Single-cell profiling reveals the metastasis-associated immune signature of hepatocellular carcinoma.

AIM: Metastasis is the leading cause of mortality in hepatocellular carcinoma (HCC). The metastasis-associated immune signature in HCC is worth exploring. METHODS: Bioinformatic analysis was conducted based on the single-cell transcriptome data derived from HCC patients in different stages. Cellular composition, pseudotime state transition, and cell-cell interaction were further analyzed and verified. RESULTS: Generally, HCC with metastasis exhibited suppressive immune microenvironment, while HCC without metastasis exhibited active immune microenvironment. Concretely, effector regulatory T cells (eTregs) were found to be enriched in HCC with metastasis. PHLDA1 was identified as one of exhaustion-specific genes and verified to be associated with worse prognosis in HCC patients. Moreover, A novel cluster of CCR7+ dendritic cells (DCs) was identified with high expression of maturation and migration marker genes. Pseudotime analysis showed that inhibition of differentiation occurred in CCR7+ DCs rather than cDC1 in HCC with metastasis. Furthermore, interaction analysis showed that the reduction of CCR7+ DCs lead to impaired CCR7/CCL19 interaction in HCC with metastasis. CONCLUSIONS: HCC with metastasis exhibited upregulation of exhaustion-specific genes of eTregs and inhibition of CCL signal of a novel DC cluster, which added new dimensions to the immune landscape and provided new immune therapeutic targets in advanced HCC.

CCR2-dependent CX3CR1+ colonic macrophages promote Enterococcus faecalis dissemination.

Enterococci are common commensal bacteria that colonize the gastrointestinal tracts of most mammals, including humans. Importantly, these bacteria are one of the leading causes of nosocomial infections. This study examined the role of colonic macrophages in facilitating Enterococcus faecalis infections in mice. We determined that depletion of colonic phagocytes resulted in the reduction of E. faecalis dissemination to the gut-draining mesenteric lymph nodes. Furthermore, we established that trafficking of monocyte-derived CX3CR1-expressing macrophages contributed to E. faecalis dissemination in a manner that was not reliant on CCR7, the conventional receptor involved in lymphatic migration. Finally, we showed that E. faecalis mutants with impaired intracellular survival exhibited reduced dissemination, suggesting that E. faecalis can exploit host immune cell migration to disseminate systemically and cause disease. Our findings indicate that modulation of macrophage trafficking in the context of antibiotic therapy could serve as a novel approach for preventing or treating opportunistic infections by disseminating enteric pathobionts like E. faecalis.

Analysis of the effect of CCR7 on the microenvironment of mouse oral squamous cell carcinoma by single-cell RNA sequencing technology.

BACKGROUND: Studies have shown that CCR7, an important inflammatory factor, can promote the proliferation and metastasis of oral squamous cell carcinoma (OSCC), but its role in the tumor microenvironment (TME) remains unclear. This paper explores the role of CCR7 in the TME of OSCC. METHODS: In this work, we constructed CCR7 gene knockout mice and OSCC mouse models. Single-cell RNA sequencing (scRNA-seq) and bioinformatics were used to analyze the differences in the OSCC microenvironment between three CCR7 gene knockout mice (KO) and three wild-type mice (WT). Immunohistochemistry, immunofluorescence staining, and flow cytometry were used to analyze the expression of key genes in significantly different cell types between the KO and WT groups. An in vitro experiment was used to verify the effect of CCR7 on M2 macrophage polarization. RESULTS: In the mouse OSCC models, the tumor growth rate in the KO group was significantly lower than that in the WT group. Eight main cell types (including tumor cells, fibroblasts, macrophages, granulocytes, T cells, endothelial cells, monocytes, and B cells) were identified by Seurat analysis. The scRNA-seq results showed that the proportion of tumor cells was lower, but the proportion of inflammatory cells was significantly higher in the KO group than in the WT group. CellPhoneDB analysis results indicated a strong interaction relationship between tumor cells and macrophages, T cells, fibroblasts, and endothelial cells. Functional enrichment results indicated that the expression level of the Dusp1 gene in the KO group was generally higher than that in the WT group in various cell types. Macrophage subclustering results indicated that the proportion of M2 macrophages in the KO group was lower than that in the WT group. In vitro experimental results showed that CCR7 can promote M2 macrophage polarization, thus promoting the proliferation, invasion and migration of OSCC cells. CONCLUSIONS: CCR7 gene knockout can significantly inhibit the growth of mouse oral squamous cell carcinoma by promoting the polarization of M2 macrophages.

