We observed that melanoma cells express both RUNX2 and CXCR4. Restored RUNX2 expression in RUNX2 KO cells increased the expression levels of CXCR4 and proteins associated with the metastatic process. The protein markers of autophagy LC3 and beclin were upregulated in response to increased CXCR4 levels. The CXCR4 inhibitor WZ811 reduced osteotropism and activated the mTOR and p70-S6 cell signalling proteins. Our data indicate that the RUNX2 transcription factor promotes the expression of the CXCR4 chemokine receptor on melanoma cells, which in turn promotes autophagy, cell invasiveness, and osteotropism, through the inhibition of the mTOR signalling pathway. Our data suggest that RUNX2 promotes melanoma progression by upregulating CXCR4, and we identify the latter as a key player in melanoma-related osteotropism.

RBP-J regulates homeostasis and function of circulating Ly6Clo monocytes.

Notch-RBP-J signaling plays an essential role in the maintenance of myeloid homeostasis. However, its role in monocyte cell fate decisions is not fully understood. Here, we showed that conditional deletion of transcription factor RBP-J in myeloid cells resulted in marked accumulation of blood Ly6Clo monocytes that highly expressed chemokine receptor CCR2. Bone marrow transplantation and parabiosis experiments revealed a cell-intrinsic requirement of RBP-J for controlling blood Ly6CloCCR2hi monocytes. RBP-J-deficient Ly6Clo monocytes exhibited enhanced capacity competing with wildtype counterparts in blood circulation. In accordance with alterations of circulating monocytes, RBP-J deficiency led to markedly increased population of lung tissues with Ly6Clo monocytes and CD16.2+ interstitial macrophages. Furthermore, RBP-J deficiency-associated phenotypes could be genetically corrected by further deleting Ccr2 in myeloid cells. These results demonstrate that RBP-J functions as a crucial regulator of blood Ly6Clo monocytes and thus derived lung-resident myeloid populations, at least in part through regulation of CCR2.

RBP-J regulates homeostasis and function of circulating Ly6Clo monocytes

Notch-RBP-J signaling plays an essential role in the maintenance of myeloid homeostasis. However, its role in monocyte cell fate decisions is not fully understood. Here, we showed that conditional deletion of transcription factor RBP-J in myeloid cells resulted in marked accumulation of blood Ly6Clo monocytes that highly expressed chemokine receptor CCR2. Bone marrow transplantation and parabiosis experiments revealed a cell-intrinsic requirement of RBP-J for controlling blood Ly6CloCCR2hi monocytes. RBP-J-deficient Ly6Clo monocytes exhibited enhanced capacity competing with wildtype counterparts in blood circulation. In accordance with alterations of circulating monocytes, RBP-J deficiency led to markedly increased population of lung tissues with Ly6Clo monocytes and CD16.2+ interstitial macrophages. Furthermore, RBP-J deficiency-associated phenotypes could be genetically corrected by further deleting Ccr2 in myeloid cells. These results demonstrate that RBP-J functions as a crucial regulator of blood Ly6Clo monocytes and thus derived lung-resident myeloid populations, at least in part through regulation of CCR2.

Ultrasonic microbubbles promote mesenchymal stem cell homing to the fibrotic liver via upregulation of CXCR4 expression

ObjectiveTo investigate the mechanism of ultrasound microbubbles (UTMB) promoting stem cells homing to fibrotic liver.MethodsBone marrow derived mesenchymal stem cells (BMSCs) were divided into 5 groups with or without ultrasound microbubbles and continuously irradiated with ultrasound conditions of frequency 1 MHZ and output power 0.6 W/cm2 for different times, and then injected into a mouse model of liver fibrosis through the tail vein with or without ultrasound microbubbles, with sound intensity. The effect of ultrasound microbubbles on MSC expression of CXC chemokine receptor 4 (CXCR4) and homing fibrotic liver was evaluated by flow cytometry (FCM), western blot (WB) and immunohistochemistry (IHC) analysis.ResultsThe level of CXCR4 expression was significantly higher in the ultrasound microbubble group than in the non-intervention group (P < 0.05), and the number of MSC and the rate of CXCR4 receptor positivity in the ultrasound microbubble-treated liver tissues were significantly higher than in the non-intervention group (P < 0.01).ConclusionUltrasonic microbubbles can promote the expression of CXCR4 on the surface of MSCs, thus improving the homing rate of MSCs in fibrotic liver.

Bioengineered MSCGFPCxcr2-Mmp13 Transplantation Alleviates Hepatic Fibrosis by Regulating Mammalian Target of Rapamycin Signaling.

