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Succinate in the tumor microenvironment affects tumor growth and modulates tumor associated macrophages
Abstract
Succinate is an important metabolite that modulates metabolism of immune cells and cancer cells in the tumor microenvironment (TME). Herein, we report that polyethylene succinate (PES) microparticles (MPs) biomaterial mediated controlled delivery of succinate in the TME modulates macrophage responses. Administering PES MPs locally with or without a BRAF inhibitor systemically in an immune-defective aging mice with clinically relevant BRAFV600E mutated YUMM1.1 melanoma decreased tumor volume three-fold. PES MPs in the TME also led to maintenance of M1 macrophages with up-regulation of TSLP and type 1 interferon pathway. Impressively, this led to generation of pro-inflammatory adaptive immune responses in the form of increased T helper type 1 and T helper type 17 cells in the TME. Overall, our findings from this challenging tumor model suggest that immunometabolism-modifying PES MP strategies provide an approach for developing robust cancer immunotherapies.
... More broadly, ontological pathway databases would likely benefit from separating the leucine, isoleucine, and valine catabolic entries to prevent statistical muting introduced from signature-based analyses, which often group BCAA degradation as a singular process. Succinate has been identified as having many prooncogenic roles, including HIF1α stabilisation [27,28], alteration of the tumour microenvironment [32], promotion of metastatic potential via enhanced EMT [29], suppression of anti-tumour immune responses [33,34] and the activation of pro-inflammatory signalling pathways [35]. Future directions should investigate the effects of HIBCH inhibition on these processes. ...
Metabolic reprogramming and energetic rewiring are hallmarks of cancer that fuel disease progression and facilitate therapy evasion. The remodelling of oxidative phosphorylation and enhanced lipogenesis have previously been characterised as key metabolic features of prostate cancer (PCa). Recently, succinate-dependent mitochondrial reprogramming was identified in high-grade prostate tumours, as well as upregulation of the enzymes associated with branched-chain amino acid (BCAA) catabolism. In this study, we hypothesised that the degradation of the BCAAs, particularly valine, may play a critical role in anapleurotic refuelling of the mitochondrial succinate pool, as well as the maintenance of intracellular lipid metabolism. Through the suppression of BCAA availability, we report significantly reduced lipid content, strongly indicating that BCAAs are important lipogenic fuels in PCa. This work also uncovered a novel compensatory mechanism, whereby fatty acid uptake is increased in response to extracellular valine deprivation. Inhibition of valine degradation via suppression of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) resulted in a selective reduction of malignant prostate cell proliferation, decreased intracellular succinate and impaired cellular respiration. In combination with a comprehensive multi-omic investigation that incorporates next-generation sequencing, metabolomics, and high-content quantitative single-cell imaging, our work highlights a novel therapeutic target for selective inhibition of metabolic reprogramming in PCa.
... It is not important that the induced intense oxidation of succinate in mitochondria under hypoxic loads, which often accompany pathological conditions, is accompanied by a significant increase in the production of free radicals and mitochondrial dysregulation processes [89]. The succinatedependent metabolism pathways during ageing and pathologies with a varied genesis are shown in Table 2. Analysis of postoperative cognitive dysfunction processes, gerontological patients after cardiac surgery, and a cognitive impairment model, people over 60 years of age, Cytoflavin containing succinic acid Cytoflavin containing inosine, nicotinamide, riboflavin, and succinic acid was used in elderly postoperative patients in a multicenter, double-blind, placebo-controlled, and randomised study Improvement of gerontological patients' condition [121] Study of steroidogenic adrenocortical cells in LPS-induced systemic inflammation processes in a murine model, succinate-succinate dehydrogenase relationship Increased succinate levels by disruption of oxidative phosphorylation and increased ATP synthesis connected with high ROS production SDHB expression via upregulation of DNA methyltransferase 1 and methylation processes in the SDHB promoter [122] Review analysis of the physiological and pathophysiological condition connected with succinic acid metabolism and SDH functions Succinate functions and hypoxia-inducible factor (HIF)-1α, development of pseudohypoxia and tumours via mutated SDH, succinate functions in metabolic or non-metabolic pathways, lysine succinylation process as proteins and immunomodulatory modification levels, blood formation or haematopoiesis Activation of succinate receptor 1 (SUCNR1), G protein-coupled receptor 91 (GPR91), or hypoxia-inducible factor-1α, (HIF)-1α [112] In vivo ischaemia-reperfusion of heart in an open-chest mouse model, metabolomic analysis of ex vivo Langendorff heart experiments Study of succinate-dependent mitochondrial superoxide production in myoblasts Inhibition of ischaemic succinate accumulation and its oxidation as an effective way in ischaemia-reperfusion conditions [123] Immune-defective ageing murine model, clinically relevant BRAF V600E mutated YUMM1.1 melanoma tumour model, cancer immunotherapies Tumour microenvironment study using polyethylene succinate microparticle biomaterial Succinate-mediated immune and cancer cell responses in a tumour model and immunotherapies [124] Hippocampus of different aged APP/PS1 double transgenic AD mice, analysis of the β-amyloid level with the immunohistochemistry method 3, 6, 9, and 12-month-old mice groups, learning and memory test analysis, mitochondrial damage, and autophagosome accumulation assays Abnormal accumulation of succinic acid and citric acid associated with age-related damage to hippocampal mitochondria in the APP/PS1 murine model [125] ...