Role of CCR1/5/7 in hepatocellular carcinoma: a study on prognostic evaluation, molecular subtyping, and association with immune infiltration.

This study aims to assess the prognostic value of the C-C motif chemokine receptor (CCR) gene family in hepatocellular carcinoma (HCC) and its relationship with immune infiltration and molecular subtypes of HCC. The evaluation of the GSE14520 dataset and TCGA database confirmed the prognostic significance of CCR. Building upon the correlation between CCR1, CCR5, and CCR7 and favorable prognosis, we further validated the prognostic importance of CCR1, CCR5, and CCR7 in ICGC database and an independent cohort from Guangxi autonomous region. Then, we constructed a risk prognosis model. Additionally, we observed significant positive correlations between CCR1, CCR5, and CCR7 and the infiltration of B cells, T cells, and macrophages in HCC. Subsequently, we conducted CCK assays, Transwell assays, and colony formation assays to evaluate the molecular biological functions of CCR1, CCR5, and CCR7. These experiments further confirmed that upregulation of CCR1, CCR5, and CCR7 can individually inhibit the proliferation, migration, and stemness of HCC cells. By analyzing the relationship between expression levels and tumor mutation frequency, we discovered that patients with high CCR1 expression were more likely to be classified as non-proliferative HCC. Similar conclusions were observed for CCR5 and CCR7. The association of CCR1, CCR5, and CCR7 with the molecular subtypes of HCC suggests that they may serve as intermediary molecules linking immune status and molecular subtypes in HCC. In summary, CCR1, CCR5, and CCR7 have the potential to serve as prognostic biomarkers for HCC and regulate HCC progression by influencing immune cell infiltration.

Tumour-retained activated CCR7+ dendritic cells are heterogeneous and regulate local anti-tumour cytolytic activity.

Tumour dendritic cells (DCs) internalise antigen and upregulate CCR7, which directs their migration to tumour-draining lymph nodes (dLN). CCR7 expression is coupled to an activation programme enriched in regulatory molecule expression, including PD-L1. However, the spatio-temporal dynamics of CCR7+ DCs in anti-tumour immune responses remain unclear. Here, we use photoconvertible mice to precisely track DC migration. We report that CCR7+ DCs are the dominant DC population that migrate to the dLN, but a subset remains tumour-resident despite CCR7 expression. These tumour-retained CCR7+ DCs are phenotypically and transcriptionally distinct from their dLN counterparts and heterogeneous. Moreover, they progressively downregulate the expression of antigen presentation and pro-inflammatory transcripts with more prolonged tumour dwell-time. Tumour-residing CCR7+ DCs co-localise with PD-1+CD8+ T cells in human and murine solid tumours, and following anti-PD-L1 treatment, upregulate stimulatory molecules including OX40L, thereby augmenting anti-tumour cytolytic activity. Altogether, these data uncover previously unappreciated heterogeneity in CCR7+ DCs that may underpin a variable capacity to support intratumoural cytotoxic T cells.

ZAP-70 augments tonic B-cell receptor and CCR7 signaling in IGHV-unmutated chronic lymphocytic leukemia.

ABSTRACT: Expression of ZAP-70 in a subset of patients with chronic lymphocytic leukemia (CLL) positively correlates with the absence of immunoglobulin heavy-chain gene (IGHV) mutations and is indicative of a more active disease and shorter treatment-free survival. We recently demonstrated that ZAP-70 regulates the constitutive expression of CCL3 and CCL4, activation of AKT, and expression of MYC in the absence of an overt B-cell receptor (BCR) signal, bona fide functions of BCR activation. We, here, provide evidence that these features relate to the presence of a constitutive tonic BCR signal, exclusively found in IGHV-unmutated CLL and dependent on the ZAP-70-mediated activation of AKT and its downstream target GSK-3ß. These findings are associated with increased steady-state activation of CD19 and SRC. Notably this tonic BCR signal is not present in IGHV-mutated CLL cells, discordantly expressing ZAP-70. Results of quantitative mass spectrometry and phosphoprotein analyses indicate that this ZAP-70-dependent, tonic BCR signal regulates CLL cell migration through phosphorylation of LCP1 on serine-5. Indeed, we show that CCL19- and CCL21-induced chemotaxis is regulated by and dependent on the expression of ZAP-70 through its function to enhance CCR7 signaling to LCP1. Thus, our data demonstrate that ZAP-70 converges a tonic BCR signal, exclusively present in IGHV-unmutated CLL and CCR7-mediated chemotaxis.

