Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis

Primary tumours influence the environment in the lungs before metastasis. However, the mechanism of metastasis is not well understood. Here, we show that the inflammatory chemoattractants S100A8 and S100A9, whose expression is induced by distant primary tumours, attract Mac 1 (macrophage antigen 1)(+)-myeloid cells in the premetastatic lung. In addition, tumour cells use this mechanism, through activation of the mitogen-activated protein kinase (MAPK) p38, to acquire migration activity with pseudopodia for invasion (invadopodia). The expression of S100A8 and S100A9 was eliminated in lung Mac 1(+)-myeloid cells and endothelial cells deprived of soluble factors, such as vascular endothelial growth factor A (VEGF-A), tumour necrosis factor alpha (TNFalpha) and transforming growth factor beta (TGFbeta) both in vitro and in vivo. Neutralizing anti-S100A8 and anti-S100A9 antibodies blocked the morphological changes and migration of tumour cells and Mac 1(+)-myeloid cells. Thus, the S100A8 and S100A9 pathway may be common to both myeloid cell recruitment and tumour-cell invasion.     

Dioscin elicits anti‐tumour immunity by inhibiting macrophage M2 polarization via JNK and STAT3 pathways in lung cancer

Tumour‐associated macrophage (TAM) is an important component in tumour microenvironment. Generally, TAM exhibits the function of M2‐like macrophage, which was closely related to angiogenesis and tumour progression. Dioscin, a natural steroidal saponin, has shown its powerful anti‐tumour activity recently. However, the mechanism of dioscin involved in immune regulation is still obscure. Here, we observed dioscin induced macrophage M2‐to‐M1 phenotype transition in vitro and inhibited IL‐10 secretion. Meanwhile, the phagocytosis of macrophages was enhanced. In subcutaneous lung tumour models, dioscin inhibited the augmentation of M2 macrophage populations. Furthermore, dioscin down‐regulated STAT3 and JNK signalling pathways in macrophages in vitro. In BMDMs, activating JNK and inhibiting STAT3 induce macrophages to M1 polarization while inhibiting JNK and activating STAT3 to M2 polarization. Additionally, condition mediums from dioscin‐pre‐treated macrophages inhibited the migration of 3LL cells and the tube‐formation capacity of HUVECs. What's more, dioscin‐mediated macrophage polarization inhibited the in vivo metastasis of 3LL cells. In conclusion, dioscin may act as a new anti‐tumour agent by inhibiting TAMs via JNK and STAT3 pathways in lung cancer.     

P2X7R promotes angiogenesis and tumour‐associated macrophage recruitment by regulating the NF‐κB signalling pathway in colorectal cancer cells

Overexpression of P2X7R has been observed in several tumours and is related to cancer advancement and metastasis. However, the role of P2X7R in colorectal cancer (CRC) patients is not well understood. In the current study, overexpression of P2X7R and the effects at the molecular and functional levels in CRC were assessed in a mouse orthotopic model. Functional assays, such as the CCK‐8 assay, wound healing and transwell assay, were used to determine the biological role of P2X7R in CRC cells. CSC‐related genes and properties were detected via sphere formation and real‐time PCR assays. The underlying mechanisms were explored by Western blotting, real‐time PCR and Flow cytometry. In this study, we found that overexpression of P2X7R increases in the in vivo growth of tumours. P2X7R overexpression also increased CD31, VEGF and concurrent angiogenesis. P2X7R up‐regulates aldehyde dehydrogenase‐1 (ALDH1) and CSC characteristics. Transplanted tumour cells with P2X7R overexpression stimulated cytokines to recruit tumour‐associated macrophage (TAMs) to increase the growth of tumours. We also found that the NF‐κB signalling pathway is involved in P2X7R‐induced cytokine up‐regulation. P2X7R promotes NF‐κB–dependent cytokine induction, which leads to TAM recruitment to control tumour growth and advancement and remodelling of the stroma. Our findings demonstrate that P2X7R plays a key role in TAM recruitment, which may be a therapeutic target for CRC patients.     

