Integrative analysis of bulk and single-cell gene expression profiles to identify tumor-associated macrophage-derived CCL18 as a therapeutic target of esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a common gastrointestinal malignancy with poor patient prognosis. Current treatment for ESCC, including immunotherapy, is only beneficial for a small subset of patients. Better characterization of the tumor microenvironment (TME) and the development of novel therapeutic targets are urgently needed. METHODS: In the present study, we hypothesized that integration of single-cell transcriptomic sequencing and large microarray sequencing of ESCC biopsies would reveal the key cell subtypes and therapeutic targets that determine the prognostic and tumorigenesis of ESCC. We characterized the gene expression profiles, gene sets enrichment, and the TME landscape of a microarray cohort including 84 ESCC tumors and their paired peritumor samples. We integrated single-cell transcriptomic sequencing and bulk microarray sequencing of ESCC to reveal key cell subtypes and druggable targets that determine the prognostic and tumorigenesis of ESCC. We then designed and screened a blocking peptide targeting Chemokine C-C motif ligand 18 (CCL18) derived from tumor associated macrophages and validated its potency by MTT assay. The antitumor activity of CCL18 blocking peptide was validated in vivo by using 4-nitroquinoline-1-oxide (4-NQO) induced spontaneous ESCC mouse model. RESULTS: Comparative gene expression and cell-cell interaction analyses revealed dysregulated chemokine and cytokine pathways during ESCC carcinogenesis. TME deconvolution and cell interaction analyses allow us to identify the chemokine CCL18 secreted by tumor associated macrophages could promote tumor cell proliferation via JAK2/STAT3 signaling pathway and lead to poor prognosis of ESCC. The peptide Pep3 could inhibit the proliferation of EC-109 cells promoted by CCL18 and significantly restrain the tumor progression in 4-NQO-induced spontaneous ESCC mouse model. CONCLUSIONS: For the first time, we discovered and validated that CCL18 blockade could significantly prevent ESCC progression. Our study revealed the comprehensive cell-cell interaction network in the TME of ESCC and provided novel therapeutic targets and strategies to ESCC treatment.

Chemokine CCL18 Promotes Phagocytosis Through Its Receptor CCR8 Rather than PITPNM3 in Human Microglial Cells.

CCL18 is a CC chemokine that exhibits diverse functions through interaction with various cell subsets with both proinflammatory anti-inflammatory properties through its receptors CCR8 (CC chemokine receptor 8) and PITPNM3 (phosphatidylinositol transfer protein 3). However, the function of CCL18 in microglia remains unclear. In this study, we show that CCL18 did not change the expression of the inflammatory factors, interleukin (IL)-1ß, IL-6, tumor necrosis factor alpha (TNF-α), or inducible nitric oxide synthase (iNOS), but significantly induced expression of the macrophage markers, MRC-1 and ARG-1 M2, in a human microglial clone 3 cell line (HMC3). Phagocytosis by HMC3 cells was significantly enhanced in the presence of CCL18, indicated by uptake of amyloid-ß and dextran. CCR8 and PITPNM3 were both expressed on HMC3 cells, but selective knockdown of CCR8 and PITPNM3 showed that only the former played a dominant role in phagocytosis of HMC3 through the nuclear factor kappa B (NF-κB)/Src signaling pathway. Our results suggest that CCL18 could have anti-inflammatory activity and activate the phagocytic function of microglia, which is involved in neural development, homeostasis, and repair mechanisms.

PARC/CCL18 is Associated with Inflammation, Emphysema Severity and Application of Inhaled Corticosteroids in Hospitalized COPD Patients.