Construction of ceRNA prognostic model based on the CCR7/CCL19 chemokine axis as a biomarker in breast cancer.

BACKGROUND: The study of CCR7/CCL19 chemokine axis and breast cancer (BC) prognosis and metastasis is a current hot topic. We constructed a ceRNA network and risk-prognosis model based on CCR7/CCL19. METHODS: Based on the lncRNA, miRNA and mRNA expression data downloaded from the TCGA database, we used the starbase website to find the lncRNA and miRNA of CCR7/CCL19 and established the ceRNA network. The 1008 BC samples containing survival data were divided into Train group (504 cases) and Test group (504 cases) using R "caret" package. Then we constructed a prognostic risk model using RNA screened by univariate Cox analysis in the Train group and validated it in the Test and All groups. In addition, we explored the correlation between riskScores and clinical trials and immune-related factors (22 immune-infiltrating cells, tumor microenvironment, 13 immune-related pathways and 24 HLA genes). After transfection with knockdown CCR7, we observed the activity and migration ability of MDA-MB-231 and MCF-7 cells using CCK8, scratch assays and angiogenesis assays. Finally, qPCR was used to detect the expression levels of five RNAs in the prognostic risk model in MDA-MB-231 and MCF-7 cell. RESULTS: Patients with high expression of CCR7 and CCL19 had significantly higher overall survival times than those with low expression. The ceRNA network is constructed by 3 pairs of mRNA-miRNA pairs and 8 pairs of miRNA-lncRNA. After multivariate Cox analysis, we obtained a risk prognostic model: riskScore= -1.544 *`TRG-AS1`+ 0.936 * AC010327.5 + 0.553 *CCR7 -0.208 *CCL19 -0.315 *`hsa-let-7b-5p. Age, stage and riskScore can all be used as independent risk factors for BC prognosis. By drug sensitivity analysis, we found 5 drugs targeting CCR7 (convolamine, amikacin, AH-23,848, ondansetron, flucloxacillin). After transfection with knockdown CCR7, we found a significant reduction in cell activity and migration capacity in MDA-MB-231 cells. CONCLUSION: We constructed the first prognostic model based on the CCR7/CCL19 chemokine axis in BC and explored its role in immune infiltration, tumor microenvironment, and HLA genes.

CCL21-CCR7 signaling promotes microglia/macrophage recruitment and chemotherapy resistance in glioblastoma.

Glioblastoma (GBM) is the most common and fatal primary tumor of the central nervous system (CNS) and current treatments have limited success. Chemokine signaling regulates both malignant cells and stromal cells of the tumor microenvironment (TME), constituting a potential therapeutic target against brain cancers. Here, we investigated the C-C chemokine receptor type 7 (CCR7) and the chemokine (C-C-motif) ligand 21 (CCL21) for their expression and function in human GBM and then assessed their therapeutic potential in preclinical mouse GBM models. In GBM patients, CCR7 expression positively associated with a poor survival. CCL21-CCR7 signaling was shown to regulate tumor cell migration and proliferation while also controlling tumor associated microglia/macrophage recruitment and VEGF-A production, thereby controlling vascular dysmorphia. Inhibition of CCL21-CCR7 signaling led to an increased sensitivity to temozolomide-induced tumor cell death. Collectively, our data indicate that drug targeting of CCL21-CCR7 signaling in tumor and TME cells is a therapeutic option against GBM.

