PD-1/CD80+ small extracellular vesicles from immunocytes induce cold tumours featured with enhanced adaptive immunosuppression.

Only a minority of cancer patients benefit from immune checkpoint blockade therapy. Sophisticated cross-talk among different immune checkpoint pathways as well as interaction pattern of immune checkpoint molecules carried on circulating small extracellular vesicles (sEV) might contribute to the low response rate. Here we demonstrate that PD-1 and CD80 carried on immunocyte-derived sEVs (I-sEV) induce an adaptive redistribution of PD-L1 in tumour cells. The resulting decreased cell membrane PD-L1 expression and increased sEV PD-L1 secretion into the circulation contribute to systemic immunosuppression. PD-1/CD80+ I-sEVs also induce downregulation of adhesion- and antigen presentation-related molecules on tumour cells and impaired immune cell infiltration, thereby converting tumours to an immunologically cold phenotype. Moreover, synchronous analysis of multiple checkpoint molecules, including PD-1, CD80 and PD-L1, on circulating sEVs distinguishes clinical responders from those patients who poorly respond to anti-PD-1 treatment. Altogether, our study shows that sEVs carry multiple inhibitory immune checkpoints proteins, which form a potentially targetable adaptive loop to suppress antitumour immunity.

Overexpression of RAB27A in Oral Squamous Cell Carcinoma Promotes Tumor Migration and Invasion via Modulation of EGFR Membrane Stability.

Oral squamous cell carcinoma (OSCC) is the most prevalent subtype of head and neck tumors, highly prone to lymph node metastasis. This study aims to examine the expression pattern of Ras-related protein Rab-27A (RAB27A) and explore its potential implications in OSCC. The expression of RAB27A was assessed through immunohistochemical analysis utilizing tissue microarrays. In vitro experiments were conducted using RAB27A-knockdown cells to investigate its impact on OSCC tumor cells. Additionally, transcriptome sequencing was performed to elucidate potential underlying mechanisms. RAB27A was significantly overexpressed in OSCC, and particularly in metastatic lymph nodes. It was positively correlated with the clinical progression and poor survival prognosis. Silencing RAB27A notably decreased the proliferation, migration, and invasion abilities of OSCC cells in vitro. A Gene Ontology (GO) enrichment analysis indicated a strong association between RAB27A and the epidermal growth factor receptor (EGFR) signaling pathway. Further investigations revealed that RAB27A regulated the palmitoylation of EGFR via zinc finger DHHC-type containing 13 (ZDHHC13). These findings provide insights into OSCC progression and highlight RAB27A as a potential therapeutic target for combating this aggressive cancer.

p-AKT/VPS4B regulates the small extracellular vesicle size in venous malformation endothelial cells.

OBJECTIVE: Small extracellular vesicle (sEV)-mediated intercellular communication is increasingly the key for the understanding of venous malformations (VMs). This study aims to clarify the detailed changes of sEVs in VMs. SUBJECTS AND METHODS: Fifteen VM patients without treatment history and twelve healthy donors were enrolled in the study. sEVs were isolated from both fresh lesions and cell supernatant, and were examined by western blotting, nanoparticle tracking analysis and transmission electron microscopy. Western blot analysis, immunohistochemistry and immunofluorescence were adopted to screening candidate regulator of sEV size. Specific inhibitors and siRNA were employed to validate the role of dysregulated p-AKT/vacuolar protein sorting-associated protein 4B (VPS4B) signaling on the size of sEVs in endothelial cells. RESULTS: The size of sEVs derived from both VM lesion tissues and cell model was significantly increased. VPS4B, whose expression level was mostly significantly downregulated in VM endothelial cells, was responsible for the size change of sEVs. Targeting abnormal AKT activation corrected the size change of sEVs by recovering the expression level of VPS4B. CONCLUSION: Downregulated VPS4B in endothelial cells, resulted from abnormally activated AKT signaling, contributed to the increased size of sEVs in VMs.

ALIX promotes cell migration and invasion of head and neck squamous cell carcinoma by regulating the expression of MMP9, MMP14, VEGF-C.

