Exosomes Derived from RM-1 Cells Promote the Recruitment of MDSCs into Tumor Microenvironment by Upregulating CXCR4 via TLR2/NF- κ B Pathway

Myeloid-derived suppressor cells (MDSCs) play a critical role in tumor immune escape because of its remarkable immunosuppressive effect. However, the mechanism of MDSCs migrated into tumor microenvironment remains unclear. In this study, we demonstrated the recruitment of MDSCs can be promoted by exosomes derived from prostate cancer cells, which could upregulate chemokine (CXC motif) receptor 4 (CXCR4) via the TLR2/NF- κ B signalling pathway. Flow cytometry detected that the percentage of MDSCs in the mice spleen and tumor tissue was significantly increased after injection with exosomes via mouse tail vein. Transwell chemotaxis assay showed the recruitment of MDSCs toward the lower chamber was enhanced after stimulation with exosomes, and the migration ability could be inhibited by AMD3100 (a CXCR4 specific inhibitor) both in vivo and in vitro. Additionally, Western blot and flow cytometry verified a remarkably increase of CXCR4 in MDSCs after incubation with exosomes; meanwhile, the protein level of TLR2 and activation of NF- κ B were also strengthened obviously. Nevertheless, after blocking TLR2 by C29 (a TLR2-specific inhibitor), the expression of p-p65 and CXCR4, which were hypothesized as the downstream target of TLR2, was prominently reduced. In conclusion, prostate cancer-derived exosomes could reinforce CXCR4 expression in MDSCs through the TLR2/NF- κ B signalling pathway, eventually promoting migration of MDSCs into tumor microenvironment in a CXCR4-CXCL12 axis-dependent manner.

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Spi-B Promotes the Recruitment of Tumor-Associated Macrophages via Enhancing CCL4 Expression in Lung Cancer

Frontiers in Oncology ◽

10.3389/fonc.2021.659131 ◽

2021 ◽

Vol 11 ◽

Author(s):

Qiumin Huang ◽

Junrong Liu ◽

Shuainan Wu ◽

Xuexi Zhang ◽

Zengtuan Xiao ◽

...

Keyword(s):

Lung Cancer ◽

Tumor Microenvironment ◽

Human Lung ◽

Immune Escape ◽

Critical Role ◽

Human Lung Cancer ◽

Clinical Samples ◽

Tumor Associated Macrophages ◽

Tumor Immune Escape ◽

Anticancer Immunotherapy

Tumor immune escape plays a critical role in malignant tumor progression and leads to the failure of anticancer immunotherapy. Spi-B, a lymphocyte lineage-specific Ets transcription factor, participates in mesenchymal invasion and favors metastasis in human lung cancer. However, the mechanism through which Spi-B regulates the tumor immune environment has not been elucidated. In this study, we demonstrated that Spi-B enhanced the infiltration of tumor-associated macrophages (TAMs) in the tumor microenvironment using subcutaneous mouse models and clinical samples of human lung cancer. Spi-B overexpression increased the expression of TAM polarization- and recruitment-related genes, including CCL4. Moreover, deleting CCL4 inhibited the ability of Spi-B promoting macrophage infiltration. These data suggest that Spi-B promotes the recruitment of TAMs to the tumor microenvironment via upregulating CCL4 expression, which contributes to the progression of lung cancer.

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What Do We Have to Know about PD-L1 Expression in Prostate Cancer? A Systematic Literature Review. Part 3: PD-L1, Intracellular Signaling Pathways and Tumor Microenvironment

International Journal of Molecular Sciences ◽

10.3390/ijms222212330 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12330

Author(s):

Andrea Palicelli ◽

Stefania Croci ◽

Alessandra Bisagni ◽

Eleonora Zanetti ◽

Dario De Biase ◽

...