Targeting CCR7-PI3Kγ overcomes resistance to tyrosine kinase inhibitors in ALK-rearranged lymphoma.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) show potent efficacy in several ALK-driven tumors, but the development of resistance limits their long-term clinical impact. Although resistance mechanisms have been studied extensively in ALK-driven non-small cell lung cancer, they are poorly understood in ALK-driven anaplastic large cell lymphoma (ALCL). Here, we identify a survival pathway supported by the tumor microenvironment that activates phosphatidylinositol 3-kinase γ (PI3K-γ) signaling through the C-C motif chemokine receptor 7 (CCR7). We found increased PI3K signaling in patients and ALCL cell lines resistant to ALK TKIs. PI3Kγ expression was predictive of a lack of response to ALK TKI in patients with ALCL. Expression of CCR7, PI3Kγ, and PI3Kδ were up-regulated during ALK or STAT3 inhibition or degradation and a constitutively active PI3Kγ isoform cooperated with oncogenic ALK to accelerate lymphomagenesis in mice. In a three-dimensional microfluidic chip, endothelial cells that produce the CCR7 ligands CCL19/CCL21 protected ALCL cells from apoptosis induced by crizotinib. The PI3Kγ/δ inhibitor duvelisib potentiated crizotinib activity against ALCL lines and patient-derived xenografts. Furthermore, genetic deletion of CCR7 blocked the central nervous system dissemination and perivascular growth of ALCL in mice treated with crizotinib. Thus, blockade of PI3Kγ or CCR7 signaling together with ALK TKI treatment reduces primary resistance and the survival of persister lymphoma cells in ALCL.

CCL19/CCR7 drives regulatory T cell migration and indicates poor prognosis in gastric cancer.

BACKGROUND: Gastric cancer is associated with significant morbidity and mortality in the world. Blocking programmed cell death protein 1 pathway have been approved for the treatment of a variety of tumors and have achieved remarkable clinical therapeutic effects. However, immune checkpoint inhibitors failed to achieve satisfactory results in gastric cancer. There is a need to identify novel immunotherapy targets in gastric cancer. METHODS: We analysed the correlation between Treg cells and CD8 + T cells in gastric cancer samples. We studied the relationship between chemokines and Treg cells or CD8 + T cells in gastric cancer. We compared CCL19/CCR7 expression in gastric cancer patients in TCGA database. We performed transwell experiments to determine the influence of CCL19 on Treg cells and CD8 + T cells migratory capacity. We conducted survival analysis of CCL19 and CCR7 in gastric cancer database. RESULTS: Treg cells show positive correlation with CD8 + T cells in gastric cancer. Treg cell expression was significantly upregulated in tumor tissues. Patients with high FOXP3 expression had worse overall survival than those with low FOXP3 expression. CCL19 had strong correlation with FOXP3 and weak correlation with CD8A. CCL19 had strong impact on the migratory capacity of Treg cells but weak impact on the migratory capacity of CD8 + T cells. Both CCL19 and CCR7 expression were significantly upregulated in gastric cancer tissues. Survival analysis demonstrated that both CCL19 and CCR7 indicate poor prognosis in gastric cancer. CONCLUSIONS: CCL19/CCR7 may be a potential novel therapeutic target in gastric cancer.

Effects of chemokine receptor CCR7 in the pathophysiology and clinical features of the immuno-inflammatory response in primary pterygium.

OBJECTIVE: Chemokine receptor 7 (CCR7) has been considered a critical biomarker in inflammation and the immune response; however, little is known about CCR7 in pterygia. This study aimed to investigate whether CCR7 participates in the pathogenesis of primary pterygia and how CCR7 affects the progression of pterygia. METHODS: This was an experimental study. Slip-lamp photographs of 85 pterygium patients were used to measure the width, extent, and area of pterygia with computer software. Pterygium blood vessels and general ocular redness were quantitatively analyzed with a specific algorithm. The expression of CCR7 and its ligands C-C motif ligand 19 (CCL19) and C-C motif ligand 21 (CCL21) in control conjunctivae and excised pterygia collected during surgery were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence staining. The phenotype of CCR7-expressing cells was identified by costaining for major histocompatibility complex II (MHC II), CD11b or CD11c. RESULTS: The CCR7 level was significantly increased by 9.6-fold in pterygia compared with control conjunctivae (p = 0.008). The higher the expression of CCR7 was, the more blood vessels appeared in pterygia (r = 0.437, p = 0.002) and the more general ocular redness was (r = 0.51, p < 0.001) in pterygium patients. CCR7 was significantly associated with pterygium extent (r = 0.286, p = 0.048). In addition, we found that CCR7 colocalized with CD11b, CD11c or MHC II in dendritic cells, and immunofluorescence staining showed that CCR7-CCL21 is a potential chemokine axis in pterygium. CONCLUSIONS: This work verified that CCR7 impacts the extent of primary pterygia invading the cornea and inflammation at the ocular surface, which may provide a possibility for a further in-depth understanding of the immunological mechanism in pterygia.

