Vitamin D binding protein-macrophage activating factor directly inhibits proliferation, migration, and uPAR expression of prostate cancer cells

Background

Vitamin D binding protein-macrophage activating factor (DBP-maf) is a potent inhibitor of tumor growth. Its activity, however, has been attributed to indirect mechanisms such as boosting the immune response by activating macrophages and inhibiting the blood vessel growth necessary for the growth of tumors.

Methods and Findings

In this study we show for the first time that DBP-maf exhibits a direct and potent effect on prostate tumor cells in the absence of macrophages. DBP-maf demonstrated inhibitory activity in proliferation studies of both LNCaP and PC3 prostate cancer cell lines as well as metastatic clones of these cells. Flow cytometry studies with annexin V and propidium iodide showed that this inhibitory activity is not due to apoptosis or cell death. DBP-maf also had the ability to inhibit migration of prostate cancer cells in vitro. Finally, DBP-maf was shown to cause a reduction in urokinase plasminogen activator receptor (uPAR) expression in prostate tumor cells. There is evidence that activation of this receptor correlates with tumor metastasis.

Conclusions

These studies show strong inhibitory activity of DBP-maf on prostate tumor cells independent of its macrophage activation.     

Oxidized Low Density Lipoprotein Induced Caspase-1 Mediated Pyroptotic Cell Death in Macrophages: Implication in Lesion Instability?

Background

Macrophage death in advanced lesion has been confirmed to play an important role in plaque instability. However, the mechanism underlying lesion macrophage death still remains largely unknown.

Methods and Results

Immunohistochemistry showed that caspase-1 activated in advanced lesion and co-located with macrophages and TUNEL positive reaction. In in-vitro experiments showed that ox-LDL induced caspase-1 activation and this activation was required for ox-LDL induced macrophages lysis, IL-1β and IL-18 production as well as DNA fragmentation. Mechanism experiments showed that CD36 and NLRP3/caspase-1/pathway involved in ox-LDL induced macrophage pyroptosis.

Conclusion

Our study here identified a novel cell death, pyroptosis in ox-LDL induced human macrophage, which may be implicated in lesion macrophages death and play an important role in lesion instability.     

Cancer Associated Fibroblasts Promote Tumor Growth and Metastasis by Modulating the Tumor Immune Microenvironment in a 4T1 Murine Breast Cancer Model

Background

Local inflammation associated with solid tumors commonly results from factors released by tumor cells and the tumor stroma, and promotes tumor progression. Cancer associated fibroblasts comprise a majority of the cells found in tumor stroma and are appealing targets for cancer therapy. Here, our aim was to determine the efficacy of targeting cancer associated fibroblasts for the treatment of metastatic breast cancer.

Methodology/Principal Findings

We demonstrate that cancer associated fibroblasts are key modulators of immune polarization in the tumor microenvironment of a 4T1 murine model of metastatic breast cancer. Elimination of cancer associated fibroblasts in vivo by a DNA vaccine targeted to fibroblast activation protein results in a shift of the immune microenvironment from a Th2 to Th1 polarization. This shift is characterized by increased protein expression of IL-2 and IL-7, suppressed recruitment of tumor-associated macrophages, myeloid derived suppressor cells, T regulatory cells, and decreased tumor angiogenesis and lymphangiogenesis. Additionally, the vaccine improved anti-metastatic effects of doxorubicin chemotherapy and enhanced suppression of IL-6 and IL-4 protein expression while increasing recruitment of dendritic cells and CD8⁺ T cells. Treatment with the combination therapy also reduced tumor-associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression.

Conclusions/Significance

Our findings demonstrate that cancer associated fibroblasts promote tumor growth and metastasis through their role as key modulators of immune polarization in the tumor microenvironment and are valid targets for therapy of metastatic breast cancer.     

Induction of M2 Polarization in Primary Culture Liver Macrophages from Rats with Acute Pancreatitis

Background and Aims

Systemic inflammatory response syndrome (SIRS), a major process of severe acute pancreatitis (SAP), usually occurs after various activated proinflammatory cytokines, which are produced by macrophages such as liver macrophages. Macrophages can secrete not only proinflammatory mediators but also inhibitory inflammatory cytokines such as IL-10, leading to two different functional states defined as “polarization”. The main purpose of this study was to demonstrate the polarization of liver macrophages during severe acute pancreatitis and to explore whether the polarization of these activated Liver macrophages could be reversed in vitro.