Keyword(s):

Prostate Cancer ◽

Tumor Microenvironment ◽

Literature Review ◽

Signaling Pathways ◽

Systematic Literature Review ◽

Immune Cells ◽

Intracellular Signaling ◽

Immune Escape ◽

Tumor Immune Escape ◽

Intracellular Signaling Pathways

The tumor microenvironment (TME) includes immune (T, B, NK, dendritic), stromal, mesenchymal, endothelial, adipocytic cells, extracellular matrix, and cytokines/chemokines/soluble factors regulating various intracellular signaling pathways (ISP) in tumor cells. TME influences the survival/progression of prostate cancer (PC), enabling tumor cell immune-evasion also through the activation of the PD-1/PD-L1 axis. We have performed a systematic literature review according to the PRISMA guidelines, to investigate how the PD-1/PD-L1 pathway is influenced by TME and ISPs. Tumor immune-escape mechanisms include suppression/exhaustion of tumor infiltrating cytotoxic T lymphocytes, inhibition of tumor suppressive NK cells, increase in immune-suppressive immune cells (regulatory T, M2 macrophagic, myeloid-derived suppressor, dendritic, stromal, and adipocytic cells). IFN-γ (the most investigated factor), TGF-β, TNF-α, IL-6, IL-17, IL-15, IL-27, complement factor C5a, and other soluble molecules secreted by TME components (and sometimes increased in patients’ serum), as well as and hypoxia, influenced the regulation of PD-L1. Experimental studies using human and mouse PC cell lines (derived from either androgen-sensitive or androgen-resistant tumors) revealed that the intracellular ERK/MEK, Akt-mTOR, NF-kB, WNT and JAK/STAT pathways were involved in PD-L1 upregulation in PC. Blocking the PD-1/PD-L1 signaling by using immunotherapy drugs can prevent tumor immune-escape, increasing the anti-tumor activity of immune cells.

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Gastric Cancer Extracellular Vesicles Tune the Migration and Invasion of Epithelial and Mesenchymal Cells in a Histotype-Dependent Manner

International Journal of Molecular Sciences ◽

10.3390/ijms20112608 ◽

2019 ◽

Vol 20 (11) ◽

pp. 2608 ◽

Cited By ~ 3

Author(s):

Sara Rocha ◽

Sara Pinto Teles ◽

Mafalda Azevedo ◽

Patrícia Oliveira ◽

Joana Carvalho ◽

...

Keyword(s):

Gastric Cancer ◽

Flow Cytometry ◽

Tumor Microenvironment ◽

Cancer Cells ◽

Extracellular Vesicles ◽

Mesenchymal Cells ◽

Migration And Invasion ◽

Dependent Manner ◽

Diffuse Type ◽

Transmission Electron

Extracellular vesicles (EVs) secreted by tumor cells modulate recipient cells’ behavior, but their effects in normal cells from the tumor microenvironment remain poorly known. In this study, we dissected the functional impact of gastric cancer cell-derived EVs (GC-EVs), representative of distinct GC histotypes, on the behavior of normal isogenic epithelial and mesenchymal cells. GC-EVs were isolated by differential centrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, and imaging flow-cytometry. Epithelial and mesenchymal cells were challenged with GC-EVs and submitted to proliferation, migration, and invasion assays. Expression of epithelial and mesenchymal markers was followed by immunofluorescence and flow-cytometry. Our results indicated that GC-EVs secreted by diffuse-type cancer cells decrease the migration of recipient cells. This effect was more prominent and persistent for mesenchymal recipient cells, which also increased Fibronectin expression in response to EVs. GC-EVs secreted by cancer cells derived from tumors with an intestinal component increased invasion of recipient epithelial cells, without changes in EMT markers. In summary, this study demonstrated that GC-EVs modulate the migration and invasion of epithelial and mesenchymal cells from the tumor microenvironment, in a histotype-dependent manner, highlighting new features of intestinal and diffuse-type GC cells, which may help explaining differential metastasis patterns and aggressiveness of GC histotypes.

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Role of the tumor microenvironment in PD-L1/PD-1-mediated tumor immune escape

Molecular Cancer ◽

10.1186/s12943-018-0928-4 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 143

Author(s):

Xianjie Jiang ◽

Jie Wang ◽

Xiangying Deng ◽

Fang Xiong ◽

Junshang Ge ◽

...

