CCL21 and CLDN11 Are Key Driving Factors of Lymph Node Metastasis in Gastric Cancer.

BACKGROUND: Gastric cancer (GC) is a leading cause of cancer-related deaths worldwide. Understanding the molecular mechanisms of GC metastasis is crucial for improving patient survival outcomes. METHODS: RNA sequencing and analysis were performed on tissue samples from primary and lymph node metastatic lesions of gastric cancer. Differential gene analysis and functional pathway analysis were conducted. Immune infiltrating environment and protein expression levels were evaluated using immunohistochemistry. Cell experiments were conducted to investigate the role of CCL21 in GC metastasis. RESULTS: ACTG2, CNN1, DES, MUC6, and PGC were significantly upregulated in primary tumor cells, while CCL21, MS4A1, CR2, CLDN11, and FDCSP were significantly upregulated in metastatic tumor cells. Functional pathway analysis revealed enrichment in pathways related to immune response. CLDN11 and CCL21 were found to play important roles in promoting gastric cancer metastasis. Cell experiments confirmed the role of CCL21 in promoting GC cell growth and metastasis. CCL21 is highly expressed in GC tissues and binds to CCR7, leading to upregulation of CLDN11. This results in GC-lymph node metastasis and abnormal activation of immune cells (B cells and CD4+ T cells). CONCLUSION: Inhibition of CCL21 and CLDN11 proteins may be a promising strategy for treating GC and preventing lymph node metastasis. These findings provide specific molecular markers for early lymph node metastases of GC, which can aid in developing treatment strategies and predicting patient prognosis.

Chemokine Analysis in Patients with Metastatic Uveal Melanoma Suggests a Role for CCL21 Signaling in Combined Epigenetic Therapy and Checkpoint Immunotherapy.

Purpose: Patients with metastatic uveal melanoma have limited therapeutic options and high mortality rate so new treatment options are needed. Patients and Methods: We previously reported that patients treated with the PD-1 inhibitor pembrolizumab and the histone deacetylase inhibitor entinostat in the PEMDAC trial, experienced clinical benefits if their tumor originated from iris or was wildtype for BAP1 tumor suppressor gene. Here we present the 2-year follow-up of the patients in the PEMDAC trial and identify additional factors that correlate with response or survival. Results: Durable responses were observed in 4 patients, with additional 8 patients exhibiting a stable disease. The median overall survival was 13.7 months. Grade 3 adverse events were reported in 62% of the patients, but they were all manageable. No fatal toxicity was observed. Activity of thymidine kinase 1 in plasma was higher in patients with stable disease or who progressed on treatment, compared with those with partial response. Chemokines and cytokines were analyzed in plasma. Three chemokines were significantly different when comparing patients with and without response. One of the factors, CCL21, was higher in the plasma of responding patients before treatment initiation but decreased in the same patients upon treatment. In tumors, CCL21 was expressed in areas resembling tertiary lymphoid structures (TLS). High plasma levels of CCL21 and presence of TLS-like regions in the tumor correlated with longer survival. Conclusions: This study provides insight into durable responses in the PEMDAC trial, and describes dynamic changes of chemokines and cytokines in the blood of these patients. Significance: The most significant finding from the 2-year follow-up study of the PEMDAC trial was that high CCL21 levels in blood was associated with response and survival. CCL21 was also expressed in TLS-like regions and presence of these regions was associated with longer survival. These analyses of soluble and tumor markers can inform on predictive biomarkers needing validation and become hypothesis generating for experimental research.

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.

Display of CCL21 on cancer cell membrane through genetic modification using a pH low insertion peptide.

CCL21, a secondary lymphoid tissue chemokine, plays an important role in generating an effective anti-tumor immune response. In this study, a genetically modified CCL21 was developed by inserting a pH low insertion peptide to establish a CCL21-rich microenvironment for tumors. The fusion tag thioredoxin (Trx) was designed and fused at the N-terminal of the recombinant protein to protect it from being irrevocably misfolded in microbial host cells. The prokaryotic expression vector pET32a-CCL21-pHLIP was constructed and successfully expressed in E. coli BL21 (DE3) with a soluble expression form and a molecular weight of ~35 kDa. The induction conditions were optimized to obtain an extremely high yield of 6.7 mg target protein from 31.1 mg total protein. The 6xHis tagged Trx-CCL21-pHLIP was purified using Ni-NTA resin, and it was confirmed using SDS-PAGE and Western blot analyses. Consequently, the Trx-CCL21-pHLIP protein was successfully displayed on the cancer cell surface in a weak acidic microenvironment and showed the same ability as CCL21 in recruiting CCR7-positive cells. Additionally, the CCL21 fusion protein with or without Trx tag showed similar functions. Therefore, the study implies the feasibility of directing a modular genetic method for the development of protein-based drugs.