Methods

Liver macrophages were isolated from rats with acute pancreatitis. These primary culture macrophages were treated with IL-4 or regulatory T cells in vitro to reverse their polarization and was evaluated by measuring M1/M2 marker expression using real time PCR and immunofluorescence staining.

Results

Acute pancreatitis was induced successfully by intra-pancreatic ductal injection of 5% sodium taurocholate. The liver macrophages demonstrated M1 polarization from 4 h to 16 h after the onset of acute pancreatitis. However, after IL-4 or Treg treatment, the polarization of the liver macrophages was reversed as indicated by increased expression of M2 markers and reduced expression of M1 markers. Furthermore, the effect of Treg on modulating macrophage polarization was slightly better than that of IL-4 in vitro.

Conclusion

Liver macrophages, a pivotal cell type in the pathogenesis of SAP, become M1 polarized during pancreatic inflammation. Treatment of these cells with IL-4 and Treg can reverse this activation in vitro. This method of altering macrophage polarization could be a prospective therapy for SAP.     

Regulation of granulocyte and macrophage populations of murine bone marrow cells by G-CSF and CD137 protein

Background

Granulocytes and monocytes/macrophages differentiate from common myeloid progenitor cells. Granulocyte colony-stimulating factor (G-CSF) and CD137 (4-1BB, TNFRSF9) are growth and differentiation factors that induce granulocyte and macrophage survival and differentiation, respectively. This study describes the influence of G-CSF and recombinant CD137-Fc protein on myelopoiesis.

Methodology/Principal Findings

Both, G-CSF and CD137 protein support proliferation and survival of murine bone marrow cells. G-CSF enhances granulocyte numbers while CD137 protein enhances macrophage numbers. Both growth factors together give rise to more cells than each factor alone. Titration of G-CSF and CD137 protein dose-dependently changes the granulocyte/macrophage ratio in bone marrow cells. Both factors individually induce proliferation of hematopoietic progenitor cells (lin^-, c-kit⁺) and differentiation to granulocytes and macrophages, respectively. The combination of G-CSF and CD137 protein further increases proliferation, and results in a higher number of macrophages than CD137 protein alone, and a lower number of granulocytes than G-CSF alone demonstrating that CD137 protein-induced monocytic differentiation is dominant over G-CSF-induced granulocytic differentiation. CD137 protein induces monocytic differentiation even in early hematopoietic progenitor cells, the common myeloid progenitors and the granulocyte macrophage progenitors.

Conclusions/Significance

This study confirms earlier data on the regulation of myelopoiesis by CD137 receptor - ligand interaction, and extends them by demonstrating the restriction of this growth promoting influence to the monocytic lineage.     

High Content Analysis of Primary Macrophages Hosting Proliferating Leishmania Amastigotes: Application to Anti-leishmanial Drug Discovery

Background/Objectives

Human leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis.

Methodology/Principal Findings

We have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline.

Conclusions/Significance

Our assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage required for parasite development and growth, such as organelle trafficking/acidification or production of microbicidal effectors. These data thus validate a novel phenotypic screening assay using virulent Leishmania amastigotes growing inside primary macrophage to identify new chemical entities with bona fide drug potential.     

CXCR4/CXCL12 participate in extravasation of metastasizing breast cancer cells within the liver in a rat model

Introduction

Organ-specific composition of extracellular matrix proteins (ECM) is a determinant of metastatic host organ involvement. The chemokine CXCL12 and its receptor CXCR4 play important roles in the colonization of human breast cancer cells to their metastatic target organs. In this study, we investigated the effects of chemokine stimulation on adhesion and migration of different human breast cancer cell lines in vivo and in vitro with particular focus on the liver as a major metastatic site in breast cancer.

