Effects Of Tumor-associated Macrophages Research Articles

Effect of tumor-associated macrophages on lncRNA PURPL/miR-363/PDZD2 axis in osteosarcoma cells

Tumor-associated macrophages (TAMs) are known to participate in osteosarcoma (OS) progression. As demonstrated in our previous research, miR-363 played a tumor inhibitory effect in OS cells via lowering the PDZ domain containing 2 (PDZD2) expression. The regulatory roles of TAMs on miR-363/PDZD2 and the internal mechanism relating to long noncoding RNA p53 upregulated regulator of P53 levels (lncRNA PURPL) are examined in this study. TAM-like macrophages were formed by inducing CD14+ peripheral blood mononuclear cells (PBMCs). The TAMs migration was detected after MG-63 cells transfected with miR-363 mimics or inhibitors. We then analyzed the regulatory activity of PURPL on miR-363 expression. We also tested the influences of PURPL overexpression/knockdown on MG-63 cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT), as well as TAMs migration. Silence in PDZD2 expression was used to confirm the effects of PURPL on MG-63 cells. We successfully induced TAM-like macrophages. MG-63 cells transfecting miR-363 mimics suppressed TAMs migration while transfecting a converse effect was seen in miR-363 inhibitor. TAMs raised PURPL expression in MG-63 cells, which was an upstream regulator of miR-363. Along with TAMs migration, PURPL overexpression promoted MG-63 cell proliferation, migration, invasion, and EMT. An opposite influence was seen due to the PURPL knockdown. The silence of PDZD2 weakened the influences of PURPL overexpression on MG-63 cells and TAMs migration. On modulating the PURPL/miR-363/PDZD2 axis, TAMs-promoted OS development might be achieved.

Autophagic tumor-associated macrophages promote the endothelial mesenchymal transition in lung adenocarcinomas through the FUT4/p-ezrin pathway.

BackgroundLung adenocarcinoma is one of the most common malignant tumors with high morbidity and mortality, but the effect of Tumor-associated macrophages (TAMs) on lung adenocarcinoma has not been studied clearly now.MethodsIn this study, TAMs were stably transfected with Atg5 silence or overexpression lentiviral vectors to inhibit or induce autophagy of TAMs. In addition, the expression of fucosyltransferase IV (FUT4) or Ezrin were interfered in TAMs with autophagy. The above treated TAMs were then co-cultured with A549 or H1299 cells. The expressions of genes were detected by qPCR, western blotting, cell immunofluorescence, and enzyme-linked immunosorbent assay. Meanwhile, cell migration and invasion were analyzed by Transwell assay and wound healing assay. Furthermore, the effects of TAMs with autophagy were explored in lung adenocarcinoma xenograft model of mice.ResultsThe results showed that overexpression of autophagy-related gene 5 (ATG5) induced autophagy in TAMs, which increased the expression of FUT4, TGF-β1, and p-ezrin, and promoted epithelial-mesenchymal transition (EMT) in lung adenocarcinoma cells. However, FUT4 silencing partially reversed the effects of TAM autophagy, specifically, the expression of TGF-β1 and p-ezrin was inhibited and EMT in lung adenocarcinoma cells was suppressed. Notably, ezrin deletion in autophagic TAMs induced by rapamycin reduced TGF-β1 expression and suppressed EMT in lung adenocarcinoma cells. Consistently, in vivo experiments also revealed that autophagic TAMs increased the expression of FUT4, TGF-β1, and p-ezrin, and promoted EMT in lung adenocarcinomas. Similarly, FUT4 silencing partially reversed the effects of autophagic TAMs on EMT in lung adenocarcinomas.ConclusionsIn conclusion, autophagic TAMs promoted TGF-β1 secretion through the FUT4/p-ezrin pathway and induced EMT in co-cultured lung adenocarcinoma cells.

The Purification and Characterization of Exosomes from Macrophages.

Macrophages play an essential role in diverse biological processes, from the immune response to inflammatory and neurodegenerative disorders, to various cancers. A macrophage subpopulation, known as tumor-associated macrophages (TAMs), has been shown to promote tumorigenesis, metastasis, and immune escape of cancer cells. Some of the pro-tumorigenic effects of TAMs are mediated via the secretion of nano-vesicles (exosomes) from macrophages to neighboring cells. In this chapter, we describe peritoneal macrophage isolation methods, polarization of TAMs, and purification and characterization of macrophage-derived exosomes.