OBJECTIVE: The poor survival rate of head and neck squamous cell carcinoma (HNSCC), one of the most prevalent human cancer, is attributed to frequent locoregional recurrence and lymph node metastases. Though it is reported that the expression of ALG-2 interacting protein X (ALIX) closely correlates with the progression of various tumors, its role in HNSCC remains unclear. The present study aims to investigate the role of ALIX in the development of HNSCC. DESIGN: With immunohistochemical staining, the expression levels of ALIX and series of related functional proteins were compared in normal mucosal (n = 18), HNSCC tissues (n = 54), and metastatic lymph nodes (n = 11). Further, the correlation analysis was performed among the proteins detected. By knocking down ALIX in HNSCC cell lines, the correlation of ALIX with the proteins was verified in vitro. The role of ALIX in proliferation, migration, and invasion of HNSCC cells was further studied by flow cytometry, wounding healing, and transwell assays, respectively. RESULTS: Higher expression level of ALIX was revealed in HNSCC samples, especially in metastatic lymph nodes, than in normal mucosal tissues. Accordingly, increasing levels of MMP9, MMP14, and VEGF-C were also discovered in metastatic lymph nodes and significantly correlated with the expression of ALIX. In vitro assays demonstrated that the knockdown of ALIX reduced both the transcriptional and protein levels of MMP9, MMP14, and VEGF-C, together with suppressed migration and weakened invasion of HNSCC cell lines. CONCLUSIONS: ALIX up-regulated the expression of MMP9, MMP14 and VEGF-C, and promoted migration and invasion of HNSCC cells.

Endothelial cells induce degradation of ECM through enhanced secretion of MMP14 carried on extracellular vesicles in venous malformation.

Venous malformations (VMs), featuring localized dilated veins, are the most common developmental vascular anomalies. Aberrantly organized perivascular extracellular matrix (ECM) is one of the prominent pathological hallmarks of VMs, accounting for vascular dysfunction. Although previous studies have revealed various proteins involved in ECM remodeling, the detailed pattern and molecular mechanisms underlying the endothelium-ECM interplay have not been fully elucidated. Our previous studies revealed drastically elevated extracellular vesicle (EV) secretion in VM lesions. Here, we identified increased EV-carried MMP14 in lesion fluids of VMs and culture medium of TIE2-L914F mutant endothelial cells (ECs), along with stronger ECM degradation. Knockdown of RAB27A, a required regulator for vesicle docking and fusion, led to decreased secretion of EV-carried MMP14 in vitro. Histochemical analysis further demonstrated a highly positive correlation between RAB27A in the endothelium and MMP14 in the perivascular environment. Therefore, our results proved that RAB27A-regulated secretion of EV-MMP14, as a new pattern of endothelium-ECM interplay, contributed to the development of VMs by promoting ECM degradation.

Surviving Inhibition Induces Cell Cycle Arrest and Disrupts Multipotency in Haemangioma Stem Cells.

OBJECTIVE: To explore the potential therapies for infantile haemangiomas by targeting survivin, a member of the inhibitor of apoptosis protein family, using its specific small molecule inhibitor YM155. METHODS: The expression of survivin in human haemangioma tissue was explored using immunohistochemistry and immunohistofluorescence. Cell cycle analysis and EdU assays were used to measure cell proliferation. Heochst33342 and Annexin V/PI double staining were performed to measure cell apoptosis. The capacity for self-renewal and multilineage differentiation potential of haemangioma stem cells (HemSCs) were measured by clone formation assays and multiple differentiation assays. Murine haemangioma models were established to explore the therapeutic efficacy of YM155 in vivo. RESULTS: Strong staining of survivin in stromal cells was observed in the proliferative haemangioma tissue. In vitro studies demonstrated that YM155 induced cell cycle arrest and proliferation suppression of HemSCs, and also caused cell apoptosis at a higher concentration. YM155 impaired the self-renewal capacities and damaged multiple differentiation potentials of HemSCs. Importantly, YM155 suppressed blood vessel formation and cell proliferation, and induced cell apoptosis in murine haemangioma models. CONCLUSION: The present study demonstrated that targeting survivin using its specific suppressant, YM155, prevented the progression of infantile haemangioma by suppressing cell proliferation, inducing cell apoptosis and disrupting the differentiation potential of HemSCs. These results indicate a novel and promising therapeutic approach for the treatment of infantile haemangioma.