Methods

Time lapse microscopy, in vitro adhesion and migration assays were performed under CXCL12 stimulation. Activation of small GTPases showed chemokine receptor signalling dependence from ECM components. The initial events of hepatic colonisation of MDA-MB-231 and MDA-MB-468 cells were investigated by intravital microscopy of the liver in a rat model and under shRNA inhibition of CXCR4.

Results

In vitro, stimulation with CXCL12 induced increased chemotactic cell motility (p<0.05). This effect was dependent on adhesive substrates (type I collagen, fibronectin and laminin) and induced different responses in small GTPases, such as RhoA and Rac-1 activation, and changes in cell morphology. In addition, binding to various ECM components caused redistribution of chemokine receptors at tumour cell surfaces. In vivo, blocking CXCR4 decreased extravasation of highly metastatic MDA-MB-231 cells (p<0.05), but initial cell adhesion within the liver sinusoids was not affected. In contrast, the less metastatic MDA-MB-468 cells showed reduced cell adhesion but similar migration within the hepatic microcirculation. Conclusion: Chemokine-induced extravasation of breast cancer cells along specific ECM components appears to be an important regulator but not a rate-limiting factor of their metastatic organ colonization.     

An Antimicrobial Peptide Regulates Tumor-Associated Macrophage Trafficking via the Chemokine Receptor CCR2, a Model for Tumorigenesis

Background

Tumor-associated macrophages (TAMs) constitute a significant part of infiltrating inflammatory cells that are frequently correlated with progression and poor prognosis of a variety of cancers. Tumor cell-produced human β-defensin-3 (hBD-3) has been associated with TAM trafficking in oral cancer; however, its involvement in tumor-related inflammatory processes remains largely unknown.

Methodology

The relationship between hBD-3, monocyte chemoattractant protein-1 (MCP-1), TAMs, and CCR2 was examined using immunofluorescence microscopy in normal and oral carcinoma in situ biopsy specimens. The ability of hBD-3 to chemoattract host macrophages in vivo using a nude mouse model and analysis of hBD-3 on monocytic cell migration in vitro, applying a cross-desensitization strategy of CCR2 and its pharmacological inhibitor (RS102895), respectively, was also carried out.

Conclusions/Findings

MCP-1, the most frequently expressed tumor cell-associated chemokine, was not produced by tumor cells nor correlated with the recruitment of macrophages in oral carcinoma in situ lesions. However, hBD-3 was associated with macrophage recruitment in these lesions and hBD-3-expressing tumorigenic cells induced massive tumor infiltration of host macrophages in nude mice. HBD-3 stimulated the expression of tumor-promoting cytokines, including interleukin-1α (IL-1α), IL-6, IL-8, CCL18, and tumor necrosis factor-α (TNF-α) in macrophages derived from human peripheral blood monocytes. Monocytic cell migration in response to hBD-3 was inhibited by cross-desensitization with MCP-1 and the specific CCR2 inhibitor, RS102895, suggesting that CCR2 mediates monocyte/macrophage migration in response to hBD-3. Collectively, these results indicate that hBD-3 utilizes CCR2 to regulate monocyte/macrophage trafficking and may act as a tumor cell-produced chemoattractant to recruit TAMs. This novel mechanism is the first evidence of an hBD molecule orchestrating an in vivo outcome and demonstrates the importance of the innate immune system in the development of tumors.     

Drug-selected human lung cancer stem cells: cytokine network, tumorigenic and metastatic properties

Background

Cancer stem cells (CSCs) are thought to be responsible for tumor regeneration after chemotherapy, although direct confirmation of this remains forthcoming. We therefore investigated whether drug treatment could enrich and maintain CSCs and whether the high tumorogenic and metastatic abilities of CSCs were based on their marked ability to produce growth and angiogenic factors and express their cognate receptors to stimulate tumor cell proliferation and stroma formation.