Keyword(s):

Tumor Microenvironment ◽

Immune Escape ◽

Tumor Immune Escape

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Elevated circulating CCL2 in prostate cancer patients.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.6_suppl.201 ◽

2020 ◽

Vol 38 (6_suppl) ◽

pp. 201-201

Author(s):

Justin Lin ◽

Amanda Caress ◽

Farzana Ahmed ◽

Nicole Taylor ◽

Yixuan Gong ◽

...

Keyword(s):

Prostate Cancer ◽

Inflammatory Cytokines ◽

Neutralizing Antibodies ◽

Critical Role ◽

Dependent Manner ◽

U937 Cells ◽

Ccl2 Mrna ◽

Pc3 Cells ◽

Cytokine Array ◽

Human Cytokine

201 Background: Monocyte chemoattractant protein 1 (CCL2 or MCP-1), a chemokine secreted by monocytic cells, is critical in recruiting Treg and MDSCs into the tumor microenvironment and in regulating prostate cancer (PCa) migration and proliferation. In this study, we examined circulating CCL2 levels in healthy vs PCa patients and used an in vitro coculture model to identify the source of the elevated CCL2. Methods: Serum CCL2 concentrations were evaluated via ELISA in 59 patients (19 health controls, 20 “treatment naïve” PCa, 20 mCRPC). Monocytic leukemia cells (U937) were either directly cocultured with PC3 PCa cell line or cultured in the PC3 conditioned medium (CM). The induction of CCL2 mRNA in the cultures was examined by qPCR. The secretion of CCL2 into cell culture supernatants was evaluated via human cytokine array. Neutralizing antibodies to several overexpressed inflammatory cytokines in PC3 cells were added into the PC3 CM to evaluate the contribution of these inflammatory cytokines to CCL2 induction. Results: Circulating CCL2 concentrations were significantly higher in prostate cancer patient serum compared to control patient serum (p=4.4e-6) (Table). To understand the potential source of elevated CCL2, we grew U937 and PC3 in coculture and evaluated with qPCR, revealing that while CCL2 was not expressed in PC3 cells, it was expressed at very low levels in U937 cells. Interestingly, coculture of PC3 with U937 increased CCL2 mRNA expression by over 10-fold, and the result was confirmed at protein levels by human cytokine array. Our results also indicated that IL-6 and GM-SCF were the two major cytokines released by PCa cells to induce CCL2 mRNA in U937 cells and MEK and JAK-STAT signaling were crucial for CCL2 induction. Conclusions: Prostate cancer cells induce CCL2 secretion from monocytes in an IL-6 and GM-CSF dependent manner. Given the critical role of CCL2 in mediating immunosuppressive tumor microenvironments, our study highlighted the CCL2 Concentrations in PCa vs Healthy Serum.[Table: see text]

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Critical role for Epac1 in inflammatory pain controlled by GRK2-mediated phosphorylation of Epac1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1516036113 ◽

2016 ◽

Vol 113 (11) ◽

pp. 3036-3041 ◽

Cited By ~ 61

Author(s):

Pooja Singhmar ◽

XiaoJiao Huo ◽

Niels Eijkelkamp ◽

Susana Rojo Berciano ◽

Faiza Baameur ◽

...

Keyword(s):

Chronic Pain ◽

Molecular Mechanism ◽

Inflammatory Pain ◽

Critical Role ◽

Specific Inhibitor ◽

Mechanical Hyperalgesia ◽

Dependent Manner ◽

Phosphorylation Event

cAMP signaling plays a key role in regulating pain sensitivity. Here, we uncover a previously unidentified molecular mechanism in which direct phosphorylation of the exchange protein directly activated by cAMP 1 (EPAC1) by G protein kinase 2 (GRK2) suppresses Epac1-to-Rap1 signaling, thereby inhibiting persistent inflammatory pain. Epac1−/− mice are protected against inflammatory hyperalgesia in the complete Freund’s adjuvant (CFA) model. Moreover, the Epac-specific inhibitor ESI-09 inhibits established CFA-induced mechanical hyperalgesia without affecting normal mechanical sensitivity. At the mechanistic level, CFA increased activity of the Epac target Rap1 in dorsal root ganglia of WT, but not of Epac1−/−, mice. Using sensory neuron-specific overexpression of GRK2 or its kinase-dead mutant in vivo, we demonstrate that GRK2 inhibits CFA-induced hyperalgesia in a kinase activity-dependent manner. In vitro, GRK2 inhibits Epac1-to-Rap1 signaling by phosphorylation of Epac1 at Ser-108 in the Disheveled/Egl-10/pleckstrin domain. This phosphorylation event inhibits agonist-induced translocation of Epac1 to the plasma membrane, thereby reducing Rap1 activation. Finally, we show that GRK2 inhibits Epac1-mediated sensitization of the mechanosensor Piezo2 and that Piezo2 contributes to inflammatory mechanical hyperalgesia. Collectively, these findings identify a key role of Epac1 in chronic inflammatory pain and a molecular mechanism for controlling Epac1 activity and chronic pain through phosphorylation of Epac1 at Ser-108. Importantly, using the Epac inhibitor ESI-09, we validate Epac1 as a potential therapeutic target for chronic pain.