Gene Expression Profiling of Peripheral Blood Mononuclear Cells in Type 2 Diabetes: An Exploratory Study.

Background and Objectives: Visceral obesity is associated with chronic low-grade inflammation that predisposes to metabolic syndrome. Indeed, infiltration of adipose tissue with immune-inflammatory cells, including 'classical' inflammatory M1 and anti-inflammatory 'alternative' M2 macrophages, causes the release of a variety of bioactive molecules, resulting in the metabolic complications of obesity. This study examined the relative expression of macrophage phenotypic surface markers, cholesterol efflux proteins, scavenger receptors, and adenosine receptors in human circulating peripheral blood mononuclear cells (PBMCs), isolated from patients with type 2 diabetes mellitus (T2DM), with the aim to phenotypically characterize and identify biomarkers for these ill-defined cells. Materials and Methodology: PBMCs were isolated from four groups of adults: Normal-weight non-diabetic, obese non-diabetic, newly diagnosed with T2DM, and T2DM on metformin. The mRNA expression levels of macrophage phenotypic surface markers (interleukin-12 (IL-12), C-X-C motif chemokine ligand 10 (CXCL10), C-C motif chemokine ligand 17 (CCL17), and C-C motif receptor 7 (CCR7)), cholesterol efflux proteins (ATP-binding cassette transporter-1 (ABCA1), ATP binding cassette subfamily G member 1 (ABCG1), and sterol 27-hydroxylase (CYP27A)), scavenger receptors (scavenger receptor-A (SR-A), C-X-C motif ligand 16 (CXCL16), and lectin-like oxidized LDL receptor-1 (LOX-1)), and adenosine receptors (adenosine A2A receptor (A2AR) and adenosine A3 receptor (A3R)) were measured using qRT-PCR. Results: In PBMCs from T2DM patients, the expression of IL-12, CCR7, ABCA1, and SR-A1 was increased, whereas the expression of CXCL10, CCL17, ABCG1,27-hydroxylase, LOX-1, A2AR and A3R was decreased. On the other hand, treatment with the antidiabetic drug, metformin, reduced the expression of IL-12 and increased the expression of 27-hydroxylase, LOX-1, CXCL16 and A2AR. Conclusions: PBMCs in the circulation of patients with T2DM express phenotypic markers that are different from those typically present in adipose tissue M1 and M2 macrophages and could be representative of metabolically activated macrophages (MMe)-like cells. Our findings suggest that metformin alters phenotypic markers of MMe-like cells in circulation.

Effects of let-7a microRNA and C-C chemokine receptor type 7 expression on cellular function and prognosis in esophageal squamous cell carcinoma.