Methodology/Findings

Treatment of lung tumor cells with doxorubicin, cisplatin, or etoposide resulted in the selection of drug surviving cells (DSCs). These cells expressed CD133, CD117, SSEA-3, TRA1-81, Oct-4, and nuclear β-catenin and lost expression of the differentiation markers cytokeratins 8/18 (CK 8/18). DSCs were able to grow as tumor spheres, maintain self-renewal capacity, and differentiate. Differentiated progenitors lost expression of CD133, gained CK 8/18 and acquired drug sensitivity. In the presence of drugs, differentiation of DSCs was abrogated allowing propagation of cells with CSC-like characteristics. Lung DSCs demonstrated high tumorogenic and metastatic potential following inoculation into SCID mice, which supported their classification as CSCs. Luminex analysis of human and murine cytokines in sonicated lysates of parental- and CSC-derived tumors revealed that CSC-derived tumors contained two- to three-fold higher levels of human angiogenic and growth factors (VEGF, bFGF, IL-6, IL-8, HGF, PDGF-BB, G-CSF, and SCGF-β). CSCs also showed elevated levels of expression of human VEGFR2, FGFR2, CXCR1, 2 and 4 receptors. Moreover, human CSCs growing in SCID mice stimulated murine stroma to produce elevated levels of angiogenic and growth factors.

Conclusions/Significance

These findings suggest that chemotherapy can lead to propagation of CSCs and prevention of their differentiation. The high tumorigenic and metastatic potentials of CSCs are associated with efficient cytokine network production that may represent a target for increased efficacy of cancer therapy.     

Comparison of Immunity in Mice Cured of Primary/Metastatic Growth of EMT6 or 4THM Breast Cancer by Chemotherapy or Immunotherapy

Purpose

We have compared cure from local/metastatic tumor growth in BALB/c mice receiving EMT6 or the poorly immunogenic, highly metastatic 4THM, breast cancer cells following manipulation of immunosuppressive CD200:CD200R interactions or conventional chemotherapy.

Methods

We reported previously that EMT6 tumors are cured in CD200R1KO mice following surgical resection and immunization with irradiated EMT6 cells and CpG oligodeoxynucleotide (CpG), while wild-type (WT) animals developed pulmonary and liver metastases within 30 days of surgery. We report growth and metastasis of both EMT6 and a highly metastatic 4THM tumor in WT mice receiving iv infusions of Fab anti-CD200R1 along with CpG/tumor cell immunization. Metastasis was followed both macroscopically (lung/liver nodules) and microscopically by cloning tumor cells at limiting dilution in vitro from draining lymph nodes (DLN) harvested at surgery. We compared these results with local/metastatic tumor growth in mice receiving 4 courses of combination treatment with anti-VEGF and paclitaxel.

Results

In WT mice receiving Fab anti-CD200R, no tumor cells are detectable following immunotherapy, and CD4+ cells produced increased TNFα/IL-2/IFNγ on stimulation with EMT6 in vitro. No long-term cure was seen following surgery/immunotherapy of 4THM, with both microscopic (tumors in DLN at limiting dilution) and macroscopic metastases present within 14 d of surgery. Chemotherapy attenuated growth/metastases in 4THM tumor-bearers and produced a decline in lung/liver metastases, with no detectable DLN metastases in EMT6 tumor-bearing mice-these latter mice nevertheless showed no significantly increased cytokine production after restimulation with EMT6 in vitro. EMT6 mice receiving immunotherapy were resistant to subsequent re-challenge with EMT6 tumor cells, but not those receiving curative chemotherapy. Anti-CD4 treatment caused tumor recurrence after immunotherapy, but produced no apparent effect in either EMT6 or 4THM tumor bearers after chemotherapy treatment.

Conclusion

Immunotherapy, but not chemotherapy, enhances CD4⁺ immunity and affords long-term control of breast cancer growth and resistance to new tumor foci.     

Novel HIV-1 MiRNAs Stimulate TNFα Release in Human Macrophages via TLR8 Signaling Pathway

Purpose

To determine whether HIV-1 produces microRNAs and elucidate whether these miRNAs can induce inflammatory response in macrophages (independent of the conventional miRNA function in RNA interference) leading to chronic immune activation.

Methods

Using sensitive quantitative Real Time RT-PCR and sequencing, we detected novel HIV-derived miRNAs in the sera of HIV+ persons, and associated with exosomes. Release of TNFα by macrophages challenged with HIV miRNAs was measured by ELISA.