Aims: Hepatic fibrosis is the pathological change during chronic liver diseases (CLD) that turns into cirrhosis if not reversed timely. Allogenic mesenchymal stem cell (MSC) therapy is an alternative to liver transplantation for CLD. However, poor engraftment of the transplanted MSCs limits their therapeutic efficacy. MSCs express chemokine receptors that regulate their physiology. We observed several-fold increased expressions of Cxcl3 and decreased expression of Mmp13 in the fibrotic liver. Therefore, we bioengineered MSCs with stable overexpression of Cxcr2 (CXCL3-cognate receptor) and Mmp13, collagenase (MSCGFPCxcr2-Mmp13). Results: The CXCL3/CXCR2 axis significantly increased migration through the activation of AKT/ERK/mTOR signaling. These bioengineered MSCs transdifferentiated into hepatocyte-like cells (MSCGFPCxcr2-Mmp13-HLCs) that endured the drug-/hepatotoxicant-induced toxicity by significantly increasing the antioxidants-Nrf2 and Sod2, while decreasing the apoptosis-Cyt C, Casp3, Casp9, and drug-metabolizing enzyme-Cyp1A1, Cyp1A2, Cyp2E1 markers. Therapeutic transplantation of MSCGFPCxcr2-Mmp13 abrogated AAP-/CCl4-induced hepatic fibrosis in mice by CXCR2-mediated targeted engraftment and MMP-13-mediated reduction in collagen. Mechanistically, induction of CXCL3/CXCR2 axis-activated mTOR-p70S6K signaling led to increased targeted engraftment and modulation of the oxidative stress by increasing the expression and activity of nuclear Nrf2 and SOD2 expression in the regenerated hepatic tissues. A marked change in the fate of transplanted MSCGFPCxcr2-Mmp13 toward hepatocyte lineage demonstrated by co-immunostaining of GFP/HNF4α along with reduced COL1α1 facilitated the regeneration of the fibrotic liver. Innovation and Conclusions: Our study suggests the therapeutic role of allogenic Cxcr2/Mmp13-bioengineered MSC transplantation decreases the hepatic oxidative stress as an effective translational therapy for hepatic fibrosis mitigation-mediated liver regeneration.

G-CSF increases calprotectin expression, liver damage and neuroinflammation in a murine model of alcohol-induced ACLF.

Background and aims: Granulocyte colony-stimulating factor (G-CSF) has been proposed as a therapeutic option for patients with ACLF, however clinical outcomes are controversial. We aimed at dissecting the role of G-CSF in an alcohol-induced murine model of ACLF. Methods: ACLF was triggered by a single alcohol binge (5g/kg) in a bile duct ligation (BDL) liver fibrosis model. A subgroup of mice received two G-CSF (200μg/kg) or vehicle injections prior to acute decompensation with alcohol. Liver, blood and brain tissues were assessed. Results: Alcohol binge administered to BDL-fibrotic mice resulted in features of ACLF indicated by a significant increase in liver damage and systemic inflammation compared to BDL alone. G-CSF treatment in ACLF mice induced an increase in liver regeneration and neutrophil infiltration in the liver compared to vehicle-treated ACLF mice. Moreover, liver-infiltrating neutrophils in G-CSF-treated mice exhibited an activated phenotype indicated by increased expression of CXC motif chemokine receptor 2, leukotriene B4 receptor 1, and calprotectin. In the liver, G-CSF triggered increased oxidative stress, type I interferon response, extracellular matrix remodeling and inflammasome activation. Circulating IL-1β was also increased after G-CSF treatment. In the cerebellum, G-CSF increased neutrophil infiltration and S100a8/9 expression, induced microglia proliferation and reactive astrocytes, which was accompanied by oxidative stress, and inflammasome activation compared to vehicle-treated ACLF mice. Conclusion: In our novel ACLF model triggered by alcohol binge that mimics ACLF pathophysiology, neutrophil infiltration and S100a8/9 expression in the liver and brain indicate increased tissue damage, accompanied by oxidative stress and inflammasome activation after G-CSF treatment.

SQLE is a promising prognostic and immunological biomarker and correlated with immune Infiltration in Sarcoma.