Tumor-associated macrophage enhances invasion and migration of breast cancer cells T47D

Objective To explore the effect of tumor-associated macrophage (TAM) on invasion and migration of breast cancer cells T47D. Methods Briefly, phorbol 12-myristate 13-acetate (PMA) was employed to treat mononuclear cells THP-1. And then interleukin 4 (IL-4) was applied to stimulate mentioned cells to establish activated TAM. Furthermore, the supernatant of M0 and M2 was used to culture T47D cells, respectively. Then, the wound healing experiment and transwell assay were carried out to investigate cells invasion and migration. Finally, epithelial-mesenchymal transition (EMT) biomarkers in T47D cells treated with M0 or M2 were determined by quantitative real time polymerase chain reaction (qRT-PCR) and western blot assays. Results Activated TAM model was successfully established by PMA following with IL-4. Comparing with M0, M2 evidently accelerated invasion and migration in T47D cells. Meanwhile, M2 upregulated N-cadherin, vimentin and β-catenin, downregulated E-cadherin at mRNA and protein expression levels. Conclusions M2 promotes invasion and migration of T47D cells via EMT. Key words: Macrophages; Breast neoplasms; Cell migration assays; In vitro

Effect of tumor-associated macrophages in promoting epithelial-mesenchymal transition of Hep3B hepatoma cells and related mechanisms

Objective: To investigate the possible mechanisms of tumor-associated macrophages (TAMs) in regulating epithelia-mesenchymal transition (EMT) of Hep3B hepatoma cells, since EMT is closely associated with the malignancy of hepatoma cells and tumor microenvironment plays an important role in regulating EMT of hepatoma cells, and to provide new regimens for the clinical studies and treatment of liver cancer. Methods: Human monocytic leukemia THP-1 cells were successfully induced to TAMs. With TAMs as target cells, they were co-cultured with the supernatant of Hep3B hepatoma cells or Hep3B hepatoma cells, and Western blot and RT-PCR were used to measure the change in the expression of Toll-like receptor 4 (TLR4) in TAMs. The expression of TLR4 in TAMs was downregulated by transient plasmid transfection with shRNA. With Hep3B hepatoma cells as target cells, the supernatants of TAMs and TAMs transfected with shRNA TLR4 plasmid were used for intervention, and Western blot was used to measure the protein expression of E-cadherin, N-cadherin, and vimentin. The two-sided t-test was used for comparison of the means of two independent samples. Results: THP-1 cells were successfully induced to TAMs. According to the results of Western blot, compared with the control-CM group, the TAM-CM group had a significant reduction in the protein expression of E-cadherin and significant increases in the protein expression of N-cadherin and vimentin in Hep3B cells. After the expression of TLR4 in TAMs was downregulated, the culture solution of TAMs was used for the intervention of Hep3B cells (shRNA group), and compared with the TAM-CM group, the shRNA group had a significant increase in the expression of E-cadherin and significant reductions in the protein expression of N-cadherin and vimentin in Hep3B cells. Western blot and RT-PCR showed that the expression of TLR4 in TAMs was influenced by Hep3B cells. Conclusion: TAMs can promote EMT of Hep3B hepatoma cells, and downregulation of the expression of TLR4 in TAMs may reduce EMT of Hep3B hepatoma cells, suggesting that TLR4 on the surface of TAMs may be a key molecule involved in the interaction between TAMs and Hep3B hepatoma cells.