Anti-ageing biology and medicine programmes are a focus of genetics, molecular biology, immunology, endocrinology, nutrition, and therapy. This paper discusses metabolic therapies aimed at prolonging longevity and/or health. Individual components of these effects are postulated to be related to the energy supply by tricarboxylic acid (TCA) cycle intermediates and free radical production processes. This article presents several theories of ageing and clinical descriptions of the top markers of ageing, which define ageing in different categories; additionally, their interactions with age-related changes and diseases related to α-ketoglutarate (AKG) and succinate SC formation and metabolism in pathological states are explained. This review describes convincingly the differences in the mitochondrial characteristics of energy metabolism in animals, with different levels (high and low) of physiological reactivity of functional systems related to the state of different regulatory systems providing oxygen-dependent processes. Much attention is given to the crucial role of AKG and SC in the energy metabolism in cells related to amino acid synthesis, epigenetic regulation, cell stemness, and differentiation, as well as metabolism associated with the development of pathological conditions and, in particular, cancer cells. Another goal was to address the issue of ageing in terms of individual characteristics related to physiological reactivity. This review also demonstrated the role of the Krebs cycle as a key component of cellular energy and ageing, which is closely associated with the development of various age-related pathologies, such as cancer, type 2 diabetes, and cardiovascular or neurodegenerative diseases where the mTOR pathway plays a key role. This article provides postulates of postischaemic phenomena in an ageing organism and demonstrates the dependence of accelerated ageing and age-related pathology on the levels of AKG and SC in studies on different species (roundworm Caenorhabditis elegans, Drosophila, mice, and humans used as models). The findings suggest that this approach may also be useful to show that Krebs cycle metabolites may be involved in age-related abnormalities of the mitochondrial metabolism and may thus induce epigenetic reprogramming that contributes to the senile phenotype and degenerative diseases. The metabolism of these compounds is particularly important when considering ageing mechanisms connected with different levels of initial physiological reactivity and able to initiate individual programmed ageing, depending on the intensity of oxygen consumption, metabolic peculiarities, and behavioural reactions.
Cuproptosis is an emerging cell death pathway that depends on the intracellular Cu ions. Elesclomol (ES) as an efficient Cu ionophore can specifically transport Cu into mitochondria and trigger cuproptosis. However, ES can be rapidly removed and metabolized during intravenous administration, leading to a short half‐life and limited tumor accumulation, which hampers its clinical application. Here, the study develops a reactive oxygen species (ROS)‐responsive polymer (PCP) based on cinnamaldehyde (CA) and polyethylene glycol (PEG) to encapsulate ES‐Cu compound (EC), forming ECPCP. ECPCP significantly prolongs the systemic circulation of EC and enhances its tumor accumulation. After cellular internalization, the PCP coating stimulatingly dissociates exposing to the high‐level ROS, and releases ES and Cu, thereby triggering cell death via cuproptosis. Meanwhile, Cu²⁺‐stimulated Fenton‐like reaction together with CA‐stimulated ROS production simultaneously breaks the redox homeostasis, which compensates for the insufficient oxidative stress treated with ES alone, in turn inducing immunogenic cell death of tumor cells, achieving simultaneous cuproptosis and immunotherapy. Furthermore, the excessive ROS accelerates the stimuli‐dissociation of ECPCP, forming a positive feedback therapy loop against tumor self‐alleviation. Therefore, ECPCP as a nanoplatform for cuproptosis and immunotherapy improves the dual antitumor mechanism of ES and provides a potential optimization for ES clinical application.