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.

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.

Epithelial-mesenchymal transition related genes in unruptured aneurysms identified through weighted gene coexpression network analysis.

Intracranial aneurysm (IA) can cause fatal subarachnoid hemorrhage (SAH) after rupture, and identifying patients with unruptured IAs is essential for reducing SAH fatalities. The epithelial-mesenchymal transition (EMT) may be vital to IA progression. Here, identified key EMT-related genes in aneurysms and their pathogenic mechanisms via bioinformatic analysis. The GSE13353, GSE75436, and GSE54083 datasets from Gene Expression Omnibus were analyzed with limma to identify differentially expressed genes (DEGs) among unruptured aneurysms, ruptured aneurysms, and healthy samples. The results revealed that three EMT-related DEGs (ADIPOQ, WNT11, and CCL21) were shared among all groups. Coexpression modules and hub genes were identified via weighted gene co-expression network analysis, revealing two significant modules (red and green) and 14 EMT-related genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses suggested that cytokine interactions were closely related. Gene set enrichment analysis revealed that unruptured aneurysms were enriched for the terms "inflammatory response" and "vascular endothelial growth". Protein-protein interaction analysis identified seven key genes, which were evaluated with the GSE54083 dataset to determine their sensitivity and specificity. In the external validation set, we verified the differential expression of seven genes in unruptured aneurysms and normal samples. Together, these findings indicate that FN1, and SPARC may help distinguish normal patients from patients with asymptomatic IAs.

Circadian clocks guide dendritic cells into skin lymphatics.

Migration of leukocytes from the skin to lymph nodes (LNs) via afferent lymphatic vessels (LVs) is pivotal for adaptive immune responses1,2. Circadian rhythms have emerged as important regulators of leukocyte trafficking to LNs via the blood3,4. Here, we demonstrate that dendritic cells (DCs) have a circadian migration pattern into LVs, which peaks during the rest phase in mice. This migration pattern is determined by rhythmic gradients in the expression of the chemokine CCL21 and of adhesion molecules in both mice and humans. Chronopharmacological targeting of the involved factors abrogates circadian migration of DCs. We identify cell-intrinsic circadian oscillations in skin lymphatic endothelial cells (LECs) and DCs that cogovern these rhythms, as their genetic disruption in either cell type ablates circadian trafficking. These observations indicate that circadian clocks control the infiltration of DCs into skin lymphatics, a process that is essential for many adaptive immune responses and relevant for vaccination and immunotherapies.

Bioinformatics analysis identified shared differentially expressed genes as potential biomarkers for Hashimoto's thyroiditis-related papillary thyroid cancer.

Background: Although the etiology of Hashimoto's thyroiditis (HT), a common autoimmune endocrine disease, is unknown, studies suggest a potential association with genetic factors and environmental conditions inducing excessive iodine intake. Additionally, HT patients have a high risk of papillary thyroid cancer (PTC), which is probably related to the chronic inflammation and autoimmune pathologic process occurring in HT, as it is thought to be associated with neoplastic transformation. Methods: Bioinformatics approaches can identify differentially expressed genes (DEGs) and analyze DEG functions in diseases. R software was used in this study to identify DEGs in HT and PTC using data in Gene Expression Omnibus (GEO). The online tools DAVID, Reactome, and AmiGO were employed for annotation, visualization, and integration of DEGs related to HT and PTC, and the STRING database and Cytoscape software were applied to predict and visualize protein-protein networks (PPIs) for DEG-encoded proteins. Coexpressed DEGs in HT and PTC were validated by reverse transcription PCR (RT-PCR). Results: In total, 326, 231, and 210 DEGs in HT specimens and samples of central PTC and PTC invasive areas, respectively, were detected. According to the PPI network, PTPN6, HLA-A, C3AR1, LCK and ITGB2 are hub genes among HT-DEGs, whereas FN1, CDH2, SERPINA1, and CYR61 are PTC-DEG hub genes. The shared DEGs LTF and CCL21 were validated by RT-PCR. Both bioinformatics and RT-PCR analyses showed LTF and CCL21 to be upregulated in HT tissues and downregulated in PTC tissues. Conclusions: We identified that expression of LTF and CCL21 are significantly different in HT and PTC, suggesting an underlying association between HT and PTC.

