Fungal polysaccharides from Inonotus obliquus are agonists for Toll-like receptors and induce macrophage anti-cancer activity.

Fungal polysaccharides can exert immunomodulating activity by triggering pattern recognition receptors (PRRs) on innate immune cells such as macrophages. Here, we evaluate six polysaccharides isolated from the medicinal fungus Inonotus obliquus for their ability to activate mouse and human macrophages. We identify two water-soluble polysaccharides, AcF1 and AcF3, being able to trigger several critical antitumor functions of macrophages. AcF1 and AcF3 activate macrophages to secrete nitric oxide and the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Combined with interferon-γ, the fungal polysaccharides trigger high production of IL-12p70, a central cytokine for antitumor immunity, and induce macrophage-mediated inhibition of cancer cell growth in vitro and in vivo. AcF1 and AcF3 are strong agonists of the PRRs Toll-like receptor 2 (TLR2) and TLR4, and weak agonists of Dectin-1. In comparison, two prototypical particulate ß-glucans, one isolated from I. obliquus and one from Saccharomyces cerevisiae (zymosan), are agonists for Dectin-1 but not TLR2 or TLR4, and are unable to trigger anti-cancer functions of macrophages. We conclude that the water-soluble polysaccharides AcF1 and AcF3 from I. obliquus have a strong potential for cancer immunotherapy by triggering multiple PRRs and by inducing potent anti-cancer activity of macrophages.

Antibody blockade of Jagged1 attenuates choroidal neovascularization.

Antibody-based blocking of vascular endothelial growth factor (VEGF) reduces choroidal neovascularization (CNV) and retinal edema, rescuing vision in patients with neovascular age-related macular degeneration (nAMD). However, poor response and resistance to anti-VEGF treatment occurs. We report that targeting the Notch ligand Jagged1 by a monoclonal antibody reduces neovascular lesion size, number of activated phagocytes and inflammatory markers and vascular leakage in an experimental CNV mouse model. Additionally, we demonstrate that Jagged1 is expressed in mouse and human eyes, and that Jagged1 expression is independent of VEGF signaling in human endothelial cells. When anti-Jagged1 was combined with anti-VEGF in mice, the decrease in lesion size exceeded that of either antibody alone. The therapeutic effect was solely dependent on blocking, as engineering antibodies to abolish effector functions did not impair the therapeutic effect. Targeting of Jagged1 alone or in combination with anti-VEGF may thus be an attractive strategy to attenuate CNV-bearing diseases.

Hacking macrophages to combat cancer and inflammatory diseases - Current advances and challenges.

Recently, immunotherapy has been served as the treatment of choice for various human pathophysiologies, including inflammatory diseases and cancer. Though most of the current approaches target the lymphoid compartment, macrophages intimately implicated in the induction or resolution of inflammation have rationally gained their place into the therapeutics arena. In this review, I discuss the past and novel ground-breaking strategies focusing on macrophages in different human diseases and highlight the current challenges and considerations underlying their translational potentials.

Reactive Species from Two-Signal Activated Macrophages Interfere with Their Oxygen Consumption Measurements.

Metabolic modulation of macrophage activation has emerged as a promising strategy lately in immunotherapeutics. However, macrophages have a broad spectrum of functions and thus, understanding the exact metabolic changes that drive a particular immune response, is of major importance. In our previous work, we have reported a key role of nitric oxide (NO●) in two(2)-signal activated macrophages [M(2-signals)]. Further characterization using metabolic analysis in intact cells, showed that the basal and maximal respiration levels of M(2-signals) were comparable, with cells being unresponsive to the injections-inducd mitochondrial stress. Here, we show that excessive NO● secretion by the M(2-signals) macrophages, interferes with the oxygen (O2) consumption measurements on cells using the seahorse metabolic analyzer. This is attributed mainly to the consumption of ambient oxygen by NO● to form NO2- and/or NO3- but also to the reduction of O2 to superoxide anion (O2●-) by stray electrons from the electron transport chain, leading to the formation of peroxynitrite (ONOO-). We found that reactive species-donors in the absence of cells, produce comparable oxygen consumption rates (OCR) with M(2-signals) macrophages. Furthermore, inhibition of NO● production, partly recovered the respiration of activated macrophages, while external addition of NO● in non-activated macrophages downregulated their OCR levels. Our findings are crucial for the accurate metabolic characterization of cells, especially in cases where reactive nitrogen or oxygen species are produced in excess.

The Role of Macrophages in Oral Squamous Cell Carcinoma.