Hypothesis: are neoplastic macrophages/microglia present in glioblastoma multiforme?

Most malignant brain tumours contain various numbers of cells with characteristics of activated or dysmorphic macrophages/microglia. These cells are generally considered part of the tumour stroma and are often described as TAM (tumour-associated macrophages). These types of cells are thought to either enhance or inhibit brain tumour progression. Recent evidence indicates that neoplastic cells with macrophage characteristics are found in numerous metastatic cancers of non-CNS (central nervous system) origin. Evidence is presented here suggesting that subpopulations of cells within human gliomas, specifically GBM (glioblastoma multiforme), are neoplastic macrophages/microglia. These cells are thought to arise following mitochondrial damage in fusion hybrids between neoplastic stem cells and macrophages/microglia.     

Macrophages and Apoptotic cells in Human Colorectal Tumours

The numerical relationship between tumour associated macrophages (TAM) and apoptotic cells in 12 human colorectal tumours was evaluated. TAM were labelled immunohistochemically and apoptotic cells were visualized by counterstaining with haematoxylin and eosin (H&E). The stereological techniques. Cavalieri's estimator of volume and the Disector were used to estimate both tumour volume and numerical density of both cell types. The occurrence of TAM per unit volume of tissue increased with increasing tumour volume to a maximum in a tumour of 110·5 cm³, after which numbers declined. Levels of apoptosis also increased with tumour volume though more erratically than levels of TAM and declined for tumour volumes greater than 80 cm³. This is the first report of an attempt to assess the relationship between apoptotic cells and TAM in human tumours.     

TGFbeta1 regulation and collagen-release-independent connective tissue re-modelling by the ruthenium complex NAMI-A in solid tumours

PURPOSE: The objective of this study is to evaluate the fibrotic process induced in vivo by NAMI-A in mice with solid tumours. In addition, the in vitro effects of NAMI-A on collagen fibres and the expression of TGFbeta1 in TS/A adenocarcinoma cells, NIH/3T3 fibroblasts and co-culture of fibroblasts and tumour cells have also been studied. METHODS: Collagen fibres release was assayed in supernatant of culture cells treated with 0.1 and 0.01 mM NAMI-A. TGFbeta1 was detected by RT-PCR and immunoblot on cellular lysates. RESULTS: NAMI-A, given to mice bearing MCa mammary carcinoma at advanced stages of growth, increased the thickness of connective tissue and induced recruitment of leukocytes, particularly in the peritumour capsule. In vitro NAMI-A stimulated collagen production by NIH/3T3 fibroblasts and decreased collagen release by TS/A tumour cells after prolonged exposure, either after single cell treatment or in co-cultures. In co-cultures, NAMI-A, in a dose-dependent manner, down-regulated the expression of TGFbeta1 mRNA and protein in tumour cells and up-regulated it in fibroblasts. The isoform of this cytokine is involved in fibrosis, invasion and metastatic processes. CONCLUSIONS: These data emphasize the ability of NAMI-A to evoke beneficial effects from healthy cells against tumour growth and metastases. The contribution of fibroblasts to the fibrosis arising in tumour masses is due to TGFbeta1, and its down-regulation in tumour cells might explain the documented reduction of gelatinase release.     

Oesophageal squamous cell carcinoma–associated IL‐33 rewires macrophage polarization towards M2 via activating ornithine decarboxylase