BACKGROUND: C-C chemokine receptor type 7 (CCR7) participates in chemotactic and metastatic responses in various cancers, including in esophageal squamous cell carcinoma (ESCC). The microRNA (miRNA) let-7a suppresses migration and invasion of various types of cancer cells by downregulating CCR7 expression. METHODS: The expression levels of CCR7 and let-7a were measured in the cell lines, tumor, and peritumoral tissues of ESCC patients. KYSE cell lines were transfected with synthetic let-7a miRNA and a let-7a miRNA inhibitor, and their CCR7 expression levels as well as invasive ability were evaluated. A highly invasive cell line was established via an invasion assay, and CCR7 expression level along with let-7a level was subsequently evaluated. Cancer cells overexpressing CCR7 were injected subcutaneously into mice, and the animals were monitored for tumor growth along with lymph node metastasis. RESULTS: A negative correlation between CCR7 and let-7a expression was observed in the ESCC cell lines as well as in tissue samples from patients. Synthetic let-7a decreased CCR7 expression level, while the let-7a inhibitor increased it. In vitro, the established highly invasive cancer cells with high and low levels of CCR7 and let-7a expression, respectively, exhibited a greater invasive ability than the wild-type cell line. The cells were associated with tumor growth and lymph node metastasis in mice. Patients in the high-CCR7/low-let-7a group had the worst prognosis, with a five-year recurrence free survival (5-RFS) rate of 37.5%, followed by the high-CCR7/high-let-7a (5-RFS: 60.0%) and low-CCR7 (5-RFS: 85.7%; p = 0.038) groups. CONCLUSIONS: The expression of CCR7 was downregulated by let-7a miRNA in esophageal cancer cells. The decrease in let-7a expression level led to the increased expression level of CCR7 in ESCC cells, consequently increasing their invasive ability and malignancy and resulting in a worse prognosis for ESCC patients. TRIAL REGISTRATION: Retrospectively registered.

Site-Specific Polyplex on CCR7 Down-Regulation and T Cell Elevation for Lymphatic Metastasis Blocking on Breast Cancer.

Tumor metastasis contributes to high cancer mortality. Tumor cells in lymph nodes (LNs) are difficult to eliminate but underlie uncontrollable systemic metastasis. The CC chemokine receptor 7 (CCR7) is overexpressed in tumor cells and interacts with CC chemokine ligand 21 (CCL21) secreted from LNs, potentiating their lymphatic migration. Here, a site-specific polyplex is developed to block the CCR7-CCL21 signal and kill tumor cells toward LNs, greatly limiting their lymphatic infiltration. A CCR7-targeting small interfering RNA (siCCR7) is condensed by mPEG-poly-(lysine) with chlorin e6 (Ce6) modification (PPLC) to form PPLC/siCCR7. The knockdown of CCR7 by siCCR7 in tumor cells significantly reduced their response on CCL21 and LN tropism. Additionally, photodynamic therapy-mediated immune activation precisely targets and kills tumor cells released from the primary foci before they reaches the LNs, reducing the number of tumor cells entering the LNs. Consequently, the PPLC/siCCR7 polyplexes inhibited up to 92% of lung metastasis in 4T1 tumor bearing mice and reduced tumor cell migration to LNs by up to 80%. This site-specific strategy optimized anti-metastasis efficacy and promotes the clinical translational development of anti-metastatic therapy.

CCR7 alterations associated with inferior outcome of adult T-cell leukemia/lymphoma under mogamulizumab treatment.

Adult T-cell leukemia/lymphoma (ATL) patients have a very poor prognosis. The humanized anti-CCR4 therapeutic monoclonal antibody, mogamulizumab, is a key agent for ATL treatment. Our previous integrated molecular analysis demonstrated that among all the driver genes in ATL, CCR7 gene alterations were significantly associated with clinical response to mogamulizumab. Accordingly, here we investigated the detailed clinical impact of CCR7 alterations in a larger cohort of ATL patients. These CCR7 alterations, most of which lead to C-terminus truncations, were observed in 27 of 223 patients (12%). For patients receiving mogamulizumab but not allogeneic hematopoietic stem cell transplantation (HSCT), CCR7 alterations were significantly associated with worse survival (median survival from the first dose of mogamulizumab of 0.7 years for 12 patients with CCR7 alterations vs. 1.6 years for 72 patients without, p = 0.020). On the other hand, the presence or absence of CCR7 alterations had no significant impact on survival in the entire cohort (median overall survival of 1.4 and 1.8 years, respectively, p = 0.901), or on the survival of patients receiving allogeneic HSCT (median survival from the day of transplantation of 0.9 years for 6 patients with CCR7 alterations and 1.4 years for 48 without, p = 0.543). Multivariate analysis indicated that patients with CCR4 alterations but lacking CCR7 alterations (n = 20) had significantly better survival after receiving mogamulizumab-containing treatments (hazard ratio for survival, 0.437, 95% confidence interval, 0.192-0.994). This study contributes to the establishment of precision medicine for ATL.

