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Alternatively activated macrophage-derived secretome stimulates ovarian cancer spheroid spreading through a JAK2/STAT3 pathway.

ABSTRACT: High-grade serous ovarian cancer (HGSOC) metastasizes when tumor spheroids detach from the primary tumor and re-attach throughout the peritoneal cavity. Once the cancer cells have implanted in these new sites, the development of metastatic lesions is dependent on the disaggregation of cancer cells from the spheroids and subsequent expansion across the collagenous extracellular matrix (ECM). As HGSOC progresses an increase in alternatively activated macrophages (AAMs) in the surrounding ascites fluid has been observed and AAMs have been shown to enhance tumor invasion and growth in a wide range of cancers. We hypothesized that soluble factors from AAMs in the peritoneal microenvironment promote the disaggregation of ovarian cancer spheroids across the underlying ECM. We determined that co-culture with AAMs significantly increased HGSOC spheroid spreading across a collagen matrix. Multivariate modeling identified AAM-derived factors that correlated with enhanced spread of HGSOC spheroids and experimental validation showed that each individual cell line responded to a distinct AAM-derived factor (FLT3L, leptin, or HB-EGF). Despite this ligand-level heterogeneity, we determined that the AAM-derived factors utilized a common signaling pathway to induce spheroid spreading: JAK2/STAT3 activation followed by MMP-9 mediated spreading. Furthermore, immunostaining demonstrated that FLT3, LEPR, EGFR, and pSTAT3 were upregulated in metastases in HGSOC patients, with substantial patient-to-patient heterogeneity. These results suggest that inhibiting individual soluble factors will not inhibit AAM-induced effects across a broad group of patients; instead, the downstream JAK2/STAT3/MMP-9 pathway should be examined as potential therapeutic targets to slow metastasis in ovarian cancer.

SUBMITTER: Fogg KC

PROVIDER: S-EPMC6659112 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Alternatively activated macrophage-derived secretome stimulates ovarian cancer spheroid spreading through a JAK2/STAT3 pathway.

Fogg Kaitlin C KC Olson Will R WR Miller Jamison N JN Khan Aisha A Renner Carine C Hale Isaac I Weisman Paul S PS Kreeger Pamela K PK

Cancer letters 20190524

High-grade serous ovarian cancer (HGSOC) metastasizes when tumor spheroids detach from the primary tumor and re-attach throughout the peritoneal cavity. Once the cancer cells have implanted in these new sites, the development of metastatic lesions is dependent on the disaggregation of cancer cells from the spheroids and subsequent expansion across the collagenous extracellular matrix (ECM). As HGSOC progresses an increase in alternatively activated macrophages (AAMs) in the surrounding ascites f ...[more]

PMID: 31129149

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Project description:BackgroundZhiqiao Gancao decoction (ZQGCD) was created by Professor Gong Zhengfeng, a renowned Chinese medicine expert. Clinical studies have shown its efficacy in alleviating pain and enhancing lumbar function in intervertebral disc degeneration (IDD) patients. However, the precise mechanism of ZQGCD in treating IDD remains unclear.MethodsThe active components of ZQGCD were identified using Liquid chromatography-tandem mass spectrometry (LC-MS/MS). A rat model of intervertebral disc degeneration was established, and rats in each group received ZQGCD for three weeks. Assessment parameters included hyperalgesia status, observation of intervertebral disc tissue degeneration and macrophage infiltration, and analysis of JAK2/STAT3 pathway protein expression in the intervertebral disc. Primary macrophage M1 polarization was induced using LPS, with cells treated using the JAK2 inhibitor (AZD1480) and ZQGCD to evaluate macrophage polarization, cellular supernatant inflammatory factors, and JAK2/STAT3 pathway expression. Macrophage supernatant served as a conditioned medium to observe its effects on the proliferation of nucleus pulposus cells (NPCs) and the expression of collagen II and MMP3 proteins.ResultsA total of 81 active components were identified in ZQGCD. Following ZQGCD treatment, infiltrating macrophages in intervertebral disc tissues of model rats decreased, the content of M1 macrophages decreased, while the content of M2 macrophages increased, the expression of proinflammatory factors and pain-inducing factors in serum decreased, and the expression of substance P in intervertebral disc tissue decreased. Consequently, the intervertebral disc degeneration and hyperalgesia of rats were improved. In vitro studies revealed that LPS induced M1 macrophage polarization. By inhibiting the JAK2/STAT3 pathway, both JAK2 inhibitors and ZQGCD effectively suppressed M1 polarization, resulting in decreased levels of IL-1β, IL-6, TNF-α, and various other inflammatory factors. Consequently, this inhibition led to a delay in the degeneration of NPCs.ConclusionThere is macrophage infiltration in the intervertebral disc tissue of IDD rats, and JAK2/STAT3 pathway is activated, macrophages are polarized to M1 type, resulting in inflammatory microenvironment, leading to intervertebral disc degeneration and hyperalgesia. ZQGCD exhibited a delaying effect on IDD and improved hyperalgesia by inhibiting the JAK2/STAT3/macrophage M1 polarization pathway.