BackgroundThe tumour microenvironment primarily constitutes macrophages in the form of an immunosuppressive M2 phenotype, which promotes tumour growth. Thus, the development of methodologies to rewire M2‐like tumour‐associated macrophages (TAMs) into the M1 phenotype, which inhibits tumour growth, might be a critical advancement in cancer immunotherapy research.MethodsThe expressions of IL‐33 and indicators related to macrophage polarization in oesophageal squamous cell carcinoma (ESCC) tissues and peripheral blood mononuclear cell (PBMC)–derived macrophages were determined. Inhibition of ornithine decarboxylase (ODC) with small interfering RNA was used to analyse the phenotype of macrophage polarization and polyamine secretory signals. CCK‐8, wound‐healing and Transwell assays were used to detect the proliferation and migration of ECA109 cells in vitro. The tumour xenograft assay in nude mice was used to examine the role of IL‐33 in ESCC development in vivo.ResultsThis study showed the substantially elevated IL‐33 expression in ESCC tissues compared with the normal tissues. Additionally, enhanced infiltration of M2‐like macrophages into the ESCC tumour tissue was also observed. We observed a strong correlation between the IL‐33 levels and the infiltration of M2‐like macrophages in ESCC tumours locally. Mechanistically, IL‐33 induces M2‐like macrophage polarization by activating ODC, a key enzyme that catalyses the synthesis of polyamines. Inhibition of ODC suppressed M2‐like macrophage polarization. Finally, in vivo, we confirmed that IL‐33 promotes tumour progression.ConclusionsThis study revealed an oncogenic role of IL‐33 by actively inducing M2‐like macrophage differentiation; thus, contributing to the formation of an immunosuppressive ESCC tumour microenvironment. Thus, IL‐33 could act as a novel target for cancer immunotherapies.     

FDPS promotes glioma growth and macrophage recruitment by regulating CCL20 via Wnt/β‐catenin signalling pathway

Glioma is one of the most lethal tumours and common malignant in the central nervous system (CNS), which exhibits diffuse invasion and aggressive growth. Several studies have reported the association of FDPS to tumour development and progression. However, the role of FDPS in progression of glioma and macrophage recruitment is not well‐elucidated. In the current study, a remarkable enhancement in FDPS level was observed in glioma tissues and associated with poor prognosis, contributed to tumour growth. FDPS was correlated with macrophage infiltration in glioma and pharmacological deletion of macrophages largely abrogated the oncogenic functions of FDPS in glioma. Mechanistically, FDPS activated Wnt/β‐catenin signalling pathway and ultimately facilitates macrophage infiltration by inducing CCL20 expression. In conclusion, overexpressed FDPS exhibits an immunomodulatory role in glioma. Therefore, targeting FDPS may be an effective therapeutic strategy for glioma.     

Murine CXCL14 is dispensable for dendritic cell function and localization within peripheral tissues

Dendritic cells (DCs) have long been recognized as key regulators of immune responses. However, the process of their recruitment to peripheral tissues and turnover during homeostasis remains largely unknown. The chemokine CXCL14 (BRAK) is constitutively expressed in skin and other epithelial tissues. Recently, the human chemokine was proposed to play a role in the homeostatic recruitment of macrophage and/or DC precursors toward the periphery, such as skin. Although so far no physiological function could be demonstrated for the murine CXCL14, it shows a remarkable homology to the human chemokine. In order to elucidate the in vivo role of CXCL14, we generated a mouse defective for this chemokine. We studied various components of the immune system with emphasis on monocytes/macrophages and DC/Langerhans cell (LC) populations in different tissues during steady state but did not find a significant difference between knockout (CXCL14(-)(/)(-)) and control mice. Functionally, LCs were able to become activated, to migrate out of skin, and to elicit a delayed type of hypersensitivity reaction. Overall, our data indicate that murine CXCL14 is dispensable for the homeostatic recruitment of antigen-presenting cells toward the periphery and for LC functionality.     

Blocking retinoic acid receptor-α enhances the efficacy of a dendritic cell vaccine against tumours by suppressing the induction of regulatory T cells