Chemokine C-C motif ligand 21 synergized with programmed death-ligand 1 blockade restrains tumor growth.

Programmed death-ligand 1 (PD-L1) blockade has revolutionized the prognosis of several cancers, but shows a weak effect on pancreatic cancer (PC) due to poor effective immune infiltration. Chemokine C-C motif ligand 21 (CCL21), a chemokine promoting T cell immunity by recruiting and colocalizing dendritic cells (DCs) and T cells, serves as a potential antitumor agent in many cancers. However, its antitumor response and mechanism combined with PD-L1 blockade in PC remain unclear. In our study, we found CCL21 played an important role in leukocyte chemotaxis, inflammatory response, and positive regulation of PI3K-AKT signaling in PC using Metascape and gene set enrichment analysis. The CCL21 level was verified to be positively correlated with infiltration of CD8+ T cells by the CIBERSORT algorithm, but no significant difference in survival was observed in either The Cancer Genome Atlas or the International Cancer Genome Consortium cohort when stratified by CCL21 expression. Additionally, we found the growth rate of allograft tumors was reduced and T cell infiltration was increased, but tumor PD-L1 abundance elevated simultaneously in the CCL21-overexpressed tumors. Then, CCL21 was further verified to increase tumor PD-L1 level through the AKT-glycogen synthase kinase-3ß axis in human PC cells, which partly impaired the antitumor T cell immunity. Finally, the combination of CCL21 and PD-L1 blockade showed superior synergistic tumor suppression in vitro and in vivo. Together, our findings suggested that CCL21 in combination with PD-L1 blockade might be an efficient and promising option for the treatment of PC.

Urinary small extracellular vesicles derived CCL21 mRNA as biomarker linked with pathogenesis for diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is a leading cause of renal failure, whereas the effective and early diagnostic biomarkers are still lacking. METHODS: Fourteen cytokines and chemokines mRNA were detected in urinary extracellular vesicles (EVs) from the screening cohort including 4 healthy controls (HC), 4 diabetes mellitus (DM) and 4 biopsy-proven DN patients, and was validated in another 16 HC and 15 DM and 28 DN patients. Correlation analysis was performed between the candidate biomarkers and clinic parameters as well as kidney histological changes. The findings were also confirmed in DN rat model with single injection of STZ. RESULTS: The number of small EVs secreted in urine was increased in DN patients compared to DM patients and healthy controls, with expression of AQP1 (a marker of proximal tubules) and AQP2 (a marker of distal/collecting tubules). Small EVs derived CCL21 mRNA increased significantly in DN patients and correlated with level of proteinuria and eGFR. Interestingly, elevated CCL21 mRNA from urine small EVs was observed in DN patients with normal renal function and could discriminate early DN patients from DM more efficiently compared to eGFR and proteinuria. CCL21 also showed an accurate diagnostic ability in distinguishing incipient from overt DN. Histologically, CCL21 mRNA expression increased progressively with the deterioration of tubulointerstitial inflammation and showed the highest level in nodular sclerosis group (class III) in DN patients. Remarkable infiltration of CD3 positive T cells including both CD4 and CD8 positive T cell population were observed in DN patients with high-CCL21 expression. Besides, accumulation of CD3 positive T cells correlated with level of urinary small EVs derived CCL21 and co-localized with CCL21 in the tubulointerstitium in DN patients. Finally, the correlation of CCL21 expression in renal cortex and urinary small EVs was confirmed in STZ-induced DN rat model. CONCLUSIONS: Urinary small EVs derived CCL21 mRNA may serve as early biomarker for identifying DN linked with pathogenesis. CCL21 mRNA mediated T cell infiltration may constitute the key mechanism of chronic inflammation in DN.

A humanized monoclonal antibody against the endothelial chemokine CCL21 for the diagnosis and treatment of inflammatory bowel disease.