Oral cancer is a common malignancy worldwide, with high disease-related death rates. Oral squamous cell carcinoma (OSCC) accounts for more than 90% of oral tumors, with surgical management remaining the treatment of choice. However, advanced and metastatic OSCC is still incurable. Thus, emphasis has been given lately in understanding the complex role of the oral tumor microenvironment (TME) in OSCC progression, in order to identify novel prognostic biomarkers and therapeutic targets. Tumor associated macrophages (TAMs) constitute a major population of the OSCC TME, with bipolar role in disease progression depending on their activation status (M1 vs. M2). Here, we provide an up to date review of the current literature on the role of macrophages during oral oncogenesis, as well as their prognostic significance in OSCC survival and response to standard treatment regimens. Finally, we discuss novel concepts regarding the potential use of macrophages as targets for OSCC immunotherapeutics and suggest future directions in the field.

Targeting the Notch Signaling Pathway in Chronic Inflammatory Diseases.

The Notch signaling pathway regulates developmental cell-fate decisions and has recently also been linked to inflammatory diseases. Although therapies targeting Notch signaling in inflammation in theory are attractive, their design and implementation have proven difficult, at least partly due to the broad involvement of Notch signaling in regenerative and homeostatic processes. In this review, we summarize the supporting role of Notch signaling in various inflammation-driven diseases, and highlight efforts to intervene with this pathway by targeting Notch ligands and/or receptors with distinct therapeutic strategies, including antibody designs. We discuss this in light of lessons learned from Notch targeting in cancer treatment. Finally, we elaborate on the impact of individual Notch members in inflammation, which may lay the foundation for development of therapeutic strategies in chronic inflammatory diseases.

Specialized Pro-Resolving Mediators as Potential Regulators of Inflammatory Macrophage Responses in COVID-19.

The recent outbreak of SARS-CoV2 has emerged as one of the biggest pandemics of our century, with outrageous health, social and economic consequences globally. Macrophages may lay in the center of COVID-19 pathogenesis and lethality and treatment of the macrophage-induced cytokine storm has emerged as essential. Specialized pro-resolving mediators (SPMs) hold strong therapeutic potentials in the management of COVID-19 as they can regulate macrophage infiltration and cytokine production but also promote a pro-resolving macrophage phenotype. In this review, we discuss the homeostatic functions of SPMs acting directly on macrophages on various levels, towards the resolution of inflammation. Moreover, we address the molecular events that link the lipid mediators with COVID-19 severity and discuss the clinical potentials of SPMs in COVID-19 immunotherapeutics.

Both Type I and Type II Interferons Can Activate Antitumor M1 Macrophages When Combined With TLR Stimulation.

Triggering or enhancing antitumor activity of tumor-associated macrophages is an attractive strategy for cancer treatment. We have previously shown that the cytokine interferon-γ (IFN-γ), a type II IFN, could synergize with toll-like receptor (TLR) agonists for induction of antitumor M1 macrophages. However, the toxicity of IFN-γ limits its clinical use. Here, we investigated whether the less toxic type I IFNs, IFN-α, and IFN-ß, could potentially replace IFN-γ for induction of antitumor M1 macrophages. We measured in vitro the ability of type I and II IFNs to synergize with TLR agonists for transcription of inducible nitric oxide synthase (iNOS) mRNA and secretion of nitric oxide (NO) by mouse bone marrow-derived macrophages (BMDMs). An in vitro growth inhibition assay was used to measure both cytotoxic and cytostatic activity of activated macrophages against Lewis lung carcinoma (LLC) cancer cells. We found that both type I and II IFNs could synergize with TLR agonists in inducing macrophage-mediated inhibition of cancer cell growth, which was dependent on NO. The ability of high dose lipopolysaccharide (LPS) to induce tumoricidal activity in macrophages in the absence of IFN-γ was shown to depend on induction of autocrine type I IFNs. Antitumor M1 macrophages could also be generated in the absence of IFN-γ by a combination of two TLR ligands when using the TLR3 agonist poly(I:C) which induces autocrine type I IFNs. Finally, we show that encapsulation of poly(I:C) into nanoparticles improved its potency to induce M1 macrophages up to 100-fold. This study reveals the potential of type I IFNs for activation of antitumor macrophages and indicates new avenues for cancer immunotherapy based on type I IFN signaling, including combination of TLR agonists.

Aiming for the Insulin-like Growth Factor-1 system in breast cancer therapeutics.