BACKGROUND: Pulmonary and activation-regulated chemokine (PARC) also named CC-chemokine ligand 18 (CCL18) is a lung-predominant inflammatory protein that is found in serum. The relationship of PARC/CCL18 with the chronic obstructive pulmonary disease (COPD) is not fully understood. The aim of the present study is to analyze the expression of PARC/CCL18 in COPD. METHODS: Ninety-eight hospitalized COPD patients and 60 healthy volunteers from January 2019 to December 2019 were recruited in this retrospective study. Gender, age, height, weight, disease duration, smoking status, blood cell classification and count, length of hospital stay (LOS), symptom score, including COPD Assessment Test (CAT) score, modified British Medical Research Council (mMRC) score, lung function and therapy were recorded and serum PARC/CCL18 was analyzed by ELISA. The correlation between symptom score, blood cell classification and count, CRP, lung function parameters and serum levels of PARC/CCL18 and ROC curves of PARC/CCL18 levels and inhaled corticosteroids (ICS) were accessed. RESULTS: It was found that serum PARC/CCL18 level in hospitalized COPD population was significantly higher than that in healthy people (p=0.003). COPD patients with emphysema had significantly higher serum level of PARC/CCL18 than those without emphysema (p=0.049). Total lung capacity (TLC) and residual volume (RV)/TLC had positive correlation with serum level of PARC/CCL18 (p=0.001, 0.020, respectively). Furthermore, serum PARC/CCL18 level was predictive for the application ICS (p=0.003) and related to C-reactive protein (p <0.0001) in hospitalized COPD patients. CONCLUSION: PARC/CCL18 is associated with the severity of inflammation and emphysema in COPD. Furthermore, PARC/CCL18 is a predictor of ICS application in the treatment of hospitalized COPD patients.

Controlling porous titanium/soft tissue interactions with an innovative surface chemical treatment: Responses of macrophages and fibroblasts.

In order to create a stable interface with the host tissue, porous implants are widely used to ensure the in-growth of the cells and the colonization of the implant. An ideal porous implant should have a 3D architecture that enables fast migration of incoming cells while not inducing a significant pro-inflammatory response by the immune cells. Moreover, in patients where the healing is impeded (patients with co-morbidities and metabolic diseases), porosity by itself is not enough for fast colonization, and the surface properties of the implant should also be controlled. In this study, we present a controlled oxidation-based surface treatment of microbead-based porous titanium implants which not only increases the colonization by connective tissue cells but also decreases the macrophage attachment. The treatment created a nanotextured surface on the implants with an acidic shift of isoelectric point (from 4.09 to 3.09) without endangering implant's mechanical integrity. The attachment and metabolic activity of activated macrophages were significantly lower on treated surfaces with an increase in the secretion of anti-inflammatory IL-1RA and a decrease in pro-fibrotic CCL-18. Human fibroblasts proliferated faster on the treated surfaces over 14 days with near complete colonization of the whole thickness of the implant with an accompanying an increase in the secretion of TGF-beta. The surface treated samples demonstrated partial filling of the entire pores. We demonstrated that the use of nanoscale surface treatments that can be applied to the whole internal surface of porous titanium implants can significantly alter both the immune response and the colonization of the implants and can be used to fine-tune and personalize implant interfaces according to patient needs.

LC-MS/MS analysis of plasma glucosylsphingosine as a biomarker for diagnosis and follow-up monitoring in Gaucher disease in the Spanish population.

Background Gaucher disease (GD), caused by a deficiency in acid ß-glucosidase, leads to the accumulation of glucosylsphingosine (GluSph), which has been used as a powerful biomarker for the diagnosis and follow-up of GD. Our aim was to perform the first retrospective study of GluSph in Spanish patients, analyzing its relationship with classical biomarkers and other parameters of disease and its utility regarding treatment monitoring. Methods Classical biomarkers were evaluated retrospectively by standard methods in a total of 145 subjects, including 47 GD patients, carriers, healthy controls and patients suffering from other lysosomal lipidoses. GluSph was also measured using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method developed as part of the present study. Results The optimized method presented intra- and inter-assay variations of 3.1 and 11.5%, respectively, overall recovery higher than 96% and linearity up to plasma concentrations of 1000 ng/mL with 100% specificity and sensitivity. Only GD patients displayed GluSph levels above 5.4 ng/mL at diagnosis and this was significantly correlated with the classical biomarkers chitotriosidase (r = 0.560) and the chemokine CCL18/PARC (CCL18/PARC) (ρ = 0.515), as well as with the Spanish magnetic resonance imaging index (S-MRI, r = 0.364), whereas chitotriosidase correlated with liver volume (r = 0.372) and CCL18/PARC increased in patients with bone manifestations (p = 0.005). GluSph levels decreased with treatment in naïve patients. Conclusions Plasma GluSph is the most disease-specific biomarker for GD with demonstrated diagnostic value and responsiveness to therapy. GluSph in the present series of patients failed to demonstrate better correlations with clinical characteristics at onset than classical biomarkers.