Project description:BackgroundAlternatively activated (M2) macrophages are phenotypically characterized by the expression of specific markers, mainly macrophage scavenger receptors (CD204 and CD163) and mannose receptor-1 (CD206), and participate in the fibrotic process by over-producing pro-fibrotic molecules, such as transforming growth factor-beta1 (TGFbeta1) and metalloproteinase (MMP)-9. Endothelin-1 (ET-1) is implicated in the fibrotic process, exerting its pro-fibrotic effects through the interaction with its receptors (ETA and ETB). The study investigated the possible role of ET-1 in inducing the transition from cultured human macrophages into M2 cells.MethodsCultured human monocytes (THP-1 cell line) were activated into macrophages (M0 macrophages) with phorbol myristate acetate and subsequently maintained in growth medium (M0-controls) or treated with either ET-1 (100nM) or interleukin-4 (IL-4, 10ng/mL, M2 inducer) for 72 hours. Similarly, primary cultures of human peripheral blood monocyte (PBM)-derived macrophages obtained from healthy subjects, were maintained in growth medium (untreated cells) or treated with ET-1 or IL-4 for 6 days. Both M0 and PBM-derived macrophages were pre-treated with ET receptor antagonist (ETA/BRA, bosentan 10-5M) for 1 hour before ET-1 stimulation. Protein and gene expression of CD204, CD206, CD163, TGFbeta1 were analysed by immunocytochemistry, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Gene expression of interleukin(IL)-10 and macrophage derived chemokine (CCL-22) was evaluated by qRT-PCR. MMP-9 production was investigated by gel zymography.ResultsET-1 significantly increased the expression of M2 phenotype markers CD204, CD206, CD163, IL-10 and CCL-22, and the production of MMP-9 in both cultures of M0 and PBM-derived macrophages compared to M0-controls and untreated cells. In cultured PBM-derived macrophages, ET-1 increased TGFbeta1 protein and gene expression compared to untreated cells. The ET-1-mediated effects were contrasted by ETA/BRA treatment in both cultured cell types.ConclusionET-1 seems to induce the M2 phenotype in cultured human macrophages, a process apparently contrasted by the action of the ETA/BRA, suggesting possible clinical implications in those fibrotic diseases characterized by increased ET-1 concentrations, such as systemic sclerosis but also type 2 diabetes.

