Heme catabolism by tumor-associated macrophages controls metastasis formation.

Although the pathological significance of tumor-associated macrophage (TAM) heterogeneity is still poorly understood, TAM reprogramming is viewed as a promising anticancer therapy. Here we show that a distinct subset of TAMs (F4/80hiCD115hiC3aRhiCD88hi), endowed with high rates of heme catabolism by the stress-responsive enzyme heme oxygenase-1 (HO-1), plays a critical role in shaping a prometastatic tumor microenvironment favoring immunosuppression, angiogenesis and epithelial-to-mesenchymal transition. This population originates from F4/80+HO-1+ bone marrow (BM) precursors, accumulates in the blood of tumor bearers and preferentially localizes at the invasive margin through a mechanism dependent on the activation of Nrf2 and coordinated by the NF-κB1-CSF1R-C3aR axis. Inhibition of F4/80+HO-1+ TAM recruitment or myeloid-specific deletion of HO-1 blocks metastasis formation and improves anticancer immunotherapy. Relative expression of HO-1 in peripheral monocyte subsets, as well as in tumor lesions, discriminates survival among metastatic melanoma patients. Overall, these results identify a distinct cancer-induced HO-1+ myeloid subgroup as a new antimetastatic target and prognostic blood marker.

Macrophage activation and polarization: nomenclature and experimental guidelines.

Description of macrophage activation is currently contentious and confusing. Like the biblical Tower of Babel, macrophage activation encompasses a panoply of descriptors used in different ways. The lack of consensus on how to define macrophage activation in experiments in vitro and in vivo impedes progress in multiple ways, including the fact that many researchers still consider there to be only two types of activated macrophages, often termed M1 and M2. Here, we describe a set of standards encompassing three principles-the source of macrophages, definition of the activators, and a consensus collection of markers to describe macrophage activation-with the goal of unifying experimental standards for diverse experimental scenarios. Collectively, we propose a common framework for macrophage-activation nomenclature.

Prolonged exposure to simulated microgravity promotes stemness impairing morphological, metabolic and migratory profile of pancreatic cancer cells: a comprehensive proteomic, lipidomic and transcriptomic analysis.

BACKGROUND: The impact of the absence of gravity on cancer cells is of great interest, especially today that space is more accessible than ever. Despite advances, few and contradictory data are available mainly due to different setup, experimental design and time point analyzed. METHODS: Exploiting a Random Positioning Machine, we dissected the effects of long-term exposure to simulated microgravity (SMG) on pancreatic cancer cells performing proteomic, lipidomic and transcriptomic analysis at 1, 7 and 9 days. RESULTS: Our results indicated that SMG affects cellular morphology through a time-dependent activation of Actin-based motility via Rho and Cdc42 pathways leading to actin rearrangement, formation of 3D spheroids and enhancement of epithelial-to-mesenchymal transition. Bioinformatic analysis reveals that SMG may activates ERK5/NF-κB/IL-8 axis that triggers the expansion of cancer stem cells with an increased migratory capability. These cells, to remediate energy stress and apoptosis activation, undergo a metabolic reprogramming orchestrated by HIF-1α and PI3K/Akt pathways that upregulate glycolysis and impair ß-oxidation, suggesting a de novo synthesis of triglycerides for the membrane lipid bilayer formation. CONCLUSIONS: SMG revolutionizes tumor cell behavior and metabolism leading to the acquisition of an aggressive and metastatic stem cell-like phenotype. These results dissect the time-dependent cellular alterations induced by SMG and pave the base for altered gravity conditions as new anti-cancer technology.

Suppressors of cytokine signaling 2 and 3 diametrically control macrophage polarization.

Suppressors of cytokine signaling (SOCS) are important regulators of lipopolysaccharide (LPS) and cytokine responses but their role in macrophage polarization is unknown. We have shown here that myeloid-restricted Socs3 deletion (Socs3(Lyz2cre)) resulted in resistance to LPS-induced endotoxic shock, whereas Socs2(-/-) mice were highly susceptible. We observed striking bias toward M2-like macrophages in Socs3(Lyz2cre) mice, whereas the M1-like population was enriched in Socs2(-/-) mice. Adoptive transfer experiments showed that responses to endotoxic shock and polymicrobial sepsis were transferable and macrophage dependent. Critically, this dichotomous response was associated with enhanced regulatory T (Treg) cell recruitment by Socs3(Lyz2cre) cells, whereas Treg cell recruitment was absent in the presence of Socs2(-/-) macrophages. In addition, altered polarization coincided with enhanced interferon-gamma (IFN-γ)-induced signal transducer and activator of transcription-1 (STAT1) activation in Socs2(-/-) macrophages and enhanced interleukin-4 (IL-4) plus IL-13-induced STAT6 phosphorylation in Socs3(Lyz2cre) macrophages. SOCS, therefore, are essential controllers of macrophage polarization, regulating inflammatory responses.