Despite the major discoveries occurred in oncology the recent years, breast malignancies remain one of the most common causes of cancer-related deaths for women in developed countries. Development of HER2-targeting drugs has been considered a breakthrough in anti-cancer approaches and alluded to the potential of targeting growth factors in breast cancer (BrCa) therapeutics. More than twenty-five years have passed since the Insulin-like Growth Factor-1 (IGF-1) system was initially recognized as a potential target candidate in BrCa therapy. To date, a growing body of studies have implicated the IGF-1 signaling with the BrCa biology. Despite the promising experimental evidence, the impression from clinical trials is rather disappointing. Several reasons may account for this and the last word regarding the efficacy of this system as a target candidate in BrCa therapeutics is probably not written yet. Herein, we provide the theoretical basis, as well as, a comprehensive overview of the current literature, regarding the different strategies targeting the various components of the IGF-1/IGF-1R axis in several pathophysiological aspects of BrCa, including the tumor micro-environment and cancer stemness. In addition, we review the rationale for targeting the IGF-1 system in the different BrCa molecular subtypes and in treatment resistant breast tumors with a focus on both the molecular mechanisms and on the clinical perspectives of such approaches in specific population subgroups. We also discuss the future challenges, as well as, the development of novel molecules and strategies targeting the system and suggest potential improvements in the field.

The Role of the Androgen Receptor Signaling in Breast Malignancies.

Breast cancer (BrCa) is the most common malignancy among women worldwide, and one of the leading causes of cancer-related deaths in females. Despite the development of novel therapeutic modalities, triple-negative breast cancer (TNBC) remains an incurable disease. Androgen receptor (AR) is widely expressed in BrCa and its role in the disease may differ depending on the molecular subtype and the stage. Interestingly, AR has been suggested as a potential target candidate in TNBC, while sex hormone levels may regulate the role of AR in BrCa subtypes. In the presence of estrogen receptor α (ERa), AR may antagonize the ERα-induced effects, whereas in the absence of estrogens, AR may act as an ERα-mimic, promoting tumor. Thus, depending on the BrCa micro-environment, both agonists and antagonists of the AR have been suggested as therapeutic approaches. Herein, we review the role of AR signaling in BrCa and the molecular cross-talk mechanisms with other molecules/pathways, as well as its therapeutic implications in the different subtypes of the disease.

Generation and Functional In Vitro Analysis of Semliki Forest Virus Vectors Encoding TNF-α and IFN-γ.

Cytokine gene delivery by viral vectors is a promising novel strategy for cancer immunotherapy. Semliki Forest virus (SFV) has many advantages as a delivery vector, including the ability to (i) induce p53-independent killing of tumor cells via apoptosis, (ii) elicit a type-I interferon (IFN) response, and (iii) express high levels of the transgene. SFV vectors encoding cytokines such as interleukin (IL)-12 have shown promising therapeutic responses in experimental tumor models. Here, we developed two new recombinant SFV vectors encoding either murine tumor necrosis factor-α (TNF-α) or murine interferon-γ (IFN-γ), two cytokines with documented immunostimulatory and antitumor activity. The SFV vector showed high infection rate and cytotoxicity in mouse and human lung carcinoma cells in vitro. By contrast, mouse and human macrophages were resistant to infection with SFV. The recombinant SFV vectors directly inhibited mouse lung carcinoma cell growth in vitro, while exploiting the cancer cells for production of SFV vector-encoded cytokines. The functionality of SFV vector-derived TNF-α was confirmed through successful induction of cell death in TNF-α-sensitive fibroblasts in a concentration-dependent manner. SFV vector-derived IFN-γ activated macrophages toward a tumoricidal phenotype leading to suppressed Lewis lung carcinoma cell growth in vitro in a concentration-dependent manner. The ability of SFV to provide functional cytokines and infect tumor cells but not macrophages suggests that SFV may be very useful for cancer immunotherapy employing tumor-infiltrating macrophages.

Toll-Like Receptor Ligands and Interferon-γ Synergize for Induction of Antitumor M1 Macrophages.