The immune system has evolved regulatory mechanisms to control immune responses to self-antigens. Regulatory T (Treg) cells play a pivotal role in maintaining immune tolerance, but tumour growth is associated with local immunosuppression, which can subvert effector immune responses. Indeed, the induction and recruitment of Treg cells by tumours is a major barrier in the development of effective immunotherapeutics and vaccines against cancer. Retinoic acid (RA) has been shown to promote conversion of naïve T cells into Treg cells. This study addresses the hypothesis that blocking RA receptor alpha (RARα) may enhance the efficacy of a tumour vaccine by inhibiting the induction of Treg cells. We found that RA significantly enhanced TGF-β-induced expression of Foxp3 on naïve and committed T cells in vitro and that this was blocked by an antagonist of RARα (RARi). In addition, RARi significantly suppressed TGF-β and IL-10 and enhanced IL-12 production by dendritic cells (DC) in response to killed tumour cells or TLR agonists. Furthermore, RARi augmented the efficacy of an antigen-pulsed and TLR-activated DC vaccine, significantly attenuating growth of B16 tumours in vivo and enhancing survival of mice. This protective effect was associated with significant reduction in tumour-infiltrating FoxP3⁺ and IL-10⁺ Treg cells and a corresponding increase in tumour-infiltrating CD4⁺ and CD8⁺ T cells that secreted IFN-γ. Our findings demonstrate that RARα is an important target for the development of effective anti-tumour immunotherapeutics and for improving the efficacy of cancer vaccines.     

CXCL12 derived from CD248-expressing cancer-associated fibroblasts mediates M2-polarized macrophages to promote nonsmall cell lung cancer progression

Nonsmall cell lung cancer (NSCLC) is among the most prevalent malignant tumours threatening human health. In the tumour microenvironment (TME), cancer-associated fibroblasts (CAFs) induce M2-polarized macrophages, which strongly regulate tumour progression. However, little is known about the association between CAFs and M2 macrophages. CD248 is a transmembrane glycoprotein found in several cancer cells, tumour stromal cells, and pericytes. Here, we isolated CAFs from tumour tissues of NSCLC patients to detect the relationship between CD248 expression and patient prognosis. We knocked down the expression of CD248 on CAFs to detect CXCL12 secretion and macrophage polarization. We then examined the effects of CD248-expressing CAF-induced M2 macrophage polarization to promote NSCLC progression in vitro and in vivo. We found that CD248 is expressed mainly in NSCLC-derived CAFs and that the expression of CD248 correlates with poor patient prognosis. Blocking CXCL12 receptor (CXCR4) drastically decreased M2 macrophage chemotaxis. CD248 promotes CAFs secreting CXCL12 to mediate M2-polarized macrophages to promote NSCLC progression both in vitro and in vivo. Collectively, our data suggest that CD248-positive CAFs induce NSCLC progression by mediating M2-polarized macrophages.     

Ovarian cancer cells polarize macrophages toward a tumor-associated phenotype

Tumor-associated macrophages (TAM) may have tumor-promoting activity, but it is not clear how their phenotype is achieved. In this study, we demonstrate that ovarian cancer cells switch cocultured macrophages to a phenotype similar to that found in ovarian tumors. Tumor cells caused dynamic changes in macrophage cytokine, chemokine, and matrix metalloprotease mRNA, and protein-inducing mediators that are found in human cancer. Macrophage mannose, mannose receptor, and scavenger receptors (SR-As) were also up-regulated by coculture, but not by conditioned medium. To further validate the model, we studied SR-A regulation on TAM in vitro and in vivo. Coculture of murine macrophages from mice deficient in TNF-alpha or its receptors revealed that TNF-alpha was key to SR-A induction via its p75 receptor. SR-A expression was also reduced in TAM from ovarian cancers treated with anti-TNF-alpha Abs or grown in TNF-alpha(-/-) mice. Chemical communication between tumor cells and macrophages may be important in regulating the cancer cytokine microenvironment.     

M-CSF (monocyte colony stimulating factor) and M-CSF receptor expression by breast tumour cells: M-CSF mediated recruitment of tumour infiltrating monocytes?