Pregnancy preparation: redistribution of CCR7-positive cells in the rat uterus.

In brief: Changes in the endometrium prior to implantation may be critical in predicting pregnancy outcomes. This study shows that the endocrine system directs positional changes in CCR7+ cells before implantation, which may be critical for developing maternal tolerance. Abstract: Suppression of the maternal immune system is vital for the implantation of the semi-allogeneic embryo. Although progress in understanding the dialogue between mother and embryo has been made, key interactions between maternal immune cells, hormones, and chemokines remain elusive. Uterine expression of the C-C chemokine receptor type 7 (CCR7) could recruit T regulatory cells and facilitate localized immune suppression. To test this concept, Ccr7 mRNA and protein were assessed in uterine tissue. Ccr7 mRNA expression peaked at day 4 in pregnant rat uteri and then declined at days 5 and 6. CCR7 protein showed similar quantitative changes. To test if female sex steroids affected the spatial distribution of CCR7-expressing cells, uteri from ovariectomized rats, progesterone-pretreated rats (2 mg daily), and progesterone-pretreated rats injected with estradiol (0.2 µg) were analyzed. Progesterone increased CCR7-positive (+) cells in the antimesometrial stroma. Progesterone and estradiol increased CCR7+ cells in the mesometrial stroma. Estradiol increased the density of cluster of differentiation 4 (CD4) positive cells in the mesometrial stromal region over progesterone alone. The density of cells expressing the T regulatory cell marker, forkhead box protein 3 (FOXP3), increased in the antimesometrial stroma in response to progesterone alone. Progesterone and estradiol increased FOXP3+ cells in the antimesometrial region of the stroma. Co-localization of CCR7, CD4, and FOXP3 in the stroma suggests CCR7+ cells are T regulatory cells. Polarization of CCR7+ cells in the endometrial stroma was an intrinsic response regulated by sex steroids and did not require the presence of an embryo.

In silico analysis and in planta production of recombinant ccl21/IL1ß protein and characterization of its in vitro anti-tumor and immunogenic activity.

CCL21 has an essential role in anti-tumor immune activity. Epitopes of IL1ß have adjuvant activity without causing inflammatory responses. CCR7 and its ligands play a vital role in the immune balance; specifically, in transport of T lymphocytes and antigen-presenting cells such as dendritic cells to the lymph nodes. This study aimed to produce epitopes of CCL21 and IL1ß as a recombinant protein and characterize its in vitro anti-tumor and immunogenic activity. A codon-optimized ccl21/IL1ß gene was designed and synthesized from human genes. Stability and binding affinity of CCL21/IL1ß protein and CCR7 receptor were examined through in silico analyses. The construct was introduced into N. tabacum to produce this recombinant protein and the structure and function of CCL21/IL1ß were examined. Purified protein from transgenic leaves generated a strong signal in SDS PAGE and western blotting assays. FTIR measurement and MALDI-TOF/TOF mass spectrography showed that ccl21/IL-1ß was correctly expressed in tobacco plants. Potential activity of purified CCL21/IL1ß in stimulating the proliferation and migration of MCF7 cancer cell line was investigated using the wound healing method. The results demonstrated a decrease in survival rate and metastasization of cancer cells in the presence of CCL21/IL1ß, and IC50 of CCL21 on MCF7 cells was less than that of non-recombinant protein. Agarose assay on PBMCsCCR7+ showed that CCL21/IL1ß has biological activity and there is a distinguishable difference between chemokinetic (CCL21) and chemotactic (FBS) movements. Overall, the results suggest that CCL21/IL1ß could be considered an effective adjuvant in future in vivo and clinical tests.

Identification of Potential Biomarkers of Platelet RNA in Glioblastoma by Bioinformatics Analysis.