Project description:Alternatively activated macrophages have been reported to be helpful to alleviate systematic lupus erythematosus (SLE), and azithromycin could serve as an immunomodulator by promoting alternatively activated macrophage phenotype. However, the effect of azithromycin in SLE and the involved mechanism remain undetermined. The aim of this study is to characterize azithromycin and the underlying mechanism contributing to SLE therapy. First, we compared monocytes from SLE patients and matched healthy donors, and found monocytes from SLE patients exhibited more CD14+CD86+ cells, impaired phagocytic activity, and elevated interleukin (IL)-1?, IL-6, and tumor necrosis factor (TNF)-? (the classical activated phenotype), which could be blocked by azithromycin. On the contrary, there were fewer CD14+CD163+ cells in SLE patients, accompanied by decreased arginase (Arg)-1 and found in inflammatory zone (Fizz)-1 (the alternatively activated phenotype). And IL-10, the crucial immune regulatory factor secreted by alternatively activated monocytes/macrophages, also showed a decreased trend in SLE patients. In addition, all these markers were up-regulated after azithromycin treatment. Next, we used activated lymphocyte-derived-DNA to imitate SLE macrophages in vitro to investigate the possible mechanism involved. Azithromycin showed the same effect in imitated SLE macrophages, with distinct Akt phosphorylation at 30?min and 12?h. After inhibiting Akt phosphorylation by LY294002, the down-regulation of CD80, IL-1?, IL-6, and TNF-? caused by azithromycin raised again, meanwhile, the up-regulation of CD206, Arg-1, Fizz-1, and IL-10 due to azithromycin was abolished. Additionally, insulin-like growth factor 1 (IGF-1), the specific agonist of Akt, played a similar role to azithromycin in imitated SLE macrophages. Taken together, our data indicated a novel role of azithromycin in alleviating SLE by promoting alternatively activated macrophage phenotype, and the PI3K/Akt signaling pathway was involved. Our findings provide a rationale for further investigation of novel therapeutic strategy for SLE patients.

Project description:BackgroundM1 macrophages (Mφs) are involved in osteogenic differentiation of ligamentum flavum (LF) cells and play an important role in heterotopic ossification. However, the mechanism by which M1 Mφs influence osteogenic differentiation of LF cells has not been studied.MethodsThe effect of conditioned medium including secretions of M1 Mφs (CM-M1) on LF cells was analyzed by GeneChip profiling and ingenuity pathway analysis (IPA). THP-1 cells were polarized into M1 Mφs and CM-M1 was used to induce LF cells. In addition, LF cells were induced by CM-M1 in the presence of cyclooxygenase 2 (COX-2) inhibitors or oncostatin M (OSM)-neutralizing antibodies. Based on the presence of OSM, knockout of OSMR or GP130 receptors, or addition of the Janus kinase 2 (JAK2) inhibitor AZD1480 or signal transducer and activator of transcription 3 (STAT3) inhibitor Stattic were examined for effects on osteogenic differentiation of LF cells. OSM secretion was quantified by ELISA, while qPCR and western blot were used to evaluate expression of osteogenic genes and receptor and signaling pathway-related proteins, respectively.ResultsGeneChip and IPA results indicate that the OSM signaling pathway and its downstream signaling molecules JAK2 and STAT3 are significantly activated. ELISA results indicate that OSM is highly expressed in cells treated with CM-M1 and lowly expressed in cells treated with CM-M1 and a COX-2 inhibitor. Besides, CM-M1 induces osteogenic differentiation of LF cells, which is weakened when COX-2 inhibitors or OSM-neutralizing antibody are added to it. Recombinant OSM could induce osteogenic differentiation of LF cells and upregulate expression of OSMR, GP130, phosphorylated (P)-JAK2, and P-STAT3. Upon knockdown of OSMR or GP130, or the addition of AZD1480 or Stattic, P-JAK2 and P-STAT3 expression were decreased and osteogenic differentiation was reduced.ConclusionM1 Mφ-derived OSM induces osteogenic differentiation of LF cells and the JAK2/STAT3 signaling pathway plays an important role.

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Alternatively activated macrophage-derived secretome stimulates ovarian cancer spheroid spreading through a JAK2/STAT3 pathway.

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Alternatively activated macrophage-derived secretome stimulates ovarian cancer spheroid spreading through a JAK2/STAT3 pathway.

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