Evolving knowledge about the tumor–immune microenvironment (TIME) is driving innovation in designing novel therapies against hard‐to‐treat breast cancer. Targeting the immune components of TIME has emerged as a promising approach for cancer therapy. While recent immunotherapies aim at restoring antitumor immunity, counteracting tumor escape remains challenging. Hence there is a pressing need to better understand the complex tumor–immune crosstalk within TIME. Considering this imperative, this study aims at investigating the crosstalk between the two abundant immune cell populations within the breast TIME—macrophages and T cells, in driving tumor progression using an organotypic 3D in vitro tumor‐on‐a‐chip (TOC) model. The TOC features distinct yet interconnected organotypic tumor and stromal entities. This triculture platform mimics the complex TIME, embedding the two immune populations in a suitable 3D matrix. Analysis of invasion, morphometric measurements, and flow cytometry results underscores the substantial contribution of macrophages to tumor progression, while the presence of T cells is associated with a deceleration in the migratory behavior of both cancer cells and macrophages. Furthermore, cytokine analyses reveal significant upregulation of leptin and RANTES (regulated on activation, normal T Cell expressed and secreted) in triculture. Overall, this study highlights the complexity of TIME and the critical role of immune cells in cancer progression.
Metabolic reprogramming is a hallmark of cancer and fundamental for disease progression. The remodelling of oxidative phosphorylation (OxPhos) and enhanced lipogenesis are key characteristics of prostate cancer (PCa). Recently, succinate-dependent mitochondrial reprogramming was identified in high-grade prostate tumours with upregulation of enzymes associated with branched-chain amino acid catabolism. We hypothesised that the degradation of branched-chain amino acids, particularly valine may play a critical role in anapleurotic refuelling of the mitochondrial succinate pool. Through suppression of valine availability, we report strongly reduced lipid content despite compensatory upregulation of fatty acid uptake, indicating valine is an important lipogenic fuel in PCa. Inhibition of the enzyme 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) resulted in selective inhibition of cellular proliferation of malignant but not benign prostate cells. In combination with this and a comprehensive multi-omic investigation of both patient and cell line data, our work highlights a therapeutic target for selective inhibition of metabolic reprogramming in PCa.
Significance
Differences in immune functioning stem from multiple factors, including sex and aging. However, the specific roles of these variables in immunity remain elusive. We profiled immunocytes from young and old males and females at single-cell resolution and constructed a precise atlas of blood-circulating immunocytes. T cell– and B cell–activated signals were higher in young females than males, while aging increased the sex-related differences in immunocytes, cellular composition, and inflammatory signaling. Additionally, males showed a higher accumulation of inflammatory factors during aging, whereas cell–cell communication analysis revealed different trends in gene expression between females and males with aging. These findings might aid in the understanding of the mechanisms underlying sex-based differences in immunity and disease susceptibility across the lifespan.
The study of cancer biology should be based around a comprehensive vision of the entire tumor ecosystem, considering the functional, bioenergetic and metabolic state of tumor cells and those of their microenvironment, and placing particular importance on immune system cells. Enhanced understanding of the molecular bases that give rise to alterations of pathways related to tumor development can open up new therapeutic intervention opportunities, such as metabolic regulation applied to immunotherapy. This review outlines the role of various oncometabolites and immunometabolites, such as TCA intermediates, in shaping pro/anti-inflammatory activity of immune cells such as MDSCs, T lymphocytes, TAMs and DCs in cancer. We also discuss the extraordinary plasticity of the immune response and its implication in immunotherapy efficacy, and highlight different therapeutic intervention possibilities based on controlling the balanced systems of specific metabolites with antagonistic functions.