Results

HIV infection of primary alveolar macrophages produced elevated levels of viral microRNAs vmiR88, vmiR99 and vmiR-TAR in cell extracts and in exosome preparations from conditioned medium. Furthermore, these miRNAs were also detected in exosome fraction of sera from HIV-infected persons. Importantly, vmiR88 and vmiR99 (but not vmiR-TAR) stimulated human macrophage TNFα release, which is dependent on macrophage TLR8 expression. These data support a potential role for HIV-derived vmiRNAs released from infected macrophages as contributing to chronic immune activation in HIV-infected persons, and may represent a novel therapeutic target to limit AIDS pathogenesis.

Conclusion

Novel HIV vmiR88 and vmiR99 are present in the systemic circulation of HIV+ persons and could exhibit biological function (independent of gene silencing) as ligands for TLR8 signaling that promote macrophage TNFα release, and may contribute to chronic immune activation. Targeting novel HIV-derived miRNAs may represent a therapeutic strategy to limit chronic immune activation and AIDS progression.     

The Ketogenic Diet and Hyperbaric Oxygen Therapy Prolong Survival in Mice with Systemic Metastatic Cancer

Introduction

Abnormal cancer metabolism creates a glycolytic-dependency which can be exploited by lowering glucose availability to the tumor. The ketogenic diet (KD) is a low carbohydrate, high fat diet which decreases blood glucose and elevates blood ketones and has been shown to slow cancer progression in animals and humans. Abnormal tumor vasculature creates hypoxic pockets which promote cancer progression and further increase the glycolytic-dependency of cancers. Hyperbaric oxygen therapy (HBO₂T) saturates tumors with oxygen, reversing the cancer promoting effects of tumor hypoxia. Since these non-toxic therapies exploit overlapping metabolic deficiencies of cancer, we tested their combined effects on cancer progression in a natural model of metastatic disease.

Methods

We used the firefly luciferase-tagged VM-M3 mouse model of metastatic cancer to compare tumor progression and survival in mice fed standard or KD ad libitum with or without HBO₂T (2.5 ATM absolute, 90 min, 3x/week). Tumor growth was monitored by in vivo bioluminescent imaging.

Results

KD alone significantly decreased blood glucose, slowed tumor growth, and increased mean survival time by 56.7% in mice with systemic metastatic cancer. While HBO₂T alone did not influence cancer progression, combining the KD with HBO₂T elicited a significant decrease in blood glucose, tumor growth rate, and 77.9% increase in mean survival time compared to controls.

Conclusions

KD and HBO₂T produce significant anti-cancer effects when combined in a natural model of systemic metastatic cancer. Our evidence suggests that these therapies should be further investigated as potential non-toxic treatments or adjuvant therapies to standard care for patients with systemic metastatic disease.     

Alveolar macrophages regulate neutrophil recruitment in endotoxin-induced lung injury

Background

Alveolar macrophages play an important role during the development of acute inflammatory lung injury. In the present study, in vivo alveolar macrophage depletion was performed by intratracheal application of dichloromethylene diphosphonate-liposomes in order to study the role of these effector cells in the early endotoxin-induced lung injury.

Methods

Lipopolysaccharide was applied intratracheally and the inflammatory reaction was assessed 4 hours later. Neutrophil accumulation and expression of inflammatory mediators were determined. To further analyze in vivo observations, in vitro experiments with alveolar epithelial cells and alveolar macrophages were performed.

Results

A 320% increase of polymorphonuclear leukocytes in bronchoalveolar lavage fluid was observed in macrophage-depleted compared to macrophage-competent lipopolysaccharide-animals. This neutrophil recruitment was also confirmed in the interstitial space. Monocyte chemoattractant protein-1 concentration in bronchoalveolar lavage fluid was significantly increased in the absence of alveolar macrophages. This phenomenon was underlined by in vitro experiments with alveolar epithelial cells and alveolar macrophages. Neutralizing monocyte chemoattractant protein-1 in the airways diminished neutrophil accumulation.