Lymphotoxin-α promotes tumor angiogenesis in HNSCC by modulating glycolysis in a PFKFB3-dependent manner.

Tumor angiogenesis is critical for tumor progression as the new blood vessels supply nutrients and facilitate metastasis. Previous studies indicate tumor associated lymphocytes, including B cells and T cells, contribute to tumor angiogenesis and tumor progression. The present study aims to identify the function of Lymphotoxin-α (LT-α), which is secreted by the activated lymphocytes, in the tumor angiogenesis of head and neck squamous cell carcinoma (HNSCC). The coculture system between HNSCC cell line Cal27 and primary lymphocytes revealed that tumor cells promoted the LT-α secretion in the cocultured lymphocytes. In vitro data further demonstrated that LT-α promoted the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) by enhancing the PFKFB3-mediated glycolytic flux. Genetic and pharmacological inhibition of PFKFB3 suppressed the enhanced proliferation and migration of HUVECs. We further identified that LT-α induced PFKFB3 expression was dependent on the TNFR/NF-κB signaling pathway. In addition, we proved that PFKFB3 blockade decreased the density of CD31 positive blood vessels in HNSCC xenografts. Finally, the results from the human HNSCC tissue array revealed that the expression of LT-α in HNSCC samples positively correlated with microvessel density, lymphocytes infiltration and endothelial PFKFB3 expression. In conclusion, infiltrated lymphocyte secreted LT-α enhances the glycolysis of ECs in a PFKFB3-dependent manner through the classical NF-κB pathway and promotes the proliferation and migration of ECs, which may contribute to the aberrant angiogenesis in HNSCCs. Our study suggests that PFKFB3 blockade is a promising therapeutic approach for HNSCCs by targeting tumor angiogenesis.

Lymphotoxins Promote the Progression of Human Lymphatic Malformation by Enhancing Lymphatic Endothelial Cell Proliferation.

Formation of inflammation-related tertiary lymphoid organs promotes human lymphatic malformation (LM) development. However, the role of lymphotoxins (LTs) and LT-related inducible ligand, the crucial mediators for tertiary lymphoid organ formation, is undetermined in LMs. Herein, we show that LTs and LT-related inducible ligand promote LM development by enhancing lymphatic endothelial cell (LEC) proliferation via activating NF-κB pathways. The expression of LTs and their receptors was increased in LMs, especially the infected ones, when compared with normal skins. Nuclear translocation of p65, p52, and RelB in the LECs of LMs indicated the activation of classic and alternative NF-κB pathways. Pearson's correlation and cluster analysis suggested the close relationship between LEC proliferation and NF-κB activation. Moreover, in vitro data demonstrated LTs accelerated the proliferation of human dermal LECs (HdLECs) through activation of NF-κB. In addition, lipopolysaccharide (LPS) up-regulated LT receptor expression in HdLECs, leading to increased sensitivity to LTs. Suppression of LT receptors hampered LPS-enhanced HdLEC proliferation, indicating the crucial role of LT pathways in inflammatory lymphangiogenesis. Besides, evidence from the LM rat models demonstrated LTα and LPS enhanced LEC proliferation, therefore promoting LM development. Blocking LT pathways by neutralizing antibodies against LTα and lymphotoxin ß receptor may decelerate the growth of the disease. In summary, our present study demonstrated activation of LT signaling pathways in LECs contributed to the progression of LMs.

Down-regulation of polycystin in lymphatic malformations: possible role in the proliferation of lymphatic endothelial cells.