T cell activation is intimately linked to metabolism, as distinct metabolic requirements support the functional and phenotypical differences between quiescent and activated T cells. Metabolic transition from mitochondrial oxidative phosphorylation to aerobic glycolysis is crucial for a proper T cell activation. However, the role of tricarboxylic acid cycle (TCA), and in particular succinate dehydrogenase (SDH) in activated T cells needs further elucidation. Here we show that inhibition of SDH during activation of T cells results in strong impairment of proliferation, expression of activation markers, and production of key inflammatory cytokines, despite a concomitant increase in glycolytic metabolic activity. Similar effect of SDH inhibition were demonstrated in pre-activated T cell. Interestingly, itaconic acid, an endogenous SDH inhibitor released from activated macrophages and dendritic cells, had no immunomodulator effect. Taken together, our findings demonstrate that SDH enzyme fitness is critical for mounting and maintaining appropriate activation and function of human T cells.
Nanoparticles (NPs) can serve as a promising vaccine delivery platform for improving pharmacological property and codelivery of antigens and adjuvants. However, NP‐based vaccines are generally associated with complex synthesis and postmodification procedures, which pose technical and manufacturing challenges for tailor‐made vaccine production. Here, modularly programmed, polyethyleneimine (PEI)‐based NP vaccines are reported for simple production of personalized cancer vaccines. Briefly, PEI is conjugated with neoantigens by facile coupling chemistry, followed by electrostatic assembly with CpG adjuvants, leading to the self‐assembly of nontoxic, sub‐50 nm PEI NPs. Importantly, PEI NPs promote activation and antigen cross‐presentation of antigen‐presenting cells and cross‐priming of neoantigen‐specific CD8+ T cells. Surprisingly, after only a single intratumoral injection, PEI NPs with optimal PEGylation elicit as high as ≈30% neoantigen‐specific CD8+ T cell response in the systemic circulation and sustain elevated CD8+ T cell response over 3 weeks. PEI‐based nanovaccines exert potent antitumor efficacy against pre‐established local tumors as well as highly aggressive metastatic tumors. PEI engineering for modular incorporation of neoantigens and adjuvants offers a promising strategy for rapid and facile production of personalized cancer vaccines. Modularly programmed, polyethyleneimine (PEI)‐based nanoparticle (NP) vaccines are developed for simple production of personalized cancer vaccines. PEI‐antigen conjugates and CpG adjuvants are mixed form nontoxic, sub‐50 nm PEI NPs. PEI NPs elicit strong, long‐lasting neoantigen‐specific CD8+ T cell response with potent antitumor efficacy against local as well as metastatic tumors. PEI NPs offer a promising strategy for personalized cancer vaccination.
By targeting the tumor microenvironment to stimulate antitumor immunity, immunotherapies have revolutionized cancer treatment. However, many patients do not respond initially or develop secondary resistance. Based on the limited resources in the tumor microenvironment and competition between tumor and immune cells, the field of immune metabolism has produced extensive knowledge showing that targeting metabolism could help to modulate antitumor immunity. However, among all the different potentially targetable metabolic pathways, it remains unclear which have more potential to overcome resistance to immune checkpoint inhibitors. Here, we explore metabolic reprogramming in cancer cells, which might inhibit antitumor immunity, and strategies that can be used to favor the antitumor response.
Immunometabolism revealed the crucial role of cellular metabolism in controlling immune cell phenotype and functions. Macrophages, key immune cells that support progression of numerous inflammatory diseases, have been well described as undergoing vast metabolic rewiring upon activation. The immunometabolite succinate particularly gained a lot of attention and emerged as a crucial regulator of macrophage responses and inflammation. Succinate was originally described as a metabolite that supports inflammation via distinct routes. Recently, studies have indicated that succinate and its receptor SUCNR1 can suppress immune responses as well. These apparent contradictory effects might be due to specific experimental settings and particularly the use of distinct succinate forms. We therefore compared the phenotypic and functional effects of distinct succinate forms and receptor mouse models that were previously used for studying succinate immunomodulation. Here, we show that succinate can suppress secretion of inflammatory mediators IL-6, tumor necrosis factor (TNF) and nitric oxide (NO), as well as inhibit Il1b mRNA expression of inflammatory macrophages in a SUCNR1-independent manner. We also observed that macrophage SUCNR1 deficiency led to an enhanced inflammatory response without addition of exogenous succinate. While our study does not reveal new mechanistic insights into how succinate elicits different inflammatory responses, it does indicate that the inflammatory effects of succinate and its receptor SUCNR1 in macrophages are clearly context dependent.