Squalene epoxidase (SQLE) is an essential enzyme involved in cholesterol biosynthesis. However, its role in sarcoma and its correlation with immune infiltration remains unclear. All original data were downloaded from The Cancer Genome Atlas (TCGA). SQLE expression was explored using the TCGA database, and correlations between SQLE and cancer immune characteristics were analyzed via the TISIDB databases. Generally, SQLE is predominantly overexpressed and has diagnostic and prognostic value in sarcoma. Upregulated SQLE was associated with poorer overall survival, poorer disease-specific survival, and tumor multifocality in sarcoma. Mechanistically, we identified a hub gene that included a total of 82 SQLE-related genes, which were tightly associated with histone modification pathways in sarcoma patients. SQLE expression was negatively correlated with infiltrating levels of dendritic cells and plasmacytoid dendritic cells and positively correlated with Th2 cells. SQLE expression was negatively correlated with the expression of chemokines (CCL19 and CX3CL1) and chemokine receptors (CCR2 and CCR7) in sarcoma. In conclusion, SQLE may be used as a prognostic biomarker for determining prognosis and immune infiltration in sarcoma.

Volumetric Parameters Derived from CXCR4-Directed PET/CT Predict Outcome in Patients with Gastrointestinal Neuroendocrine Carcinomas

BackgroundGastro-entero-pancreatic neuroendocrine carcinomas (GEP-NECs) are an aggressive subgroup of neuroendocrine neoplasms (NENs). In patients affected with NEN, there is a growing body of evidence that increased C-X-C motif chemokine receptor (CXCR4) expression is linked to decreasing overall survival (OS) in an ex-vivo setting. Thus, we aimed to determine whether the in-vivo-derived CXCR4-directed whole-body PET signal can also determine GEP-NEC patients with shorter OS.MethodsWe retrospectively included 16 patients with histologically proven GEP-NEC, who underwent CXCR4-directed PET/CT for staging and therapy planning. We assessed maximum, peak, and mean standardized uptake values as well as whole-body tumor volume (TV) and total-lesion uptake (TLU = SUVmean × TV) using a semi-automatic segmentation tool with a 50% threshold. Association of PET-based biomarkers and OS or radiographic progression-free survival (rPFS; according to RECIST 1.1 criteria) was analyzed using univariable and multivariable cox regression.ResultsMedian OS and rPFS was 7.5 and 7 months, respectively. A significant correlation between TV and TLU was found for OS (TV: hazard ratio (HR) 1.007 95% confidence interval (CI) 1.000–1.014, p = 0.0309; TLU: HR 1.002 95% CI 1.000–1.003, p = 0.0350) and rPFS (TV: HR 1.010 95% CI 1.002–1.021; p = 0.0275; TLU: HR 1.002 95% CI 1.000–1.004, p = 0.0329), respectively. No significant correlation with OS or rPFS was found for non-volumetric parameters (p > 0.4). TV remained a significant predictive marker for OS and rPFS in multivariable analysis (OS: HR 1.012 95%, CI 1.003–1.022, p = 0.0084; rPFS: HR 1.009, 95% CI 0.9999–1.019, p = 0.0491), whereas TLU remained only prognostic for OS (HR 1.009, 95% CI 0.9999–1.019, p = 0.0194) but narrowly failed significance for rPFS (p = 0.0559).ConclusionIn-vivo assessment of CXCR4 PET-derived volumetric parameters is predictive for outcome of patients with GEP-NEC and could be used as a risk stratification tool, which detects patients prone to early progression.

Abstract B032: Compromised TGFβ signaling and DNA repair primes response to immune checkpoint inhibition by activating NK cells in immunologically cold breast cancers