Infiltrating immune cells in 30 primary human epithelial breast tumours were studied using specific anti-CD3 (T cells), anti-CD68 (macrophages), anti-CD57 (NK cells), and an anti-pan-B cell antibody (L26). The majority of tumour infiltrating inflammatory cells are T cells (40-50%) and monocytes/macrophages (15-35%). The macrophage specific chemo-attractant and growth factor CSF-1 is detected by immunohistochemical techniques (IHC) at the level of invasive breast cancer cells in 46/50 tumours but not at the level of in-situ (pre-invasive) cancer. A mosaic staining pattern was usually observed, with a very high expression in areas of obvious stromal invasion (90% cells positive) and absent or trace staining in intraductal carcinoma. Macrophages and plasma cells are equally intensely positive. In-situ hybridisation experiments confirm the production of CSF-1 (mRNA) by tumour cells and show the same pattern of expression. Expression of the CSF-1 receptor protein (fms) was also observed by IHC in 41/48 invasive tumours, albeit at weaker intensities than in tumour infiltrating monocytes/macrophages. A concomitant expression of both CSF-1 and fms in in-situ carcinoma was never seen (n = 14). It is therefore proposed that the associated expression of CSF-1 and its receptor may be linked to the invasive potential of breast cancer, the monocytic infiltrate being an indication of the quantitative importance of CSF-1 production by the tumour.     

Protective immunity to UV radiation-induced skin tumours induced by skin grafts and epidermal cells

There is little evidence that cutaneous dendritic cells (DC), including epidermal Langerhans cells (LC), can induce immunity to UV radiation (UVR)-induced skin tumours. Here, it is shown that cells within skin can induce protective antitumour immunity against a UVR-induced fibrosarcoma. Transplantation of the skin overlying subcutaneous tumours onto na?ve recipients could induce protective antitumour immunity, probably because the grafting stimulated the tumour Ag-loaded DC to migrate to local lymph nodes. This suggests that cutaneous APC can present tumour Ag to induce protective antitumour immunity. Previously, it has been shown that immunization of mice with MHC class II+ epidermal cells (EC) pulsed with tumour extracts could induce delayed-type hypersensitivity against tumour cells. Here, this same immunization protocol could induce protective immunity against a minimum tumorigenic dose of UVR-induced fibrosarcoma cells, but not higher doses. Epidermal cells obtained from semiallogeneic donors and pulsed with tumour extract could also induce protective immunity. However, presentation of BSA Ag from the culture medium was found to contribute to this result using semiallogeneic EC. The results suggest that LC overlying skin tumours may be able to induce protective immunity to UVR-induced tumours if stimulated to migrate from the skin.     

The colony-stimulating factor 1 receptor is expressed on dendritic cells during differentiation and regulates their expansion

The lineage of dendritic cells (DC), and in particular their relationship to monocytes and macrophages, remains obscure. Furthermore, the requirement for the macrophage growth factor CSF-1 during DC homeostasis is unclear. Using a transgenic mouse in which the promoter for the CSF-1R (c-fms) directs the expression of enhanced GFP in cells of the myeloid lineage, we determined that although the c-fms promoter is inactive in DC precursors, it is up-regulated in all DC subsets during differentiation. Furthermore, plasmacytoid DC and all CD11c(high) DC subsets are reduced by 50-70% in CSF-1-deficient osteopetrotic mice, confirming that CSF-1 signaling is required for the optimal differentiation of DC in vivo. These data provide additional evidence that the majority of tissue DC is of myeloid origin during steady state and supports a close relationship between DC and macrophage biology in vivo.     

Three-dimensional microfluidic tumor–macrophage system for breast cancer cell invasion

The recrudescence of breast cancer can partly be attributed to poor understanding of the early steps and the mechanisms involved in breast cancer metastasis, especially how tumor inflammatory cells including tumor-associated macrophages (TAM) affect invasion process. However, invasion-related biological studies in traditional in vitro assays or in vivo models are challenging due to the arduousness in establishing models that precisely reproduce the tumor invasion environment. To this end, we proposed a juxtaposed dual-layer cell-loaded hydrogels biomimetic microfluidic system and formed monolayer size-selective permeable vascular endothelial barriers besides the dual layer to mimic mammalian blood vessels. We clarified that in this system, TAM promoted the invasion of breast cancer cells, whereas breast cancer cells maintained the phenotype of TAM cells and promoted the differentiation of U937 cells into TAM. It formed a tumor–macrophage bidirectional crosstalk system. This system could be used for drug screening. So finally, through the calculation of the survival rate of breast cancer cells when cocultured with different macrophages under paclitaxel treatment, we analyzed the antagonism of tumor–macrophage bidirectional crosstalk on anticancer drugs.     