Tumor metastasis is the major cause of mortality from cancer. Metabolic rewiring and the metastatic cascade are highly intertwined, co-operating to promote multiple steps of cancer metastasis. Metabolites generated by cancer cells influence the metastatic cascade, encompassing epithelial-mesenchymal transition (EMT), survival of cancer cells in circulation, and metastatic colonization at distant sites. A variety of molecular mechanisms underlie the prometastatic effect of tumor-derived metabolites, such as epigenetic deregulation, induction of matrix metalloproteinases (MMPs), promotion of cancer stemness, and alleviation of oxidative stress. Conversely, metastatic signaling regulates expression and activity of rate-limiting metabolic enzymes to generate prometastatic metabolites thereby reinforcing the metastasis cascade. Understanding the complex interplay between metabolism and metastasis could unravel novel molecular targets, whose intervention could lead to improvements in the treatment of cancer. In this review, we summarized the recent discoveries involving metabolism and tumor metastasis, and emphasized the promising molecular targets, with an update on the development of small molecule or biologic inhibitors against these aberrant situations in cancer.
Elderly individuals are the most susceptible to an aggressive form of coronavirus disease (COVID-19), caused by SARS-CoV-2. The remodeling of immune response that is observed among the elderly could explain, at least in part, the age gradient in lethality of COVID-19. In this review, we will discuss the phenomenon of immunosenescence, which entails changes that occur in both innate and adaptive immunity with aging. Furthermore, we will discuss inflamm-aging, a low-grade inflammatory state triggered by continuous antigenic stimulation, which may ultimately increase all-cause mortality. In general, the elderly are less capable of responding to neo-antigens, because of lower naïve T cell frequency. Furthermore, they have an expansion of memory T cells with a shrinkage of the T cell diversity repertoire. When infected by SARS-CoV-2, young people present with a milder disease as they frequently clear the virus through an efficient adaptive immune response. Indeed, antibody-secreting cells and follicular helper T cells are thought to be effectively activated in young patients that present a favorable prognosis. In contrast, the elderly are more prone to an uncontrolled activation of innate immune response that leads to cytokine release syndrome and tissue damage. The failure to trigger an effective adaptive immune response in combination with a higher pro-inflammatory tonus may explain why the elderly do not appropriately control viral replication and the potential clinical consequences triggered by a cytokine storm, endothelial injury, and disseminated organ injury. Enhancing the efficacy of the adaptive immune response may be an important issue both for infection resolution as well as for the appropriate generation of immunity upon vaccination, while inhibiting inflamm-aging will likely emerge as a potential complementary therapeutic approach in the management of patients with severe COVID-19.
The immune system is the key target for vaccines and immunotherapeutic approaches aimed at blunting infectious diseases, cancer, autoimmunity, and implant rejection. However, systemwide immunomodulation is undesirable due to the severe side effects that typically accompany such strategies. In order to circumvent these undesired, harmful effects, scientists have turned to tailorable biomaterials that can achieve localized, potent release of immune‐modulating agents. Specifically, “stimuli‐responsive” biomaterials hold a strong promise for delivery of immunotherapeutic agents to the disease site or disease‐relevant tissues with high spatial and temporal accuracy. This review provides an overview of stimuli‐responsive biomaterials used for targeted immunomodulation. Stimuli‐responsive or “environmentally responsive” materials are customized to specifically react to changes in pH, temperature, enzymes, redox environment, photo‐stimulation, molecule‐binding, magnetic fields, ultrasound‐stimulation, and electric fields. Moreover, the latest generation of this class of materials incorporates elements that allow for response to multiple stimuli. These developments, and other stimuli‐responsive materials that are on the horizon, are discussed in the context of controlling immune responses.
Vascularized composite allotransplantation (VCA) encompasses face and limb transplantation, but as with organ transplantation, it requires lifelong regimens of immunosuppressive drugs to prevent rejection. To achieve donor-specific immune tolerance and reduce the need for systemic immunosuppression, we developed a synthetic drug delivery system that mimics a strategy our bodies naturally use to recruit regulatory T cells (T reg ) to suppress inflammation. Specifically, a microparticle-based system engineered to release the T reg -recruiting chemokine CCL22 was used in a rodent hindlimb VCA model. These “Recruitment-MP” prolonged hindlimb allograft survival indefinitely (>200 days) and promoted donor-specific tolerance. Recruitment-MP treatment enriched T reg populations in allograft skin and draining lymph nodes and enhanced T reg function without affecting the proliferative capacity of conventional T cells. With implications for clinical translation, synthetic human CCL22 induced preferential migration of human T reg in vitro. Collectively, these results suggest that Recruitment-MP promote donor-specific immune tolerance via local enrichment of suppressive T reg .