Precision Medicine Approaches with Metabolomics and Artificial Intelligence.

Recent technological innovations in the field of mass spectrometry have supported the use of metabolomics analysis for precision medicine. This growth has been allowed also by the application of algorithms to data analysis, including multivariate and machine learning methods, which are fundamental to managing large number of variables and samples. In the present review, we reported and discussed the application of artificial intelligence (AI) strategies for metabolomics data analysis. Particularly, we focused on widely used non-linear machine learning classifiers, such as ANN, random forest, and support vector machine (SVM) algorithms. A discussion of recent studies and research focused on disease classification, biomarker identification and early diagnosis is presented. Challenges in the implementation of metabolomics-AI systems, limitations thereof and recent tools were also discussed.

Influence of Innate Immunity on Cancer Cell Stemness.

Even if cancer stem cells (CSCs) represent only a small proportion of the tumor mass, they significantly account for tumor maintenance, resistance to therapies, relapse and metastatic spread, due to their increased capacity of self-renewal, multipotency, tumorigenicity and quiescence. Emerging evidence suggests that the immune contexture within the tumor microenvironment (TME) determines both the response to therapy and the clinical outcome. In this context, CSCs acquire immune evasion skills by editing immune cell functions and sculpting the immunosuppressive landscape of TME. Reciprocally, infiltrating immune cells influence CSCs self-renewal, tumorigenicity and metastasis. In this review, we summarize the immunomodulatory properties of CSCs, as well as the impact of innate immune cells on cancer cells stemness in the different phases of cancer immunoediting process and neoplastic progression.

Lipid Metabolism and Cancer Immunotherapy: Immunosuppressive Myeloid Cells at the Crossroad.

Cancer progression generates a chronic inflammatory state that dramatically influences hematopoiesis, originating different subsets of immune cells that can exert pro- or anti-tumor roles. Commitment towards one of these opposing phenotypes is driven by inflammatory and metabolic stimuli derived from the tumor-microenvironment (TME). Current immunotherapy protocols are based on the reprogramming of both specific and innate immune responses, in order to boost the intrinsic anti-tumoral activity of both compartments. Growing pre-clinical and clinical evidence highlights the key role of metabolism as a major influence on both immune and clinical responses of cancer patients. Indeed, nutrient competition (i.e., amino acids, glucose, fatty acids) between proliferating cancer cells and immune cells, together with inflammatory mediators, drastically affect the functionality of innate and adaptive immune cells, as well as their functional cross-talk. This review discusses new advances on the complex interplay between cancer-related inflammation, myeloid cell differentiation and lipid metabolism, highlighting the therapeutic potential of metabolic interventions as modulators of anticancer immune responses and catalysts of anticancer immunotherapy.

The Macrophages-Microbiota Interplay in Colorectal Cancer (CRC)-Related Inflammation: Prognostic and Therapeutic Significance.

Tumor-associated macrophages (TAMs) are the main population of myeloid cells infiltrating solid tumors and the pivotal orchestrators of cancer-promoting inflammation. However, due to their exceptional plasticity, macrophages can be also key effector cells and powerful activators of adaptive anti-tumor immunity. This functional heterogeneity is emerging in human tumors, colorectal cancer (CRC) in particular, where the dynamic co-existence of different macrophage subtypes influences tumor development, outcome, and response to therapies. Intestinal macrophages are in close interaction with enteric microbiota, which contributes to carcinogenesis and affects treatment outcomes. This interplay may be particularly relevant in CRC, one of the most prevalent and lethal cancer types in the world. Therefore, both macrophages and intestinal microbiota are considered promising prognostic indicators and valuable targets for new therapeutic approaches. Here, we discuss the current understanding of the molecular circuits underlying the interplay between macrophages and microbiota in CRC development, progression, and response to both conventional therapies and immunotherapies.

A Proteomic Analysis of GSD-1a in Mouse Livers: Evidence for Metabolic Reprogramming, Inflammation, and Macrophage Polarization.