Tumor-associated macrophages may either promote or suppress tumor growth depending on their activation status. Interferon-γ (IFN-γ) has been identified as a key factor for inducing tumoricidal M1 phenotype in macrophages. However, it remains unclear whether IFN-γ is sufficient or if additional stimuli are required. Here, we tested IFN-γ and a panel of toll-like receptor (TLR) agonists for the ability to activate murine macrophages toward a tumoricidal M1 phenotype. The following TLR ligands were used: TLR1/TLR2 agonist Pam3CSK4, TLR2/TLR6 agonist lipotechoic acid, TLR3 agonist poly(I:C), TLR4 agonist lipopolysaccharide (LPS), TLR5 agonist flagellin, TLR7 agonist CL264, and TLR9 agonist CpG. We used an in vitro growth inhibition assay to measure both cytotoxic and cytostatic activity of mouse macrophages against Lewis lung carcinoma (LLC) and MOPC315 plasmacytoma tumor cells. Production of nitric oxide (NO) and cytokines by activated macrophages was quantified. We found that IFN-γ alone was not able to render macrophages tumoricidal. Similarly, macrophage activation with single TLR agonists was inefficient. In sharp contrast, IFN-γ was shown to synergize with TLR agonists for induction of macrophage tumoricidal activity and production of both NO and pro-inflammatory cytokines (TNF-α, IL-12p40, and IL-12p70). Furthermore, IFN-γ was shown to suppress macrophage IL-10 secretion induced by TLR agonists. NO production was necessary for macrophage tumoricidal activity. We conclude that two signals from the microenvironment are required for optimal induction of antitumor M1 macrophage phenotype. Combination treatment with IFN-γ and TLR agonists may offer new avenues for macrophage-based cancer immunotherapy.

Clinical studies in humans targeting the various components of the IGF system show lack of efficacy in the treatment of cancer.

The insulin-like growth factors (IGFs) system regulates cell growth, differentiation and energy metabolism and plays crucial role in the regulation of key aspects of tumor biology, such as cancer cell growth, survival, transformation and invasion. The current focus for cancer therapeutic approaches have shifted from the conventional treatments towards the targeted therapies and the IGF system has gained a great interest as anti-cancer therapy. The proliferative, anti-apoptotic and transformation effects of IGFs are mainly triggered by the ligation of the type I IGF receptor (IGF-IR). Thus, aiming at developing novel and effective cancer therapies, different strategies have been employed to target IGF system in human malignancies, including but not limited to ligand or receptor neutralizing antibodies and IGF-IR signaling inhibitors. In this review, we have focused on the clinical studies that have been conducted targeting the various components of the IGF system for the treatment of different types of cancer, providing a description and the challenges of each targeting strategy and the degree of success.

The COOH-terminus of the IGF-1Ec Isoform Enhances the Proliferation and Migration of Human MCF-7 Breast Cancer Cells.

BACKGROUND/AIM: The insulin-like growth factor-1 (IGF-1) signaling is well implicated in cancer biology, however the potential roles of the distinct IGF-1 isoforms in human malignancies are largely unknown. Recently, the carboxyl-terminal of the IGF-1Ec variant (hEc; 24aa) has been associated with osteosarcoma and prostate cancer. Herein, we investigated the potential role of hEc in breast cancer. MATERIALS AND METHODS: Synthetic hEc peptide was administrated to MCF-7 and MDA-MB-231 breast cancer cells. In addition MCF-7 cells were engineered to overexpress hEc. The proliferation and migratory capacities in response to hEc were analyzed using MTT, trypan blue and wound healing/scratch assays, while the activation of the ERK/AKT signaling pathways were investigated using phospho western blotting. RESULTS: We found that exogenous administration of hEc stimulated the proliferation of estrogen-responsive MCF-7, but not that of hormone-resistant MDA-MB-231 cells. In addition, MCF-7 cells stably-overexpressing hEc acquired an increased proliferation rate and migratory capacity, as well as, enhanced ERK1/2 phosphorylation, compared to mock and wild-type cells. CONCLUSION: hEc stimulates the proliferation and migration of MCF-7 breast cancer cells and enhances the intracellular ERK1/2 signaling.

Cadherin-11 as a therapeutic target in chronic, inflammatory rheumatic diseases.

Cadherin-11 has been identified as a key regulator of synovial architecture mediating contact between Fibroblast-like Synoviocytes and organization in the lining layer. A central role for cadherin-11 has also been suggested in the formation of the rheumatoid pannus. Therapeutic targeting of cadherin-11 results in amelioration of autoimmune experimental arthritis, as well as of experimental fibrosis. In addition, cadherin-11 expression is upregulated in the synovium of patients with chronic inflammatory arthritis, whereas detection of cadherin-11 mRNA transcripts in the peripheral blood has been associated with more severe disease phenotypes in two prototypic conditions of chronic joint inflammation and fibrosis, namely, rheumatoid arthritis and systemic sclerosis, respectively. Currently, a monoclonal antibody against cadherin-11 is in early phases of clinical trials in patients with rheumatoid arthritis. Herein, we review the current knowledge regarding the potential role of cadherin-11 in pathogenesis, as well as a biomarker and therapeutic target in chronic inflammatory rheumatic diseases.