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Butyrate Impedes the Recruitment of MDSCs to Alleviate CAC Development by Inhibition of the TLR2/MyD88/NF-κB Signaling Pathway

10.21203/rs.3.rs-1186467/v1 ◽

2021 ◽

Author(s):

Tengfei Xiao ◽

Yi Chang ◽

Hongmei Chen ◽

Jingjing Kang ◽

Mingzhong Sun

Keyword(s):

Tumor Microenvironment ◽

Mouse Model ◽

Signaling Pathway ◽

Immune Escape ◽

Suppressor Cells ◽

Short Chain Fatty Acids ◽

Inhibitory Effect ◽

Tumor Immune Escape ◽

Tnf Α ◽

Anal Prolapse

Abstract Background: The colitis-associated colorectal cancer (CAC) with inflammatory bowel disease (IBD) serving as its prelude often has a poor prognosis due to the hysteretic diagnosis. As a representative of short chain fatty acids (SCFAs), butyrate has been proved to have obvious antitumor effect. Here, we aimed to examine its effect on CAC and possible mechanism in tumor microenvironment (TME).Method: The establishment of CAC mouse model was mainly based on the combination of AOM intraperitoneal injection and DSS three cycle. HE staining was used to analyze the degree of colonic inflammation and tumor dysplasia. The proportion of MDSCs population was mainly evaluated by flow cytometry assay. RT-PCR, immunohistochemical staining and western blot analysis was carried out to detect protein molecular expression.Results: In our current study, the AOM-DSS induced CAC mouse model was utilized to evaluate the effect of butyrate on CAC. The administration of butyrate significantly improved the weight loss, falling survival rate, higher DAI index and anal prolapse caused by the AOM-DSS during the CAC modeling process. Anatomical results including the size and number of tumors and histological results including the abnormal hyperplasia shown by HE staining also confirmed the inhibitory effect of butyrate on CAC. In addition, the proportion of myeloid-derived suppressor cells (MDSCs) assisting tumor immune escape in tumor microenvironment (TME) decreased under the intervention of butyrate. And inflammatory mediators including CCL2, IL-6 and TNF-α in TME that induce the recruitment of MDSCs showed the same trend as MDSCs. Toll-like receptor 2 (TLR2) as a receptor molecule related to inflammation and immune function was also up-regulated in CAC, accompanied by the synchronous up-regulation of downstream Myd88 and NF-κB molecules, while the use of butyrate significantly inhibited the up-regulation of these molecules.Conclusions: Butyrate might reduce the release of CCL2, IL-6 and TNF-α in TME by inhibiting TLR2/MyD88/NF-κB signaling pathway to reduce the recruitment of MDSCs in TME, which eventually weakened the immune escape of tumors and retarded the progress of CAC.

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Circulating CD62E Occurs in Soluble Form as Well as Bound to Endothelial Microparticles (EMP): Functional Differences in Platelet Aggregation in Thrombotic Thrombocytopenic Purpura (TTP).

Blood ◽

10.1182/blood.v104.11.2624.2624 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2624-2624

Author(s):

Joaquin J. Jimenez ◽

Wenche Jy ◽

Lucia M. Mauro ◽

Michael N. Markou ◽

George W. Burke ◽

...