Glycogen storage disease type 1a (GSD-1a) is a rare genetic disease caused by mutations in the catalytic subunit of the enzyme glucose-6-phosphatase-alpha (G6Pase-α). The majority of patients develop long-term complications including renal failure and hepatocellular adenoma/carcinoma. The purpose of this study was to ascertain the proteomic changes in the liver of LS- G6pc-/- mice, a murine model of GSD-1a, in comparison with wild type mice to identify potential biomarkers of the pathophysiology of the affected liver. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze liver lysates from a total of 20 LS- G6pc-/- and 18 wild type (WT) mice. We compared the proteomic expression profile of LS- G6pc-/- and WT mice. We identified 4138 significantly expressed proteins, 1243 of which were differentially represented. Network and pathway analyses indicate that LS- G6pc-/- livers display an age-dependent modulation of the expression of proteins involved in specific biological processes associated with increased progression of liver disease. Moreover, we found upregulation of proteins involved in the process of tissue inflammation and macrophage polarization toward the M2 phenotype in LS- G6pc-/- mice with adenomas. Our results identify a metabolic reprogramming of glucose-6-P and a pathologic environment in the liver compatible with tumor development and progression.

Molecular and epigenetic basis of macrophage polarized activation.

Macrophages are unique cells for origin, heterogeneity and plasticity. At steady state most of macrophages are derived from fetal sources and maintained in adulthood through self-renewing. Despite sharing common progenitors, a remarkable heterogeneity characterized tissue-resident macrophages indicating that local signals educate them to express organ-specific functions. Macrophages are extremely plastic: chromatin landscape and transcriptional programs can be dynamically re-shaped in response to microenvironmental changes. Owing to their ductility, macrophages are crucial orchestrators of both initiation and resolution of immune responses and key supporters of tissue development and functions in homeostatic and pathological conditions. Herein, we describe current understanding of heterogeneity and plasticity of macrophages using the M1-M2 dichotomy as operationally useful simplification of polarized activation. We focused on the complex network of signaling cascades, metabolic pathways, transcription factors, and epigenetic changes that control macrophage activation. In particular, this network was addressed in sepsis, as a paradigm of a pathological condition determining dynamic macrophage reprogramming.

Metabolic influence on the differentiation of suppressive myeloid cells in cancer.

New evidences indicate that the metabolic instruction of immunity (immune metabolism) results from the integration of cell metabolism and whole-body metabolism, which are both influenced by nutrition, microbiome metabolites and disease-driven metabolism (e.g. cancer metabolism). Cancer metabolism influences the immunological homeostasis and promotes immune alterations that support disease progression, hence influencing the clinical outcome. Cancer cells display increased glucose uptake and fermentation of glucose to lactate, even in the presence of completely functioning mitochondria. A major side effect of this event is immunosuppression, characterized by limited immunogenicity of cancer cells and restriction of the therapeutic efficacy of anticancer immunotherapy. Here, we discuss how the metabolism of myeloid cells associated with cancer contributes to the differentiation of their suppressive phenotype and therefore to cancer immune evasion.

Chronic exposure to TGFß1 regulates myeloid cell inflammatory response in an IRF7-dependent manner.

Tissue microenvironment influences the function of resident and infiltrating myeloid-derived cells. In the central nervous system (CNS), resident microglia and freshly recruited infiltrating monocyte-derived macrophages (mo-MΦ) display distinct activities under pathological conditions, yet little is known about the microenvironment-derived molecular mechanism that regulates these differences. Here, we demonstrate that long exposure to transforming growth factor-ß1 (TGFß1) impaired the ability of myeloid cells to acquire a resolving anti-inflammatory phenotype. Using genome-wide expression analysis and chromatin immunoprecipitation followed by next-generation sequencing, we show that the capacity to undergo pro- to anti-inflammatory (M1-to-M2) phenotype switch is controlled by the transcription factor interferon regulatory factor 7 (IRF7) that is down-regulated by the TGFß1 pathway. RNAi-mediated perturbation of Irf7 inhibited the M1-to-M2 switch, while IFNß1 (an IRF7 pathway activator) restored it. In vivo induction of Irf7 expression in microglia, following spinal cord injury, reduced their pro-inflammatory activity. These results highlight the key role of tissue-specific environmental factors in determining the fate of resident myeloid-derived cells under both physiological and pathological conditions.

Cholangiocarcinoma stem-like subset shapes tumor-initiating niche by educating associated macrophages.