Chemokines are small proteins that promote leukocyte migration during development, infection, and inflammation. We and others isolated the unique chemokine CCL21, a potent chemo-attractant for naïve T-cells, naïve B-cells, and immature dendritic cells. CCL21 has a 37 amino acid carboxy terminal extension that is distinct from the rest of the chemokine family, which is thought to anchor it to venule endothelium where the amino terminus can interact with its cognate receptor, CCR7. We and others have reported that venule endothelium expressing CCL21 plays a crucial role in attracting naïve immune cells to sites of antigen presentation. In this study we generated a series of monoclonal antibodies to the amino terminus of CCL21 in an attempt to generate an antibody that blocked the interaction of CCL21 with its receptor CCR7. We found one humanized clone that blocked naïve T-cell migration towards CCL21, while memory effector T-cells were less affected. Using this monoclonal antibody, we also demonstrated that CCL21 is expressed in the mucosal venule endothelium of the large majority of inflammatory bowel diseases (IBD), including Crohn's disease, ulcerative colitis, and also in celiac disease. This expression correlated with active IBD in 5 of 6 cases, whereas none of 6 normal bowel biopsies had CCL21 expression. This study raises the possibility that this monoclonal antibody could be used to diagnose initial or recurrent of IBD. Significantly, this antibody could also be used for therapeutic intervention in IBD by selectively interfering with recruitment of naïve immune effector cells to sites of antigen presentation, without harming overall memory immunity.

Revisiting CCL-type chemokines in breast cancer and its milieu: prominent targetable chemokines, CCL8 and CCL21.

The patterns of chemokine expression play a decisive role in both breast cancer prognosis and metastasis. In a recent article published in Bioscience Reports, 'Bioinformatics identification of CCL8/21 as potential prognostic biomarkers in breast cancer microenvironment', Chen et al. presented that expression of both CCL8 and CCL21 among CCL-type chemokines is prominent for prognosis of the breast cancer, metastasis and chemoresistance (Biosci Rep (2020) 40(11); DOI: 10.1042/BSR20202042). Identifying the sources of the CCL8 and CCL21 in the tumor microenvironment and developing targeting strategies for these chemokines to prevent tumor growth will improve both prognosis and therapeutic outcomes.

The relation between ACKR4 and CCR7 genes expression and breast cancer metastasis.

AIMS: Breast cancer is the most severe malignant tumor in women. Chemokines and their receptors appear to be implicated in tumorigenesis and metastatic pattern. Also the scavenger atypical chemokine receptors are emerging as crucial regulators for the availability of chemokines. Therefore the aim of the present study is to evaluate the expression of CCR7, ACKR4 and their ligand; CCL21 in human breast cancer. MAIN METHODS: In this study, RT-PCR was done to detect the expression of CCR7 and ACKR4 in 50 non-metastatic and 30 metastatic breast cancer tissue. Also CCL21 level in the serum of study group was detected by ELISA. The expression of all markers is compared to 80 control healthy individual. KEY FINDINGS: Our results revealed the increase in expression of CCR7 and CCL21 level in metastatic group compared to non-metastatic and control groups while ACKR4 expression is significantly increased in breast tissues of non-metastatic patients compared to both control and metastatic groups. Also there was significant positive correlation between CCR7 expression and CCL21 level in cancer patients and significant negative correlation between ACKR4 and both CCR-7 and CCL21 in both non-metastatic and metastatic cancer groups. SIGNIFICANCE: Thus, it might be elucidating that ACKR4 and CCR7 could be a novel target for tumor therapy as targeting the chemokine-receptors axis might represent a powerful tool to prevent infiltration and metastasis and consequently improve cancer prognosis and treatment.

CD9 Upregulation-Decreased CCL21 Secretion in Mesenchymal Stem Cells Reduces Cancer Cell Migration.

Tetraspanin CD9 is widely expressed on various cell types, such as cancer cells and mesenchymal stem cells (MSCs), and/or cell-released exosomes. It has been reported that exosomal CD9 plays an important role in intercellular communications involved in cancer cell migration and metastasis. However, reports on the effect of the CD9 of MSCs or MSC-derived exosomes on cancer cell migration are still lacking. In this study, using a transwell migration assay, we found that both dextran-coated iron oxide nanoparticles (dex-IO NPs) and ionomycin stimulated exosomal CD9 expression in human MSCs (hMSCs); however, hMSCs could not deliver them to melanoma cells to affect cell migration. Interestingly, a reduced migration of melanoma cell line was observed when the ionomycin-incubated hMSC-conditioned media but not dex-IO NP-labeled hMSC-conditioned media were in the bottom chamber. In addition, we found that dex-IO NPs decreased cellular CD9 expression in hMSCs but ionomycin increased this. Simultaneously, we found that ionomycin suppressed the expression and secretion of the chemokine CCL21 in hMSCs. The silencing of CD9 demonstrated an inhibitory role of cellular CD9 in CCL21 expression in hMSCs, suggesting that ionomycin could upregulate cellular CD9 to decrease CCL21 expression and secretion of hMSCs, which would reduce the migration of B16F10, A549 and U87MG cancer cell lines due to chemoattraction reduction of CCL21. The present study not only highlights the important role of bone marrow-derived hMSCs' CD9-mediated CCL21 regulation in cancer bone metastasis but also suggests a new distinct pharmaceutical strategy for prevention or/and therapy of cancer metastasis.