The human Ec peptide: the active core of a progression growth factor with species-specific mode of action.

OBJECTIVE: Preferential IGF-1Ec expression has been firmly associated with skeletal muscle repair mechanisms, post-infarction remodeling of the myocardium, the pathophysiology of endometriosis and prostate cancer biology. Therefore, we have studied the possible biological significance of synthetic Ec peptide, a putative cleavage product of IGF-1Ec in PC-3 cells and C2C12 myoblasts. DESIGN: We had previously designed and synthesized commercially peptides corresponding to the human Ec and its mouse igf1 counterpart as well as synthetic peptides that correspond to parts of the hEc. Using proliferation and mitogenic signaling assays, we tested their effect on PC-3 cells and C2C12 myoblasts at different doses and in different culture conditions. RESULTS: Human Ec, hEc, was documented as exerting progression but not competence growth factor actions, activating ERK1/2 without affecting Akt phosphorylation in PC-3 cells. A narrow concentration range of hEc (5-50nM) stimulated the growth of PC-3 cells grown in culture media supplemented with 10% FBS. hEc did not stimulate the growth of PC-3 cells cultured with media containing 0.5% FBS or in mouse C2C12 myoblasts under any culture conditions. The activity of hEc was blocked by a neutralizing anti-human IGF-1Ec antibody but not by a neutralizing anti-human IGF-1 receptor antibody. The synthetic mouse Ec was inactive in human PC-3 cells; however, it stimulated significantly the proliferation of mouse C2C12. By analyzing the bioactivity of synthetic hEc fragments, we documented that hEc's active core is located in the last 4aa of its C-terminal end. CONCLUSION: The hEc peptide is an important progression factor for human PC-3 prostate cancer cells.

Increased messenger RNA levels of the mesenchymal cadherin-11 in the peripheral blood of systemic sclerosis patients correlate with diffuse skin involvement.

OBJECTIVES: Cadherin-11 is a cell-cell adhesion molecule also involved in cellular migration and invasion. Experimental studies implicated this molecule in inflammatory arthritis and fibrosing conditions. Moreover, cadherin-11 protein is hyper-expressed on fibroblasts and macrophages in the skin of systemic sclerosis (SSc) patients, whereas the respective mRNA levels correlate with skin thickness. Herein, we searched for possible cadherin-11 expression also in cells that circulate in SSc peripheral blood. METHODS: Cadherin-11 mRNA was quantified by real-time reverse transcription-polymerase chain reaction in 3 ml blood samples obtained from 71 SSc patients (aged 53±2 years, 65 women) and 35 control non-SSc patients with Raynaud's phenomenon. RESULTS: Cadherin-11 mRNA transcripts were detected in blood samples from 39% of patients with diffuse SSc, versus 16% of those with limited SSc, versus 6% and 16% of patients with idiopathic or associated with other connective tissue diseases Raynaud's phenomenon, respectively (p=0.049). Cadherin-11 mRNA levels in SSc patients were increased by 3.74-fold comparing to controls (p=0.036). By multivariate logistic regression analysis we found that diffuse skin involvement correlated, independently of age, gender, disease duration, lung involvement, digital ulcers, inflammatory indices or anti-Scl-70 autoantibody presence, with cadherin-11 mRNA positivity (p=0.028), but also with increased cadherin-11 mRNA levels (≥3-fold of non-SSc levels, p=0.011). CONCLUSIONS: Cadherin-11 may be hyper-expressed in the peripheral blood of diffuse SSc patients. Studies on the origin and possible pathogenic function of these circulating cells may shed light into the complex disease pathogenesis and further support the notion that cadherin-11 is a potential therapeutic target in SSc.

The role of the insulin-like growth factor-1 system in breast cancer.

IGF-1 is a potent mitogen of major importance in the mammary gland. IGF-1 binding to the cognate receptor, IGF-1R, triggers a signaling cascade leading to proliferative and anti-apoptotic events. Although many of the relevant molecular pathways and intracellular cascades remain to be elucidated, a growing body of evidence points to the important role of the IGF-1 system in breast cancer development, progression and metastasis. IGF-1 is a point of convergence for major signaling pathways implicated in breast cancer growth. In this review, we provide an overview and concise update on the function and regulation of IGF-1 as well as the role it plays in breast malignancies.