Keyword(s):

Flow Cytometry ◽

Endothelial Cells ◽

Platelet Aggregation ◽

Acute Phase ◽

Critical Role ◽

In Vivo Studies ◽

Soluble Form ◽

Dependent Manner

Abstract Injured endothelial cells (EC) are believed to play a critical role in the pathophysiology of TTP. Soluble markers of endothelial disturbance measured by enzyme-linked immunoassay (ELISA) have been found elevated in TTP. We have recently demonstrated an increase in the release of CD31/42b- EMP, and CD62E+ EMP. Moreover, we have observed that CD62E+ EMP also express vWF. The aim of this study was to quantitate soluble (s) vs. EMP-bound CD62E (bCD62E) in vitro and in vivo, in relation to the functional activity of vWF+ EMP. METHODS: Brain and renal microvascular endothelial cells (MVEC) were cultured and treated with 10ng/mL TNF-α to induce activation, or deprived of serum and growth factors (GFD) to induce apoptosis. Culture supernatants were collected and evaluated in a time-dependent manner. For in vivo studies, platelet-poor plasma was obtained from 4 TTP patients during the acute phase and upon remission. Filtration through 0.1μm, which retains most EMP, was employed to discriminate between (s) and bCD62E. sCD62E was measured by ELISA post-filtration and bCD62E by ELISA pre-filtration. Additionally, CD62E+ and CD62E+/vWF+ EMP were measured by flow cytometry. To assess pro-aggregatory function, EMP were added to washed platelets in the presence of 1 mg/mL ristocetin and aggregates were measured by flow cytometry. RESULTS: In vitro: Activation did not induce release of sCD62E at 3 hours, although bCD62E was present (1.5±0.5X106 EMP/mL). At 6 hours, some sCD62E was detected in the filtrate (0.09±0.02 ng/mL), but most was present in the unfiltered medium (3.5±0.85 ng/mL), signifying that the majority was bCD62E, confirmed by a doubling of CD62E+ EMP (3.0±0.6X106/mL). Subsequently, sCD62E levels were 1.0±0.2 ng/mL at 12 hr, 3.5±0.7 ng/mL at 18 hr, and 5±0.9 ng/mL at 24 hr. In contrast, EMP counts at 12, 18 and 24 hours were 4.6±1, 7±1.3 and 9±1.8 X106/mL (p=0.01, p=0.01, p=0.02, respectively). For all time periods, 40-60% of CD62E were positive for vWF. In control or GFD cultures, there was not a significant increase in sCD62E or CD62E+ EMP at any time period. MVEC from renal gave similar results. In acute TTP plasma samples, CD62E measured by ELISA was significantly increased (65±22 ng/mL) vs. remission (30±6 ng/mL). bCD62E accounted for 50% in acute and 15% in remission. CD62E+/vWF+ EMP were significantly elevated in plasma from acute TTP patients vs. remission (15±4.5 vs. 3±0.5, p=0.01). Sample filtration resulted in a decrease of >95% EMP in both acute and remission TTP plasma. MVEC-derived CD62E+/vWF+ EMP resulted in a dose-dependent increase in platelet aggregation. Additionally, plasma from 4 TTP patients with elevated CD62E+/vWF+ EMP obtained during the acute phase enhanced the formation of platelet aggregates by 48±12% (p=0.02) above remission plasma with low EMP counts. CONCLUSIONS: The results demonstrate that CD62E heretofore regarded as a soluble marker of endothelial dysfunction, in reality exists in both a soluble and EMP-bound form. Indeed, this distinction is highly relevant because CD62E+ EMP also express vWF and are pro-aggregatory to platelets. These EMP have been shown to be elevated during the acute phase of TTP and decrease upon remission. Thus, CD62E+/vWF+ EMP may be active participants in the formation of platelet-rich thrombi in TTP.

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HO-1 Interactors Involved in the Colonization of the Bone Niche: Role of ANXA2 in Prostate Cancer Progression

Biomolecules ◽

10.3390/biom10030467 ◽

2020 ◽

Vol 10 (3) ◽

pp. 467 ◽

Cited By ~ 1

Author(s):

Nicolás Anselmino ◽

Juan Bizzotto ◽

Pablo Sanchis ◽

Sofia Lage-Vickers ◽

Emiliano Ortiz ◽

...