The Important Role of the Chemokine Axis CCR7-CCL19 and CCR7-CCL21 in the Pathophysiology of the Immuno-inflammatory Response in Dry Eye Disease.

Purpose: To explore whether CCR7-CCL19 and CCR7-CCL21 affect the pathophysiology of the dry eye disease (DED) immuno-inflammatory response using a murine model.Methods: The mRNA expression levels of CCR7, CCL19, CCL21 and VEGF-C within corneas in DED mice were detected by real-time PCR. Immunofluorescence and flow cytometric analyses were performed to mark dendritic cells (DCs) and detect correlations among CCR7, CCL19, CCL21 and lymphatic vessels.Results: CCR7, CCL19 and CCL21 expression was dramatically increased during the development of DED. In addition, CCR7, which is expressed in DCs, was located inside and around lymphatic vessels and colocalized with CCL19 or CCL21. Positive correlations were observed between CCR7 and CCL19 (P < .01, r = 0.862), CCL21 (P < .01, r = 0.759), and VEGF-C (P < .05, r = 0.607).Conclusions: Our study revealed that both the CCR7-CCL19 and CCR7-CCL21 chemokine axis are important for DC migration to lymphatic vessels, but CCL19 may have a greater effect on DED than CCL21.

Lymphatic Metastasis of NSCLC Involves Chemotaxis Effects of Lymphatic Endothelial Cells through the CCR7-CCL21 Axis Modulated by TNF-α.

Metastasis and recurrence are the main causes of lung adenocarcinoma patients' death. Lymphatic metastasis is the main way of non-small cell lung cancer (NSCLC) metastasis. C-C chemokine receptor type 7 (CCR7) overexpression has been demonstrated to mediate occurrence and progression of NSCLC. Moreover, Chemokine ligand 21 (CCL21) was used to activate CCR7. The CCR7-CCL21 axis is one of the most common "chemokine-receptor" modes of action in the development and metastasis of multiple tumors. However, the role of the CCR7-CCL21 axis in lymphatic metastasis of NSCLC is poorly understood. The study was conducted to investigate the molecular mechanism underlying CCR7-CCL21 axis-mediated lymphatic metastasis of NSCLC A549 cells. Tumor necrosis factor α (TNF-α) could regulate the tumor microenvironment balance by promoting chemokine secretion. Our study demonstrated that TNF-α promoted CCL21 production in human lymphatic endothelial cells (HLEC). Results further showed that TNF-α significantly activated the NF-κB pathway in HLEC. NF-κB pathway inhibition with ammonium pyrrolidinedithiocarbamate (PDTC) caused a significant decrease in CCL21 secretion, suggesting that TNF-α-induced CCL21 secretion in HLEC was through NF-κB pathway. Co-culture of A549 cells and TNF-α-treated HLEC confirmed that the metastasis of A549 cells was enhanced, meanwhile, apoptosis-related proteins were hardly affected. The data proved that a co-culture system prevented cell apoptosis while inducing the lymphatic metastasis of A549 cells. However, the situation was reversed after neutralizing CCL21 expression, suggesting that TNF-α-induced CCL21 secretion in HLEC is involved in A549 cells metastasis. Collectively, our finding demonstrated that NF-κB pathway-controlled CCL21 secretion of HLEC contributing to the lymphatic metastasis of A549 cells via the CCR7-CCL21 axis, validating the CCR7-CCL21 axis as a potential target to inhibit metastasis of NSCLC.

Bioinformatics identification of CCL8/21 as potential prognostic biomarkers in breast cancer microenvironment.