Conclusion

These data suggest that alveolar macorphages play an important role in early endotoxin-induced lung injury. They prevent neutrophil influx by controlling monocyte chemoattractant protein-1 production through alveolar epithelial cells. Alveolar macrophages might therefore possess robust anti-inflammatory effects.     

Poractant alfa (Curosurf?) increases phagocytosis of apoptotic neutrophils by alveolar macrophages in vivo

Background

Clearance of apoptotic neutrophils in the lung is an essential process to limit inflammation, since they could become a pro-inflammatory stimulus themselves. The clearance is partially mediated by alveolar macrophages, which phagocytose these apoptotic cells. The phagocytosis of apoptotic immune cells by monocytes in vitro has been shown to be augmented by several constituents of pulmonary surfactant, e.g. phospholipids and hydrophobic surfactant proteins. In this study, we assessed the influence of exogenous poractant alfa (Curosurf^®) instillation on the in vivo phagocytosis of apoptotic neutrophils by alveolar macrophages.

Methods

Poractant alfa (200 mg/kg) was instilled intratracheally in the lungs of three months old adult male C57/Black 6 mice, followed by apoptotic neutrophil instillation. Bronchoalveloar lavage was performed and alveolar macrophages and neutrophils were counted. Phagocytosis of apoptotic neutrophils was quantified by determining the number of apoptotic neutrophils per alveolar macrophages.

Results

Exogenous surfactant increased the number of alveolar macrophages engulfing apoptotic neutrophils 2.6 fold. The phagocytosis of apoptotic neutrophils was increased in the presence of exogenous surfactant by a 4.7 fold increase in phagocytosed apoptotic neutrophils per alveolar macrophage.

Conclusions

We conclude that the anti-inflammatory properties of surfactant therapy may be mediated in part by increased numbers of alveolar macrophages and increased phagocytosis of apoptotic neutrophils by alveolar macrophages.     

Oxygen Consumption Can Regulate the Growth of Tumors,a New Perspective on the Warburg Effect

Background

The unique metabolism of tumors was described many years ago by Otto Warburg, who identified tumor cells with increased glycolysis and decreased mitochondrial activity. However, “aerobic glycolysis” generates fewer ATP per glucose molecule than mitochondrial oxidative phosphorylation, so in terms of energy production, it is unclear how increasing a less efficient process provides tumors with a growth advantage.

Methods/Findings

We carried out a screen for loss of genetic elements in pancreatic tumor cells that accelerated their growth as tumors, and identified mitochondrial ribosomal protein L28 (MRPL28). Knockdown of MRPL28 in these cells decreased mitochondrial activity, and increased glycolysis, but paradoxically, decreased cellular growth in vitro. Following Warburg''s observations, this mutation causes decreased mitochondrial function, compensatory increase in glycolysis and accelerated growth in vivo. Likewise, knockdown of either mitochondrial ribosomal protein L12 (MRPL12) or cytochrome oxidase had a similar effect. Conversely, expression of the mitochondrial uncoupling protein 1 (UCP1) increased oxygen consumption and decreased tumor growth. Finally, treatment of tumor bearing animals with dichloroacetate (DCA) increased pyruvate consumption in the mitochondria, increased total oxygen consumption, increased tumor hypoxia and slowed tumor growth.

Conclusions

We interpret these findings to show that non-oncogenic genetic changes that alter mitochondrial metabolism can regulate tumor growth through modulation of the consumption of oxygen, which appears to be a rate limiting substrate for tumor proliferation.     

Hypothyroidism Enhances Tumor Invasiveness and Metastasis Development

Background

Whereas there is increasing evidence that loss of expression and/or function of the thyroid hormone receptors (TRs) could result in a selective advantage for tumor development, the relationship between thyroid hormone levels and human cancer is a controversial issue. It has been reported that hypothyroidism might be a possible risk factor for liver and breast cancer in humans, but a lower incidence of breast carcinoma has been also reported in hypothyroid patients

Methodology/Principal Findings

In this work we have analyzed the influence of hypothyroidism on tumor progression and metastasis development using xenografts of parental and TRβ1–expressing human hepatocarcinoma (SK-hep1) and breast cancer cells (MDA-MB-468). In agreement with our previous observations tumor invasiveness and metastasis formation was strongly repressed when TRβ–expressing cells were injected into euthyroid nude mice. Whereas tumor growth was retarded when cells were inoculated into hypothyroid hosts, tumors had a more mesenchymal phenotype, were more invasive and metastatic growth was enhanced. Increased aggressiveness and tumor growth retardation was also observed with parental cells that do not express TRs.