Keyword(s):

Prostate Cancer ◽

Cancer Progression ◽

Critical Role ◽

Culture Conditions ◽

Heme Oxygenase 1 ◽

Mrna Levels ◽

Dependent Manner ◽

Annexin 2 ◽

Raw264.7 Cell ◽

Pc3 Cells

Background: Prostate cancer (PCa) dissemination shows a tendency to develop in the bone, where heme oxygenase 1 (HO-1) plays a critical role in bone remodeling. Previously by LC/ESI-MSMS, we screened for HO-1 interacting proteins and identified annexin 2 (ANXA2). The aim of this study was to analyze the relevance of ANXA2/HO-1 in PCa and bone metastasis. Methods: We assessed ANXA2 levels using a co-culture transwell system of PC3 cells (pre-treated or not with hemin, an HO-1 specific inducer) and the pre-osteoclastic Raw264.7 cell line. Results: Under co-culture conditions, ANXA2 mRNA levels were significantly modulated in both cell lines. Immunofluorescence analysis unveiled a clear ANXA2 reduction in cell membrane immunostaining for Raw264.7 under the same conditions. This effect was supported by the detection of a decrease in Ca2+ concentration in the conditioned medium. HO-1 induction in tumor cells prevented both, the ANXA2 intracellular relocation and the decrease in Ca2+ concentration. Further, secretome analysis revealed urokinase (uPA) as a key player in the communication between osteoclast progenitors and PC3 cells. To assess the clinical significance of ANXA2/HO-1, we performed a bioinformatics analysis and identified that low expression of each gene strongly associated with poor prognosis in PCa regardless of the clinico-pathological parameters assessed. Further, these genes appear to behave in a dependent manner. Conclusions: ANXA2/HO-1 rises as a critical axis in PCa.

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Tissue factor procoagulant activity of plasma microparticles is increased in patients with early-stage prostate cancer

Thrombosis and Haemostasis ◽

10.1160/th08-10-0654 ◽

2009 ◽

Vol 101 (06) ◽

pp. 1147-1155 ◽

Cited By ~ 51

Author(s):

Brigitte Spath ◽

Martin Friedrich ◽

Felix Kyoung-Hwan Chun ◽

Guy Marx ◽

Ali Amirkhosravi ◽

...

Keyword(s):

Prostate Cancer ◽

Tissue Factor ◽

Thrombin Generation ◽

Early Stage ◽

Critical Role ◽

Procoagulant Activity ◽

Dependent Manner ◽

Phase Reaction ◽

Coagulation Activation ◽

D Dimer

SummaryTissue factor (TF) plays a critical role in tumour growth and metastasis, and its enhanced release into plasma in association with cellular microparticles (MPs) has recently been associated with pathological cancer progression. We have previously demonstrated significantly elevated levels of plasma TF antigen as well as systemic coagulation and platelet activation in patients with localised prostate cancer. In this prospective study, we used a highly sensitive one-stage clotting assay to measure preoperative TF-specific procoagulant activity (PCA) of plasma MPs in 68 consecutive patients with early-stage prostate cancer to further explore the relevance of circulating TF in this tumour entity. Automated calibrated thrombography was used to monitor thrombin generation in cell-free plasma samples in the absence of exogenous TF or phospholipids. Compared to healthy male controls (n=20), patients had significantly increased levels of both D-dimer and TF-specific PCA of plasma MPs (p<0.001). Furthermore, MP-associated TF PCA was higher in patients with (n=29) than in those without (n=39) laboratory evidence of an acute-phase reaction (p=0.004) and decreased to normal levels within one week after radical prostatectomy. Overall, we found a significant correlation between TF-specific PCA of plasma MPs and plasma D-dimer (p=0.002), suggesting that plasma MPs contributed to in-vivo coagulation activation in a TF-dependent manner. Thrombin generation in plasma was also significantly increased in patients compared to controls (p<0.01). Collectively, our findings suggest that TF-specific PCA of plasma MPs contributes to intravascular coagulation activation in patients with early-stage prostate cancer and may represent a potential link between hypercoagulability, inflammation, and disease progression.

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