Modulation of dendritic cell trafficking to and from the airways

We investigated the fate of latex (LX) particles that were introduced into mice intranasally. Macrophages acquired the vast majority of particles and outnumbered LX particle-bearing airway dendritic cells (DCs) by at least two orders of magnitude. Yet alveolar macrophages were refractory to migration to the draining lymph node (DLN), and all transport to the DLN could be ascribed to the few LX(+) airway DCs. Upon macrophage depletion, markedly greater numbers of DCs were recruited into the alveolar space. Consequently, the number of DCs that carried particles to the DLN was boosted by 20-fold. Thus, a so far overlooked aspect of macrophage-mediated suppression of airway DC function stems from the modulation of DC recruitment into the airway. This increase in DC recruitment permitted the development of a robust assay to quantify the subsequent migration of DCs to the DLN. Therefore, we determined whether lung DCs use the same molecules that skin DCs use during migration to DLNs. Like skin DCs, lung DCs used CCR7 ligands and CCR8 for emigration to DLN, but the leukotriene C(4) transporter multidrug resistance-related protein 1 did not mediate lung DC migration as it does in skin, indicating that pathways governing DC migration from different tissues partially differ in molecular regulation.     

Somatostatin Derivate (smsDX) Attenuates the TAM-Stimulated Proliferation,Migration and Invasion of Prostate Cancer via NF-κB Regulation

Tumor development and progression are influenced by macrophages of the surrounding microenvironment. To investigate the influences of an inflammatory tumor microenvironment on the growth and metastasis of prostate cancer, the present study used a co-culture model of prostate cancer (PCa) cells with tumor-associated macrophage (TAM)-conditioned medium (MCM). MCM promoted PCa cell (LNCaP, DU145 and PC-3) growth, and a xenograft model in nude mice consistently demonstrated that MCM could promote tumor growth. MCM also stimulated migration and invasion in vitro. Somatostatin derivate (smsDX) significantly attenuated the TAM-stimulated proliferation, migration and invasion of prostate cancer. Immunohistochemistry revealed that NF-κB was over-expressed in PCa and BPH with chronic inflammatory tissue specimens and was positively correlated with macrophage infiltration. Further investigation into the underlying mechanism revealed that NF-κB played an important role in macrophage infiltration. SmsDX inhibited the paracrine loop between TAM and PCa cells and may represent a potential therapeutic agent for PCa.     

SARI suppresses colitis‐associated cancer development by maintaining MCP‐1‐mediated tumour‐associated macrophage recruitment

SARI (suppressor of AP‐1, regulated by IFN) impaired tumour growth by promoting apoptosis and inhibiting cell proliferation and tumour angiogenesis in various cancers. However, the role of SARI in regulating tumour‐associated inflammation microenvironment is still elusive. In our study, the colitis‐dependent and ‐independent primary model were established in SARI deficiency mice and immuno‐reconstructive mice to investigate the functional role of SARI in regulating tumour‐associated inflammation microenvironment and primary colon cancer formation. The results have shown that SARI deficiency promotes colitis‐associated cancer (CAC) development only in the presence of colon inflammation. SARI inhibited tumour‐associated macrophages (TAM) infiltration in colon tissues, and SARI deficiency in bone marrow cells has no observed role in the promotion of intestinal tumorigenesis. Mechanism investigations indicated that SARI down‐regulates p‐STAT1 and STAT1 expression in colon cancer cells, following inhibition of MCP‐1/CCR2 axis activation during CAC development. Inverse correlations between SARI expression and macrophage infiltration, MCP‐1 expression and p‐STAT1 expression were also demonstrated in colon malignant tissues. Collectively, our results prove the inhibition role of SARI in colon cancer formation through regulating TAM infiltration.