Abstract We investigated the interplay between immunosuppressive TGFβ signaling and immunogenic DNA repair in response to immune checkpoint inhibition (ICI) in a new model of breast cancer. Transforming growth factor beta (TGFβ) is a canonical suppressor. Cancer cells evade TGFβ either by selectively overcoming TGFβ proliferative barrier or completely shutting down signaling. Loss of TGFβ signaling is accompanied by high immunosuppressive TGFβ activity. We have shown that loss of TGFβ is critical to genomic integrity. It endorses classical DNA repair and inhibits error-prone repair. We described a score, βAlt, that reports the TGFβ/DNA repair functional relationship. Pan-cancer analysis showed that high βAlt cancers, in which the TGFβ target signature is low and DNA repair signature is high, use error-prone DNA repair, are sensitive to chemoradiation and have a greater fraction of the genome altered compared to low βAlt cancers, which maintain TGFβ signaling. We established a novel murine tumor derived transplant model (mTDT) of triple-negative breast cancer, similar to a human PDX, in immunocompetent BALB/cJ mice. Transcriptomics analysis showed that mTDT range from high to low βAlt and from immune poor to immune rich, which was confirmed by multiplex immunstaining. Unsupervised clustering of mTDT based on βAlt score demonstrated that low βAlt was associated with immune rich tumors while high βAlt was associated with immune poor tumors. A similar relationship is evident in the TCGA breast cancers. We then asked how mTDT respond to ICI. Mice bearing mTDT were randomized to monotherapy consisting of single fraction 10 Gy radiation, anti-PD-L1 or TGFβ inhibition, dual or triple treatment. Neither anti-PD-L1 or TGFβ inhibition affected survival, alone or in combination, whereas radiation significantly increased survival. Unexpectedly immune poor, high βAlt mTDT were the most responsive. To evaluate biological correlates, mice were stratified as a function of treatment as responders, in which tumor growth was controlled for 7 days post-treatment, or non-responders, in which tumor growth was uncontrolled. Tumor infiltrating CD45+ cells increased (P=0.02) as did circulating CD8 T cells (P&amp;lt;0.001) in responders. Natural killer (NK) cells were also significantly expanded in responders (P&amp;lt;0.001). TGFβ broadly downregulates chemokine receptor expression necessary to recruit NK cells and impairs NK cell cytotoxic effector function. We depleted NK cells to test whether they were necessary for the pronounced response to triple treatment. NK depletion was completely abrogated response (P&amp;lt;0.00001) indicating NK cells can provoke an ICI response in high βAlt mammary tumors devoid of lymphocytes. This preclinical model and human breast cancer shows that loss of TGFβ signaling resulting in error-prone DNA damage response is strongly associated with immune poor tumors, and suggests that TGFβ inhibition can override NK cell dysfunction to create vulnerability to ICI and anti-tumor immunity. Citation Format: Jade Moore, Jim Gkantalis, Ines Guix, Colin Foster, Mary Helen Barcellos-Hoff. Compromised TGFβ signaling and DNA repair primes response to immune checkpoint inhibition by activating NK cells in immunologically cold breast cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B032.

CCR10-mediated Enhancement of T Cell Trafficking for Improved Tumor Immunotherapy.

The effectiveness of adoptive T cell therapy for solid tumors remains suboptimal, partly attributed to insufficient T cell infiltration into the tumor site. A promising strategy involves directing T cells towards the tumor utilizing tumor-specific chemokine receptors. We analyzed chemokine receptor expression in activated T cells and chemokine expression in breast and lung cancer using The Cancer Genome Atlas (TCGA) data. Subsequently, we generated 1G4 T cell receptor-engineered T (TCR-T) cells with CCR10 and performed in vitro and in vivo efficacy tests. CCR10 exhibited insufficient expression in various human T cells. Analysis of TCGA RNA sequencing data revealed elevated expression of the chemokine CCL28, the corresponding chemokine for CCR10, in breast and lung cancer. Consequently, we generated CCR10-1G4 TCR-T cells. CCR10-1G4 dual expressing TCR-T cells exhibited comparable cellular cytotoxicity but increased mobility compared to 1G4 TCR-T cells in vitro. Furthermore, injecting CCR10-1G4 dual expressing TCR-T cells into a xenograft tumor model demonstrated enhanced in vivo trafficking and a greater reduction of tumor burden. This study highlights the potential of CCR10 for developing efficient adoptive T-cell treatments targeting solid tumors.

Dynamics of Whole Transcriptome Analysis (WTA) and Surface markers expression (AbSeq) in Immune Cells of COVID-19 Patients and Recovered captured through Single Cell Genomics.

Single-cell multi-omics studies, such as multidimensional transcriptomics (whole transcriptomic analysis, WTA), and surface marker analysis (antibody sequencing, AbSeq), have turned out to be valuable techniques that offer inaccessible possibilities for single-cell profiling of mRNA, lncRNA, and proteins. We used this technique to understand the dynamics of mRNA and protein-level differences in healthy, COVID-19-infected and recovered individuals using peripheral blood mononuclear cells (PBMCs). Our results demonstrate that compared to mRNA expression, protein abundance is a better indicator of the disease state. We demonstrate that compared to mRNA expression, protein abundance is a better indicator of the disease state. We observed high levels of cell identity and regulatory markers, CD3E, CD4, CD8A, CD5, CD7, GITR, and KLRB1 in healthy individuals, whereas markers related to cell activation, CD38, CD28, CD69, CD62L, CD14, and CD16 elevated in the SARS-CoV-2 infected patients at both WTA and AbSeq levels. Curiously, in recovered individuals, there was a high expression of cytokine and chemokine receptors (CCR5, CCR7, CCR4, CXCR3, and PTGRD2). We also observed variations in the expression of markers within cell populations under different states. Furthermore, our study emphasizes the significance of employing an oligo-based method (AbSeq) that can help in diagnosis, prognosis, and protection from disease/s by identifying cell surface markers that are unique to different cell types or states. It also allows simultaneous study of a vast array of markers, surpassing the constraints of techniques like FACS to query the vast repertoire of proteins.