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Tumor associated neutrophils promote the metastasis of pancreatic ductal adenocarcinoma.

ABSTRACT: The aim of this study was to investigate the role of tumor-associated neutrophils (TANs) in the metastasis of pancreatic ductal adenocarcinoma (PDAC), to explore the regulation of TANs, and to determine the mechanisms governing the metastasis of PDAC. The correlation between neutrophils and the patient's clinical pathological data was first evaluated. Then, the effects of neutrophils on the invasion of PDAC were analyzed using a combination of conditioned media, direct and indirect coculture of human peripheral blood neutrophils, and PDAC cell lines (Panc-1, MiaPaCa-2 and AsPC-1). The cytokines secreted by neutrophils were detected through ELISA. TAN density was significantly correlated with poor metastasis-free survival (P < .05). Through coculture, it was found that the effect of neutrophils on pancreatic cancer cells was dependent on concentration, and a high concentration of neutrophils showed a lethal effect, while a low concentration of neutrophils primarily promoted the migration ability of cancer cells. The results of the wound-healing assay, the Transwell invasion assay, and laser confocal microscopy confirmed the promoting effect and indicated that the effect of neutrophils toward cancer cells may function indirectly by releasing a series of cytokines. The results of ELISA show that this effect may be achieved through the secretion of a large amount of TNF-α and TGF-β1 by neutrophils. Our study indicated that neutrophils may increase the metastasis of PDAC by releasing a series of cell cytokines, such as TNF-α and TGF-β1.

SUBMITTER: Lianyuan T

PROVIDER: S-EPMC7583704 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionPancreatic ductal adenocarcinoma (PDAC) is projected to rise to the second leading cause of U.S. cancer-related deaths by 2020. Novel therapeutic targets are desperately needed. MicroRNAs (miRs) are small noncoding RNAs that function by suppressing gene expression and are dysregulated in cancer. miR-21 is overexpressed in PDAC tumor cells (TC) and is associated with decreased survival, chemoresistance and invasion. Dysregulation of miR regulatory networks in PDAC tumor-associated fibroblasts (TAFs) have not been previously described. In this study, we show that miR-21 expression in TAFs promotes TC invasion.MethodsIn-situ hybridization for miR-21 was performed on the 153 PDAC patient UCLA tissue microarray and 23 patient-matched lymph node metastases. Stromal and TC histoscores were correlated with clinicopathologic parameters by univariate and multivariate Cox regression. miR-21 positive cells were further characterized by immunofluorescence for mesenchymal/epithelial markers. For in vitro studies, TAFs were isolated from freshly resected human PDAC tumors by the outgrowth method. miR-21 was overexpressed/inhibited in fibroblasts and then co-cultured with GFP-MiaPaCa TCs to assess TC invasion in modified Boyden chambers.ResultsmiR-21 was upregulated in TAFs of 78% of tumors, and high miR-21 significantly correlated with decreased overall survival (P = 0.04). Stromal miR-21 expression was also significantly associated with lymph node invasion (P = 0.004), suggesting that it is driving TC spread. Co-immunofluorescence revealed that miR-21 colocalized with peritumoral fibroblasts expressing ?-smooth muscle actin. Moreover, expression of miR-21 in primary TAFs correlated with miR-21 in TAFs from patient-matched LN metastases; evidence that PDAC tumor cells induce TAFs to express miR-21. miR-21 expression in TAFs and TCs promotes invasion of TCs and is inhibited with anti-miR-21.ConclusionsmiR-21 expression in PDAC TAFs is associated with decreased overall survival and promotes TC invasion. Anti-miR-21 may represent a novel therapeutic strategy for dual targeting of both tumor and stroma in PDAC.

Project description:BackgroundPancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and is highly malignant due to its late diagnosis and early metastasis. Lung metastasis of PDAC occurs in a significant number of diagnosed patients and represents high severity of disease and poor clinical outcome. However, the molecular regulation of lung metastasis of PDAC is still not fully understood. Tumor-associated macrophages (TAMs) have recently been found to play an important role in cancer initiation, proliferation, progression, and metastasis. The proliferation, differentiation, and polarization of macrophages has been shown to be regulated by interleukin 1β (IL-1β), which is generated by NLR family pyrin domain containing 3 (NLRP3)-induced formation of inflammasome. Herein we investigated whether NLRP3 plays a role in lung metastasis of PDAC through regulation of macrophage polarization.MethodsGene profiles for NLRP3 (+/+) and NLRP3 (-/-) macrophages obtained from the Gene Expression Omnibus (GEO) public database were compared and analyzed for altered genes related to macrophage polarization. The regulation of macrophage polarization by NLRP3 was examined in a coculture system with naïve NLRP3 (+/+) or NLRP3 (-/-) macrophages and PDAC cells. Cell growth was analyzed by a Cell Counting Kit-8 (CCK-8) assay. Cell invasiveness and migratory potential were analyzed by transwell cell invasion assay and cell migration assay, respectively. PDAC formation and lung metastasis were analyzed in a mouse model of PDAC with and without NLRP3 knockout.ResultsGEO database analysis revealed significant alteration in genes that regulate macrophage polarization in NLRP3-depleted macrophages. NLRP3-depletion in macrophages seemed to favor an M1/M2b polarization. In vitro, the presence of NLRP3 in macrophages led to M2a/c/d TAM-like polarization when they were cocultured with PDAC cells. Conversely, NLRP3 depletion in macrophages led to M1/M2b polarization when they were cocultured with PDAC cells. NLRP3-depletion significantly inhibited tumor growth and stage progression in a mouse model of PDAC and significantly reduced the occurrence of lung metastasis.ConclusionsOur results suggested that NLRP3 activation in TAM enhanced lung metastasis of PDAC through regulation of TAM polarization.

Project description:The prognosis for pancreatic ductal adenocarcinoma (PDAC) remains poor despite decades of effort. The abundant extracellular matrix (ECM) in PDAC comprises a major fraction of the tumor mass and plays various roles in promoting resistance to therapies. However, nonselective depletion of ECM has led to poor patient outcomes. Consistent with that observation, we previously showed that individual matrisome proteins derived from stromal cells correlate with either long or short patient survival. In marked contrast, those derived from cancer cells correlate strongly with poor survival. Here, we studied three cancer cell-derived matrisome proteins that are significantly overrepresented during PDAC progression, AGRN (agrin), SERPINB5 (serine protease inhibitor B5), and CSTB (cystatin B). Using both overexpression and knockdown experiments, we demonstrate that all three are promoters of PDAC metastasis. Furthermore, these proteins operate at different metastatic steps. AGRN promoted epithelial-to-mesenchymal transition in primary tumors, whereas SERPINB5 and CSTB enhanced late steps in the metastatic cascade by elevating invadopodia formation and in vivo extravasation. All three genes were associated with a poor prognosis in human patients and high levels of SERPINB5, secreted by cancer cells and deposited in the ECM, correlated with poor patient prognosis. This study provides strong evidence that cancer cell-derived matrisome proteins can be causal in promoting tumorigenesis and metastasis and lead to poor patient survival. Therefore, compared with the bulk matrix, mostly made by stromal cells, precise interventions targeting cancer cell-derived matrisome proteins, such as AGRN, SERPINB5, and CSTB, may represent preferred potential therapeutic targets. SIGNIFICANCE: This study provides insights into the biological roles of cancer cell-derived matrisome proteins in PDAC and supports the notion that these proteins are protumorigenic and better therapeutic targets.

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Tumor associated neutrophils promote the metastasis of pancreatic ductal adenocarcinoma.

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