Possible immunomodulatory effects of tumor-associated macrophages in RANKL expressing apocrine-origin cancers

Possible immunomodulatory effects of tumor-associated macrophages in RANKL expressing apocrine-origin cancers

Effect of tumor-associated macrophages on invasion and metastasis of gastric cancer cells

To explore the influence of tumor-associated macrophages(TAMs) on the ability of invasion and metastasis of gastric cancer cells, and its associated mechanism. Immunohistochemistry was used to examine the expression of TAM in 10 samples of normal gastric mucosa and 15 samples of gastric cancer tissues from sample bank of Department of Pathology, Union Hospital. Phorbol 12-myristate 13-acetate(PMA) and macrophage colony stimulating factor (M-CSF) were used to make THP-1 monocytes differentiate into TAMs. AGS gastric cancer cells were divided into two groups: experiment group was cultured with RPMI/1640 condition medium containing 50% TAM and control group was cultured with RPMI/1640 complete medium. The ability of invasion and metastasis of gastric cancer cells was measured by Transwell assays. Real-time PCR and Western blot were applied to detect the expression of MMPs and its inhibitor TIMPs before and after stimulation of TAMs. Immunohistochemistry results showed that CD68(+) cell number in normal gastric mucosa tissue was significantly less than that in gastric cancer tissue [(11.3±0.8)/HP vs. (31.6±1.4)/HP, P<0.000 1]. When treated with PMA and M-CSF, THP-1 cells were differentiated into type M2 TAMs with high expression of specific markers CD68, CD163, CD204 and CD206. Transwell test revealed that the number of piercing cells in the experimental group was significantly more than that in control group [(36.8±1.1)/HP vs. (12.8±0.9)/HP, t=17.5, P=0.000). Compared to control group, the expression of MMP-2, MMP-9 mRNA in experimental group respectively increased by 1.61 and 1.87 folds(P=0.017 and P=0.009). Protein level of MMP-2, MMP-9 was up-regulated accordingly. The expression of TIMP-1 and TIMP-3 mRNA was not significantly different between two groups(P=0.120 and P=0.096). TAMs may promote the invasion and metastasis of gastric cancer cells through increasing expression of MMP-9 and MMP-2, which may be one of the mechanisms of gastric cancer development.

Histological perspective on the effects of tumor-associated macrophages in the tumor microenvironment surrounding papillary thyroid carcinoma

Tumor-associated macrophages (TAMs) are one of the most noticeable elements of the tumor microenvironment. The present study investigated the relationships between the density of CD163 immunolabeled M2-like TAMs with other histological properties of the tumor microenvironment and clinipathological features in 90 patients with papillary thyroid carcinomas (PTC). The percentage of TAMs was higher in tumors with significant lymphocytic tumor response (p = 0.020), in tumors with a significant degree of stromal tumor response (p = 0.014), those with infiltrative tumor borders (p = 0.029), in conventional variant papillary carcinoma (p = 0.032), and in patients with autoantibodies for thyroid peroxidase (p = 0.014). The tumors associated with lymphocytic thyroiditis had lower numbers of TAMs (p = 0.027). In conclusion, for the first time, the present study attempts to establish a full assessment of interactions of CD163 expressing M2-like TAMs with the triad of primary tumor- tumor microenvironment- tumor behavior and above all, with markers of autoimmunity. Thus, these alternatively polarized macrophages may act in tumor progression and dissemination according to their various products, which may be ordered by tumor cells or neighboring immune cells. The molecular studies may reveal their roles in various tumors and may improve the therapy strategies targeting TAMs in various malignant tumors, including PTCs.

Effect of tumor-associated macrophages on neoplastic progression in sepsis-surviving mice through CXCL12/CXCR4.

e22107 Background: Sepsis surviving mice (SSM) present increased tumor progression. We assessed the role of tumor-associated macrophage (TAM) and CXCL12/CXCR4 in that scenario. Methods: C57/BL6 mic...

Abstract 3865: HAS2 promotes tumor progression by stimulating interaction of cancer stem-like cells with tumor associated macrophage and stromal cells through a paracrine loop of growth factors in the bone.