The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4+ helper T (TH) cells and CD8+ cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of TH1 and TH2 signature cytokines, e.g., interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3. However, rather than conforming to stringent TH1 or TH2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed TH1/TH2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid TH1/TH2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients. Keywords: Single-cell transcriptomics, Single-cell proteomics, CAR-T, T cell activation
Succinate is a metabolic intermediate of the tricarboxylic acid (TCA) cycle within host cells. Succinate is also produced in large amounts during bacterial fermentation of dietary fiber. Elevated succinate levels within the gut lumen have been reported in association with microbiome disturbances (dysbiosis), as well as in patients with inflammatory bowel disease (IBD) and animal models of intestinal inflammation. Recent studies indicate that succinate can activate immune cells via its specific surface receptor, succinate receptor 1(SUCNR1), and enhance inflammation. However, the role of succinate in inflammatory processes within the gut mucosal immune system is unclear. This review includes current literature on the association of succinate with intestinal inflammation and the potential role of succinate–SUCNR1 signaling in gut immune functions.
Metabolism: Three steps to sensing your environment A simple, three-part model provides a systematic view of how cells sense sugars, lipids, amino acids and metabolic intermediates. Cells quickly and accurately perceive changes in intra- and extracellular molecules such as nutrients to respond to changing environments. Drawing on existing knowledge about AMPK and MTORC1 signaling, Yi-Ping Wang and Qun-Ying Lei at Fudan University in Shanghai propose a model in which three components: a sensor, transducer and effector enable metabolic sensing and signaling to proceed. The sensor detects the metabolite, and, through conjugation, conformational changes or protein–protein interactions, transmits this information to the transducer, which decides the appropriate response. The transducer then issues orders to effector proteins which coordinate the action. The future identification of novel metabolic sensors through systematic screening could lead to new therapeutic interventions for metabolic and age-related diseases.
Effectively activating macrophages that can ‘eat’ cancer cells is challenging. In particular, cancer cells secrete macrophage colony stimulating factor (MCSF), which polarizes tumour-associated macrophages from an antitumour M1 phenotype to a pro-tumorigenic M2 phenotype. Also, cancer cells can express CD47, a ‘don’t eat me’ signal that ligates with the signal regulatory protein alpha (SIRPα) receptor on macrophages to prevent phagocytosis. Here, we show that a supramolecular assembly consisting of amphiphiles inhibiting the colony stimulating factor 1 receptor (CSF-1R) and displaying SIRPα-blocking antibodies with a drug-to-antibody ratio of 17,000 can disable both mechanisms. The supramolecule homes onto SIRPα on macrophages, blocking the CD47–SIRPα signalling axis while sustainedly inhibiting CSF-1R. The supramolecule enhances M2-to-M1 repolarization within the tumour microenvironment, and significantly improves antitumour and antimetastatic efficacies in two aggressive animal models of melanoma and breast cancer, with respect to clinically available small-molecule and biologic inhibitors of CSF-1R signalling. Simultaneously blocking the CD47–SIRPα and MCSF–CSF-1R signalling axes may constitute a promising immunotherapy.
The tricarboxylic acid (TCA) cycle is a central route for oxidative phosphorylation in cells, and fulfills their bioenergetic, biosynthetic, and redox balance requirements. Despite early dogma that cancer cells bypass the TCA cycle and primarily utilize aerobic glycolysis, emerging evidence demonstrates that certain cancer cells, especially those with deregulated oncogene and tumor suppressor expression, rely heavily on the TCA cycle for energy production and macromolecule synthesis. As the field progresses, the importance of aberrant TCA cycle function in tumorigenesis and the potentials of applying small molecule inhibitors to perturb the enhanced cycle function for cancer treatment start to evolve. In this review, we summarize current knowledge about the fuels feeding the cycle, effects of oncogenes and tumor suppressors on fuel and cycle usage, common genetic alterations and deregulation of cycle enzymes, and potential therapeutic opportunities for targeting the TCA cycle in cancer cells. With the application of advanced technology and in vivo model organism studies, it is our hope that studies of this previously overlooked biochemical hub will provide fresh insights into cancer metabolism and tumorigenesis, subsequently revealing vulnerabilities for therapeutic interventions in various cancer types.