Lymphatic malformations (LMs) are composed of aberrant lymphatic vessels and regarded as benign growths of the lymphatic system. Recent studies have demonstrated that the mutant embryos of PKD1 and PKD2, encoding polycystin-1 (PC-1) and polycystin-2 (PC-2), respectively, result in aberrant lymphatic vessels similar to those observed in LMs. In this study, for the first time, we investigated PC-1 and PC-2 expression and assessed their roles in the development of LMs. Our results demonstrated that PC-1 and PC-2 gene and protein expressions were obviously decreased in LMs compared with normal skin tissues. In addition, the expression of phosphorylated ERK but not total ERK was up-regulated in LMs and negatively correlated with the expression of PC-1 and PC-2. Moreover, up-regulation of Ki67 was detected in LMs and positively correlated with ERK phosphorylation levels. Furthermore, cluster analysis better reflected close correlation between these signals. All of the above results provided strong evidence suggesting that the hyperactivation of the ERK pathway may be caused by down-regulation of PC-1 and PC-2 in LMs, contributing to increased proliferation of lymphatic endothelial cells in LMs. Our present study sheds light on novel potential mechanisms involved in LMs and may help to explore novel treatments for LMs.

Blockage of glycolysis by targeting PFKFB3 suppresses tumor growth and metastasis in head and neck squamous cell carcinoma.

BACKGROUND: Many cancers including head and neck squamous cell carcinoma (HNSCC) are characterized by a metabolic rewiring with increased glucose uptake and lactate production, termed as aerobic glycolysis. Targeting aerobic glycolysis presents a promising strategy for cancer therapy. This study investigates the therapeutic potential of glycolysis blockage by targeting phosphofructokinase-2/fructose-2, 6-bisphosphatase 3 (PFKFB3) in HNSCC. METHODS: 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15) was used as a selective antagonist of PFKFB3. Glycolytic flux was determined by measuring glucose uptake, lactate production and ATP yield. PFKFB3 expression was examined using HNSCC tissue arrays. Cell proliferation, apoptosis and motility were analysed. HNSCC xenograft mouse model and metastasis mouse model were established to examine the therapeutic efficacy of PFK15 in vivo. RESULTS: HNSCC showed an increased PFKFB3 expression compared with adjacent mucosal tissues (P < 0.01). Targeting PFKFB3 via PFK15 significantly reduced the glucose uptake, lactate production and ATP generation in HNSCC cell lines. PFK15 suppressed cell proliferation, halted cell cycle progression and induced cell apoptosis. The invadopodia of HNSCC cells was markedly reduced after PFK15 treatment, thereby impairing cell motility and extracellular matrix degradation ability. The in vivo data from the xenograft mice models proved that PFK15 administration suppressed the tumor growth. And the results from the metastatic mice models showed administration of PFK15 alleviated the lung metastasis of HNSCC and extended the life expectancy of mice. CONCLUSIONS: The pharmacological inhibition of PFKFB3 via PFK15 suppressed tumor growth and alleviated metastasis in HNSCC, offering a promising strategy for cancer therapy.

CCL2/EGF positive feedback loop between cancer cells and macrophages promotes cell migration and invasion in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) represents the most frequent malignancy in the head and neck region, and the survival rate has not been improved significantly over the past three decades. It has been reported the infiltrated macrophages contribute to the malignant progression of HNSCC. However, the crosstalk between macrophages and cancer cells remains poorly understood. In the present study, we explored interactions between monocytes/macrophages and HNSCC cells by establishing the direct co-culture system, and found that the crosstalk promoted the migration and invasion of cancer cells by enhancing the invadopodia formation through a CCL2/EGF positive feedback loop. Our results demonstrated HNSCC cells educated monocytes into M2-like macrophages by releasing C-C motif chemokine ligand 2 (CCL2, or MCP-1). And the M2-like macrophages secreted epithelial growth factor (EGF), which increased the motility of HNSCC cells by enhancing the invadopodia formation. These subcellular pseudopodia degraded extracellular matrix (ECM), facilitating tumor local invasion and distant metastasis. Moreover, EGF up-regulated CCL2 expression in HNSCC cells, which recruited monocytes and turned them into M2-like macrophages, thus forming a positive feedback paracrine loop. Finally, we reported that curcumin, a powerful natural drug, suppressed the production of EGF and CCL2 in macrophages and cancer cells, respectively, blocking the feedback loop and suppressing the migration and invasion of HNSCC cells. These results shed light on the possibilities and approaches based on targeting the crosstalk between cancer cells and monocytes/macrophages in HNSCC for potential cancer therapy.