Conclusions/Significance

These results show that changes in the stromal cells secondary to host hypothyroidism can modulate tumor progression and metastatic growth independently of the presence of TRs on the tumor cells. On the other hand, the finding that hypothyroidism can affect differentially tumor growth and invasiveness can contribute to the explanation of the confounding reports on the influence of thyroidal status in human cancer.     

Macrophages Mediate a Switch between Canonical and Non-Canonical Wnt Pathways in Canine Mammary Tumors

Objective

According to the current hypothesis, tumor-associated macrophages (TAMs) are “corrupted” by cancer cells and subsequently facilitate, rather than inhibit, tumor metastasis. Because the molecular mechanisms of cancer cell–TAM interactions are complicated and controversial we aimed to better define this phenomenon.

Methods and Results

Using microRNA microarrays, Real-time qPCR and Western blot we showed that co-culture of canine mammary tumor cells with TAMs or treatment with macrophage-conditioned medium inhibited the canonical Wnt pathway and activated the non-canonical Wnt pathway in tumor cells. We also showed that co-culture of TAMs with tumor cells increased expression of canonical Wnt inhibitors in TAMs. Subsequently, we demonstrated macrophage-induced invasive growth patterns and epithelial–mesenchymal transition of tumor cells. Validation of these results in canine mammary carcinoma tissues (n = 50) and xenograft tumors indicated the activation of non-canonical and canonical Wnt pathways in metastatic tumors and non-metastatic malignancies, respectively. Activation of non-canonical Wnt pathway correlated with number of TAMs.

Conclusions

We demonstrated that TAMs mediate a “switch” between canonical and non-canonical Wnt signaling pathways in canine mammary tumors, leading to increased tumor invasion and metastasis.Interestingly, similar changes in neoplastic cells were observed in the presence of macrophage-conditioned medium or live macrophages. These observations indicate that rather than being “corrupted” by cancer cells, TAMs constitutively secrete canonical Wnt inhibitors that decrease tumor proliferation and development, but as a side effect, they induce the non-canonical Wnt pathway, which leads to tumor metastasis.These data challenge the conventional understanding of TAM–cancer cell interactions.     

Comparative Study of Nanosecond Electric Fields In Vitro and In Vivo on Hepatocellular Carcinoma Indicate Macrophage Infiltration Contribute to Tumor Ablation In Vivo

Background and Aim

Recurrence and metastasis are associated with poor prognosis in hepatocellular carcinoma even in the patients who have undergone radical resection. Therefore, effective treatment is urgently needed for improvement of patients'' survival. Previously, we reported that nanosecond pulse electric fields (nsPEFs) can ablate melanoma by induction of apoptosis and inhibition of angiogenesis. This study aims to investigate the in vivo ablation strategy by comparing the dose effect of nanosecond electric fields in vitro and in vivo on hepatocellular carcinoma.

Materials and Methods

Four hepatocellular carcinoma cell lines HepG2, SMMC7721, Hep1-6, and HCCLM3 were pulsed to test the anti-proliferation and anti-migration ability of 100 ns nsPEFs in vitro. The animal model of human subdermal xenograft HCCLM3 cells into BALB/c nude mouse was used to test the anti-tumor growth and macrophage infiltration in vivo.

Results

In vitro assays showed anti-tumor effect of nsPEFs is dose-dependant. But the in vivo study showed the strategy of low dose and multiple treatments is superior to high dose single treatment. The macrophages infiltration significantly increased in the tumors which were treated by multiple low dose nsPEFs.

Conclusion

The low dose multiple nsPEFs application is more efficient than high dose single treatment in inhibiting the tumor volume in vivo, which is quite different from the dose-effect relationship in vitro. Beside the electric field strength, the macrophage involvement must be considered to account for effect variability and toxicology in vivo.