Interleukin-32θ inhibits tumor-promoting effects of macrophage-secreted CCL18 in breast cancer.

BACKGROUND: Tumor-associated macrophages can promote breast cancer metastasis by secreting cytokines and growth factors. Interleukin (IL)-32θ, a newly identified IL-32 isoform, was previously shown to down-regulate various proinflammatory factors of macrophages. Here, we report the presence of IL-32θ in breast cancer tissues and evaluate its effects on macrophage-regulated breast cancer metastasis. METHODS: RT-qPCR was used to analyze the mRNA expression of IL-32θ, Chemokine (C-C motif) ligand 18 (CCL18) in breast cancer tissues. In vitro cell-based experiments using IL-32θ-expressing MDA-MB-231 cells were conducted to examine the effects of IL-32θ on metastasis and its molecular signaling. In vivo xenograft, immunohistochemistry, and optical imaging models were generated to support in vitro and clinical findings. RESULTS: The clinical data displayed opposite expression patterns of CCL18 and IL-32θ mRNA in macrophage-infiltrated breast tumor tissues compared with those in the other tissues tested. In MDA-MB-231 cells, IL-32θ overexpression attenuated migration, invasion, tumor-promoting factors, and increased epithelial markers levels upon treatment with conditioned media from THP-1-derived macrophages. Additionally, IL-32θ expression in a xenograft model led to a remarkable decrease in tumor size and macrophage-stimulated tumor promotion. This inhibition was mediated through a direct interaction with protein kinase C-δ (PKCδ), subsequently eliminating the downstream factors STAT3 and NF-κB. Blocking CCL18 during co-culture of macrophages and breast cancer cells reduced the levels of breast cancer progression-related factors and PKCδ downstream signaling suggesting CCL18 as the main macrophage-secreted factors triggering the signaling pathway inhibited by IL-32θ. CONCLUSIONS: Our findings demonstrate a novel role of IL-32θ as an intracellular modulator to suppress macrophage-promoted breast cancer progression by targeting CCL18-dependent signaling.

Idiopathic Pulmonary Fibrosis: Prospective, Case-Controlled Study of Natural History and Circulating Biomarkers.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with 3 to 5 years' survival. Although FVC is used to assess disease progression and treatment response, identifying predictive circulating blood biomarkers could help identify specific biologic pathways for treatment. An international, prospective, noninterventional, case-controlled, 52-week study was therefore conducted to identify a clinical and biomarker baseline profile predictive of longitudinal disease behavior. METHODS: Patients with IPF and control subjects had lung function tests and blood sampling for biomarker quantification (control subjects at baseline only). The primary end point was disease progression rate (composite end point: decrease ≥ 10% from baseline in FVC % predicted, decrease ≥ 15% from baseline in diffusing capacity of the lung for carbon monoxide % predicted, lung transplantation, death) at week 52 and its relationship to selected biomarkers at baseline. RESULTS: Altogether, 211 subjects (154 patients with IPF and 57 control subjects) were enrolled; one-third of patients (n = 47) with IPF had progressed by week 52. Biomarkers CC-chemokine ligand 18 (CCL18), intercellular adhesion molecule 1, Krebs von den Lungen-6, surfactant protein (SP)-A, SP-D, matrix metallopeptidase 7, urokinase-type plasminogen activator receptor, and two novel biomarkers, human epididymis protein-4 (HE4) and prostasin, discriminated patients with IPF vs control subjects. There was no difference in baseline CCL18 concentration between progressors and nonprogressors at week 52 (area under the receiver operating characteristic curve, 0.62; corrected P = .161). No biomarkers were predictive for disease progression. CONCLUSIONS: Several biomarkers, including CCL18, were associated with IPF, but none predicted disease progression. Two novel biomarkers, HE4 and prostasin, were identified and warrant further investigation.