Abstract The molecular mechanism of metastatic progression, particularly the roles of microenvironment of the metastasized organs, is as yet poorly understood. In this study, we have isolated cancer stem cell-like cells (CSC) from human breast cancer cell lines, MDA-MB231, MDA231BoM and MDA231BrM. CSC were isolated from each line using surface markers (CD24-, CD44+, ESA+) and their tumor initiating abilities were tested in animal. We then performed an analysis of global expression profile for these CSCs using the Affymetrix expression array and found that among all up-regulated genes, two genes (HAS2 and MMP1) were significantly correlated with overall- and metastasis-free survival when they were cross-examined in GEO database. To further examine the role of HAS2 in tumor metastasis in vivo, we prepared CSC from MDA231BoM with or without carrying a shRNA expression vector for HAS2. The CSC were then implanted into nude mice by intra-cardiac injection followed by oral administration of 4MU, a specific inhibitor of HAS genes. We found that the injection of CSC of MDA231BoM which expresses shRNA to HAS2 significantly improved the survival rate of animals. In addition, administration of 4MU significantly suppressed tumor metastasis of CSC, suggesting that inhibition of HAS2 can indeed reduce the incidence of metastasis in vivo. We also examined the effect of tumor associated macrophage (TAM) on growth of CSC in animals by injecting CSC of MDA231-BoM with or without TAM into the tibial bone. We found that co-injection of TAM with CSC significantly augmented the growth of tumor in their legs. Moreover, the interaction between cancer cell and TAMs enhanced secretion of PDGF-BB from TAMs which then activated stromal cells and in turn enhanced self-renewal of CSCs by secretion of FGF9 and FGF7. We also found that treatment of bone marrow stem cell (BMSC) with PDGF followed by co-culture of CSC significantly promoted proliferation of CSC. Collectively, our results suggest that TAM and BMSC promote self-renewal of CSC in bone microenvironment via direct interaction between hyaluronan and CD44. Our results also provide strong rationale to target HAS2 by using 4MU in order to block the metastatic progression of CSC in breast cancer. Citation Format: Sambad Sharma, Hiroshi Okuda, Fei Xing, Aya Kobayashi, Misako Watabe, Puspa R. Pandey, Sudha K. Pai, Wen Liu, Koji Fukuda, Megumi Iiizumi, Kounosuke Watabe. HAS2 promotes tumor progression by stimulating interaction of cancer stem-like cells with tumor associated macrophage and stromal cells through a paracrine loop of growth factors in the bone. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3865. doi:10.1158/1538-7445.AM2013-3865

The biological activity of macrophages in health and disease

Macrophages are involved in immune response as phagocytes, antigen presenting cells and as effector cells of delayed-type hypersensitivity. Moreover, the activity of macrophages is associated with modulation of many biological processes during the whole life and depends on the actual macrophage phenotype induced under the influence of various microenvironmental stimuli. In pregnancy, placental macrophages induce the development of maternal tolerance to fetal antigens, while fetal macrophages are responsible for proper formation of tissues and organs. Residual macrophages play a very important role in tissue homeostasis, apoptotic cell clearance to prevent autoimmunization and first defense in infections. The inflammatory response of macrophages may be modulated by pathogens. Their suppressive activity is observed in immunologically privileged organs such as testes. In pathologies, macrophages are responsible for tissue damage in a case of nonspecific activation followed by overproduction of proinflammatory factors. Suppression of a specific immune response against tumors is mainly the effect of tumor associated macrophage (TAM) action. On the other hand, presentation of allergens or self-antigens by macrophages and their nonspecific activation by necrotic adipocytes leads to the induction of a chronic inflammatory response and impairment of immunity. Therefore, modulation of macrophage functions may be the key for improvement of therapy of cancer and allergic, autoimmune, metabolic, cardiovascular and Alzheimer's diseases.

Abstract 3323: Hyaluronan synthase 2 promotes breast cancer bone metastasis by stimulating interaction of cancer stem-like cells with tumor-associated macrophage