Objective: Glioblastoma is one of the most common and fatal malignancies in adults. Current treatment is still not optimistic. Glioblastoma (GBM) transports RNA to platelets in the blood system via microvesicles, suggesting that platelet RNA can be a potential diagnostic and therapeutic target. The roles of specific platelet RNAs in treatment of GBM are not well understood. Methods: Platelet RNA profiling of 8 GBM and 12 normal samples were downloaded from the GEO database. Differentially expressed genes (DEGs) were identified between tumors and normal samples. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to elucidate the functions of up- and downregulated genes. miRNA was predicted by miRTarBase, TargetScan, and miRDB databases. circBase and circBank were used for circRNA prediction. ceRNA (circRNA-mRNA-miRNA) network was constructed to investigate the potential interactions. Results: 22 genes were upregulated and 9 genes were downregulated. There are only two genes (CCR7 and FAM102A) that connect to miRNAs (hsa-let-7a-5p, hsa-miR-1-3p). We assessed the overall survival rates by Kaplan-Meier plotter, and relative expression of GBM and subtypes for overlapped mRNA (CCR7 and FAM102A) were evaluated, and further, we obtained circRNAs (has-circ-0015164, hsa-circ-0003243) by circBank and circBase and bind sites through the CSCD database. Finally, a ceRNA network (circRNA-mRNA-miRNA) was constructed based on 2 miRNAs, 2 mRNAs, and 2 circRNAs by Cytoscape. This study focused on potential mRNA and ceRNA biomarkers to targeted treatment of GBM and provided ideas for clinical treatment through the combination of hematology and oncology. Conclusion: The findings of this study contribute to better understand the relationship between GBM and the blood system (platelets) and might lay a solid foundation for improving GBM molecule and gene diagnosis and prognosis.

EXPRESSION OF CHEMOKINE (C-C MOTIF) RECEPTOR 7 IN PROSTATE CANCER TISSUE OF YOUNG PATIENTS AND IN METASTATIC CANCER CELLS.

BACKGROUND: Chemokine (C-C motif) receptor 7 (CCR7) is a chemokine receptor involved in the carcinogenesis of several types of tumors due to its promoting action in epithelial-mesenchymal transition events, invasion, angiogenesis and metastasis. However, its role in prostate cancer (PCa) remains unclear. AIM: To evaluate CCR7 expression by immunohistochemistry in prostate tumors from young patients and to determine the possible relationship with the clinicopathological characteristics. MATERIALS AND METHODS: We analyzed retrospectively paraffin-embedded tissue sections from 23 young PCa (≤ 55 years old) patients and evaluated the transcriptomic expression in the TCGA database. RESULTS: Expression of CCR7 was observed in 15 cases (65%). The tissue samples from younger patients (≤ 50 years) were mostly positive in 72.7% (8/11) of cases. High grade GS (≥ 3) tumors were CCR7-positive in 71% cases. The malignant cells present in lymph nodes were CCR7 positive in 100% cases. The bioinformatic analysis showed a high CCR7 expression associated with the presence of metastasis (FC = 2.6, p = 0.03) in the Cancer Genome Atlas (TCGA) PCa cohort (PRAD). CONCLUSION: We showed that CCR7 expression in tumors from young patients is associated with the early onset of the disease and could also be related to lymph node metastasis.

Shifting CCR7 towards Its Monomeric Form Augments CCL19 Binding and Uptake.

The chemokine receptor CCR7, together with its ligands, is responsible for the migration and positioning of adaptive immune cells, and hence critical for launching adaptive immune responses. CCR7 is also induced on certain cancer cells and contributes to metastasis formation. Thus, CCR7 expression and signalling must be tightly regulated for proper function. CCR7, like many other members of the G-protein coupled receptor superfamily, can form homodimers and oligomers. Notably, danger signals associated with pathogen encounter promote oligomerisation of CCR7 and is considered as one layer of regulating its function. Here, we assessed the dimerisation of human CCR7 and several single point mutations using split-luciferase complementation assays. We demonstrate that dimerisation-defective CCR7 mutants can be transported to the cell surface and elicit normal chemokine-driven G-protein activation. By contrast, we discovered that CCR7 mutants whose expression are shifted towards monomers significantly augment their capacities to bind and internalise fluorescently labelled CCL19. Modeling of the receptor suggests that dimerisation-defective CCR7 mutants render the extracellular loops more flexible and less structured, such that the chemokine recognition site located in the binding pocket might become more accessible to its ligand. Overall, we provide new insights into how the dimerisation state of CCR7 affects CCL19 binding and receptor trafficking.