BACKGROUND & AIMS: A therapeutically challenging subset of cells, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages (TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche. METHODS: CCA cells were cultured in 3D conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14+ with CCA-sphere conditioned medium. RESULTS: CCA spheres engrafted in 100% of transplanted mice and revealed a significant 20.3-fold increase in tumor-initiating fraction (p=0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14+ macrophages expressed key markers (CD68, CD115, human leukocyte antigen-D related, CD206) indicating that CCA sphere conditioned medium was a strong macrophage-activator. Gene expression profile of CCA sphere activated macrophages revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated macrophages from CCA resections recapitulated a similar molecular phenotype of in vitro-educated macrophages. Consistent with invasive features, the largest CD163+ set was found in the tumor front of human CCA specimens (n=23) and correlated with a high level of serum cancer antigen 19.9 (n=17). Among mediators released by CCA spheres, only interleukin (IL)13, IL34 and osteoactivin were detected and further confirmed in CCA patient sera (n=12). Surprisingly, a significant association of IL13, IL34 and osteoactivin with sphere stem-like genes was provided by a CCA database (n=104). In vitro combination of IL13, IL34, osteoactivin was responsible for macrophage-differentiation and invasion, as well as for in vivo tumor-promoting effect. CONCLUSION: CCA-CSCs molded a specific subset of stem-like associated macrophages thus providing a rationale for a synergistic therapeutic strategy for CCA-disease. LAY SUMMARY: Immune plasticity represents an important hallmark of tumor outcome. Since cancer stem cells are able to manipulate stromal cells to their needs, a better definition of the key dysregulated immune subtypes responsible for cooperating in supporting tumor initiation may facilitate the development of new therapeutic approaches. Considering that human cholangiocarcinoma represents a clinical emergency, it is essential to move to predictive models in order to understand the adaptive process of macrophage component (imprinting, polarization and maintenance) engaged by tumor stem-like compartment.

Tumor-associated myeloid cells as guiding forces of cancer cell stemness.

Due to their ability to differentiate into various cell types and to support tissue regeneration, stem cells simultaneously became the holy grail of regenerative medicine and the evil obstacle in cancer therapy. Several studies have investigated niche-related conditions that favor stemness properties and increasingly emphasized their association with an inflammatory environment. Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) are major orchestrators of cancer-related inflammation, able to dynamically express different polarized inflammatory programs that promote tumor outgrowth, including tumor angiogenesis, immunosuppression, tissue remodeling and metastasis formation. In addition, these myeloid populations support cancer cell stemness, favoring tumor maintenance and progression, as well as resistance to anticancer treatments. Here, we discuss inflammatory circuits and molecules expressed by TAMs and MDSCs as guiding forces of cancer cell stemness.

Myeloid suppressor cells in cancer and autoimmunity.

A bottleneck for immunotherapy of cancer is the immunosuppressive microenvironment in which the tumor cells proliferate. Cancers harness the immune regulatory mechanism that prevents autoimmunity from evading immunosurveillance and promoting immune destruction. Regulatory T cells, myeloid suppressor cells, inhibitory cytokines and immune checkpoint receptors are the major components of the immune system acting in concert with cancer cells and causing the subversion of anti-tumor immunity. This redundant immunosuppressive network poses an impediment to efficacious immunotherapy by facilitating tumor progression. Tumor-associated myeloid cells comprise heterogeneous populations acting systemically (myeloid-derived suppressor cells/MDSCs) and/or locally in the tumor microenvironment (MDSCs and tumor-associated macrophages/TAMs). Both populations promote cancer cell proliferation and survival, angiogenesis and lymphangiogenesis and elicit immunosuppression through different pathways, including the expression of immunosuppressive cytokines and checkpoint inhibitors. Several evidences have demonstrated that myeloid cells can express different functional programs in response to different microenvironmental signals, a property defined as functional plasticity. The opposed extremes of this functional flexibility are generally represented by the classical macrophage activation, which identifies inflammatory and cytotoxic M1 polarized macrophages, and the alternative state of macrophage activation, which identifies M2 polarized anti-inflammatory and immunosuppressive macrophages. Functional skewing of myeloid cells occurs in vivo under physiological and pathological conditions, including cancer and autoimmunity. Here we discuss how myeloid suppressor cells can on one hand support tumor growth and, on the other, limit autoimmune responses, indicating that their therapeutic reprogramming can generate opportunities in relieving immunosuppression in the tumor microenvironment or reinstating tolerance in autoimmune conditions.

Macrophage polarization in pathology.

Macrophages are cells of the innate immunity constituting the mononuclear phagocyte system and endowed with remarkable different roles essential for defense mechanisms, development of tissues, and homeostasis. They derive from hematopoietic precursors and since the early steps of fetal life populate peripheral tissues, a process continuing throughout adult life. Although present essentially in every organ/tissue, macrophages are more abundant in the gastro-intestinal tract, liver, spleen, upper airways, and brain. They have phagocytic and bactericidal activity and produce inflammatory cytokines that are important to drive adaptive immune responses. Macrophage functions are settled in response to microenvironmental signals, which drive the acquisition of polarized programs, whose extremes are simplified in the M1 and M2 dichotomy. Functional skewing of monocyte/macrophage polarization occurs in physiological conditions (e.g., ontogenesis and pregnancy), as well as in pathology (allergic and chronic inflammation, tissue repair, infection, and cancer) and is now considered a key determinant of disease development and/or regression. Here, we will review evidence supporting a dynamic skewing of macrophage functions in disease, which may provide a basis for macrophage-centered therapeutic strategies.