Abstract The molecular mechanism of metastatic progression, particularly the roles of microenvironment of the metastasized organs, is as yet poorly understood. In this study, we have isolated cancer stem cell-like cells (CSC) from human breast cancer cell lines, MDA-MB231, MDA231BoM and MDA231BrM. CSC were isolated from each line using surface markers (CD24-, CD44+, ESA+) and their tumor initiating abilities were tested in animal. We then performed an analysis of global expression profile for these CSCs using the Affymetrix expression array and found that among all up-regulated genes, two genes (HAS2 and MMP1) were significantly correlated with overall- and metastasis-free survival when they were cross-examined in GEO database. To further examine the role of HAS2 in tumor metastasis in vivo, we prepared CSC from MDA231BoM with or without carrying a shRNA expression vector for HAS2. The CSC were then implanted into nude mice by intra-cardiac injection followed by oral administration of 4MU, a specific inhibitor of HAS genes. We found that the injection of CSC of MDA231BoM which expresses shRNA to HAS2 significantly improved the survival rate of animals. In addition, administration of 4MU significantly suppressed tumor metastasis of CSC, suggesting that inhibition of HAS2 can indeed reduce the incidence of metastasis in vivo. We also examined the effect of tumor associated macrophage (TAM) on growth of CSC in animals by injecting CSC of MDA231-BoM with or without TAM into the tibial bone. We found that co-injection of TAM with CSC significantly augmented the growth of tumor in their legs. Moreover, the interaction between cancer cell and TAMs enhanced secretion of PDGF-BB from TAMs which then activated stromal cells and in turn enhanced self-renewal of CSCs by secretion of FGF9 and FGF7. We also found that treatment of bone marrow stem cell (BMSC) with PDGF followed by co-culture of CSC significantly promoted proliferation of CSC. Collectively, our results suggest that TAM and BMSC promote self-renewal of CSC in bone microenvironment via direct interaction between hyaluronan and CD44. Our results also provide strong rationale to target HAS2 by using 4MU in order to block the metastatic progression of CSC in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3323. doi:1538-7445.AM2012-3323

Abstract 3409: Hyaluronan synthase 2 promotes tumor progression by stimulating interaction of cancer stem-like cells with tumor associated macrophage and stromal cells in the bone

Abstract According to the recent cancer stem theory, metastatic cells must have stem cell-like capability for their self-renewal and invasive ability. Understanding how the cancer stem cells adapt themselves to the microenvironment of the distant organs and how the “niche” at the metastatic site promotes the proliferation of these cells is of paramount importance to develop more effective therapeutic strategy for metastatic disease. In this study, we have isolated cancer stem cell-like cells (CSC) from human breast cancer cell lines, MDA-MB231, MDA231BoM and MDA231BrM. The latter two cell lines are derivatives of MDA-MB231 and they preferentially metastasize to bone (MDA231BoM) or brain (MDA231BrM) when they are transplanted into mice. CSC were isolated from each line using surface markers (CD24-, CD44+, ESA+) and their tumor initiating abilities were tested in animal. We then performed an analysis of global expression profile for these CSCs using the Affymetrix expression array and found that among all up-regulated genes, two genes (HAS2 and MMP1) were significantly correlated with overall- and metastasis-free survival when they were cross-examined in GEO database. To further examine the role of HAS2 in tumor metastasis in vivo, we prepared CSC from MDA231BoM which carries shRNA expression vector for HAS2. The CSC were then implanted into nude mice by intra-cardiac injection followed by oral administration of 4MU, a specific inhibitor of HAS genes. We found that the injection of CSC of MDA231BoM which expresses shRNA significantly improved the survival rate of animals. In addition, administration of 4MU significantly suppressed tumor metastasis of CSC, suggesting that inhibition of HAS2 can indeed reduce the incidence of metastasis in vivo. We also examined the effect of tumor associated macrophage (TAM) on growth of CSC in animals by injecting CSC of MDA231-BoM with or without TAM into the tibial bone. We found that co-injection of TAM with CSC significantly augmented the growth of tumor in their legs. These results strongly support the idea that TAM plays a critical role in CSC growth in breast cancer. To understand how TAM promotes CSC proliferation, we attempted to identify secretory factor(s) from TAM by using a cytokine/growth factor antibody array system. We found that the secretion of PDGF from TAM was significantly augmented in conditioned medium of co-culture of CSC and TAM. Furthermore, we found that treatment of bone marrow stem cell (BMSC) with PDGF followed by co-culture of CSC significantly promoted proliferation of CSC. Collectively, our results suggest that TAM and BMSC promote self-renewal of CSC in bone microenvironment via direct interaction between hyaluronan and CD44. Our results also provide strong rationale to target HAS2 by using 4MU in order to block the metastatic progression of CSC in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3409. doi:10.1158/1538-7445.AM2011-3409