BACKGROUND: Breast cancer (BC) is the most common malignancy among females worldwide. The tumor microenvironment usually prevents effective lymphocyte activation and infiltration, and suppresses infiltrating effector cells, leading to a failure of the host to reject the tumor. CC chemokines play a significant role in inflammation and infection. METHODS: In our study, we analyzed the expression and survival data of CC chemokines in patients with BC using several bioinformatics analyses tools. RESULTS: The mRNA expression of CCL2/3/4/5/7/8/11/17/19/20/22 was remarkably increased while CCL14/21/23/28 was significantly down-regulated in BC tissues compared with normal tissues. Methylation could down-regulate expression of CCL2/5/15/17/19/20/22/23/24/25/26/27 in BC. Low expression of CCL3/4/23 was found to be associated with drug resistance in BC. Results from Kaplan-Meier plotter and BC Gene-Expression Miner v4.2 (bcGenExMiner) v4.2 demonstrated that BC patients with high CCL8 and low CCL19/21/22 expression were more likely to have a worse prognosis. CCL8 expression was significantly up-regulated in BC tissues compared with normal tissues. High CCL8 expression was significantly correlated with negative PR, negative ER, positive nodal status, triple-negative BC subtype, basal-like BC subtype, triple-negative and basal-like BC subtype and high grades. CCL21 was down-regulated in BC, while high levels of CCL21 was associated with negative PR, triple-negative subtype, basal-like subtype and low tumor grade. Functional analysis demonstrated that CCL8 and CCL21 were involved in carcinogenesis, tumor immune escape and chemoresistance in BC. CONCLUSION: Integrative bioinformatics analysis demonstrated CCL8/21 as potential prognostic biomarkers in BC microenvironment.

Increased expression of cystathionine beta-synthase and chemokine ligand 21 is closely associated with poor prognosis in extrahepatic cholangiocarcinoma.

The expression of Cystathionine beta-synthase (CBS) and Chemokine ligand 21 (CCL21) is associated with the tumorigenesis and progression of a variety of tumors, but whether alterations in their expression levels correlates with the carcinogenesis and progression of EHCC is still unknown. This study investigated the clinicopathological significance of CBS and CCL21 expression in EHCC.We investigated the correlations between the expression of CBS and CCL21 and clinicopathological characteristics in EHCC using EnVision immunohistochemistry.The expression of CBS and CCL21 was significantly higher in EHCC tumors than in nontumor tissues (P < .05 and P < .01). EHCC patients with CBS and CCL21 expression combined with lymph node metastasis, tumor cell invasion, and TNM III/IV stage had more severe conditions than those with no lymph node metastasis, distant invasion and TNM I/II stage (P < .01). Kaplan-Meier survival analysis showed that the overall survival rates for EHCC patients with negative CBS or CCL21 reaction were significantly higher than those for patients with positive CBS or CCL21 reaction((P < .01). CBS or CCL21 expression was revealed as an independent poor prognostic factor for EHCC patients by Cox multivariate analysis.The present study indicates that CBS and CCL21 expression is closely associated with the pathogenesis of clinical, pathological and biological behaviors and poor prognosis in EHCC.

Coexpression of IL7 and CCL21 Increases Efficacy of CAR-T Cells in Solid Tumors without Requiring Preconditioned Lymphodepletion.

PURPOSE: T-cell recruitment, survival, and proliferation are the important limitations to chimeric antigen receptor (CAR) T cells therapy in the treatment of solid tumors. In this study, we engineered CAR-T cells to coexpress cytokines IL7 and CCL21 (7 × 21 CAR-T), a cytokine combination in order to improve proliferation and chemotaxis of CAR-T cells. EXPERIMENTAL DESIGN: CLDN18.2-specific second-generation CAR-T cells coexpressing cytokines were prepared using retroviral vector transduction. The proliferation and migration of genetically engineered CAR-T cells were evaluated in vitro. The antitumor activities of genetically engineered CAR-T cells were evaluated against multiple solid tumors in C57BL/6 mice in vivo. RESULTS: In vitro, the proliferation and chemotaxis of 7 × 21 CAR-T cells are significantly improved when compared with those of the conventional CAR-T cells. In vivo, 7 × 21 CAR-T cells revealed superior therapeutic effects to either conventional CAR-T cells or 7 × 19 CAR-T cells which coexpress IL7 and CCL19 as previously reported in three different solid tumors without cyclophosphamide precondition. Interestingly, 7 × 21 CAR-T cells could also suppress the tumor growth with heterogeneous antigen expression and even induce tumor complete remission. Mechanistically, IL7 and CCL21 significantly improved survival and infiltration of CAR-T cells and dendritic cells in tumor. In addition, CCL21 also inhibited the tumor angiogenesis as proved by IHC. CONCLUSIONS: Coexpression of IL7 and CCL21 could boost CAR-T cells' antitumor activity, and 7 × 21 CAR-T cells may be served as a promising therapy strategy for solid tumors.