One hundred years have passed since the death of Élie Metchnikoff (1845–1916). He was the first to observe the uptake of particles by cells and realized the importance of this process for the host response to injury and infection. He also was a strong advocate of the role of phagocytosis in cellular immunity, and with this he gave us the basis for our modern understanding of inflammation and the innate and acquired immune responses. Phagocytosis is an elegant but complex process for the ingestion and elimination of pathogens, but it is also important for the elimination of apoptotic cells and hence fundamental for tissue homeostasis. Phagocytosis can be divided into four main steps: (i) recognition of the target particle, (ii) signaling to activate the internalization machinery, (iii) phagosome formation, and (iv) phagolysosome maturation. In recent years, the use of new tools of molecular biology and microscopy has provided new insights into the cellular mechanisms of phagocytosis. In this review, we present a general view of our current knowledge on phagocytosis. We emphasize novel molecular findings, particularly on phagosome formation and maturation, and discuss aspects that remain incompletely understood.
Recent clinical trials have demonstrated the ability to durably control cancer in some patients by manipulating T lymphocytes. These immunotherapies are revolutionizing cancer treatment but benefit only a minority of patients. It is thus a crucial time for clinicians, cancer scientists and immunologists to determine the next steps in shifting cancer treatment towards better cancer control. This Review describes recent advances in our understanding of tumour-associated myeloid cells. These cells remain less studied than T lymphocytes but have attracted particular attention because their presence in tumours is often linked to altered patient survival. Also, experimental studies indicate that myeloid cells modulate key cancer-associated activities, including immune evasion, and affect virtually all types of cancer therapy. Consequently, targeting myeloid cells could overcome limitations of current treatment options.
Cancer is a disease of ageing. Clinically, aged cancer patients tend to have a poorer prognosis than young. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumour progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression, we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. Here we find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signalling cascade in melanoma cells that results in a decrease in β-catenin and microphthalmia-associated transcription factor (MITF), and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to DNA damage induced by reactive oxygen species, rendering the cells more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumour progression, offering new possibilities for the design of therapy for the elderly.
Experimental vaccine adjuvants are being designed to target specific toll-like receptors (TLRs) alone or in combination, expressed by antigen presenting cells, notably dendritic cells (DCs). There is a need for high-content screening (HCS) platforms to explore how DC activation is affected by adjuvant combinations. Presented is a cell-based microarray approach, “immunoarray”, exposing DCs to a large number of adjuvant combinations. Microparticles encapsulating TLR ligands are printed onto arrays in a range of doses for each ligand, in all possible dose combinations. Dendritic cells are then co-localized with physisorbed microparticles on the immunoarray, adherent to isolated islands surrounded by a non-fouling background, and DC activation is quantified. Delivery of individual TLR ligands was capable of eliciting high levels of specific DC activation markers. For example, either TLR9 ligand, CpG, or TLR3 ligand, poly I:C, was capable of inducing among the highest 10% expression levels of CD86. In contrast, MHC-II expression in response to TLR4 agonist MPLA was among the highest, whereas either MPLA or poly I:C, was capable of producing among the highest levels of CCR7 expression, as well as inflammatory cytokine IL-12. However, in order to produce robust responses across all activation markers, adjuvant combinations were required, and combinations were more represented among the high responders. The immunoarray also enables investigation of interactions between adjuvants, and each TLR ligand suggested antagonism to other ligands, for various markers. Altogether, this work demonstrates feasibility of the immunoarray platform to screen microparticle-encapsulated adjuvant combinations for the development of improved and personalized vaccines.
Activation of macrophages and dendritic cells (DCs) by pro-inflammatory stimuli causes them to undergo a metabolic switch towards glycolysis and away from oxidative phosphorylation (OXPHOS), similar to the Warburg effect in tumors. However, it is only recently that the mechanisms responsible for this metabolic reprogramming have been elucidated in more detail. The transcription factor hypoxia-inducible factor-1α (HIF-1α) plays an important role under conditions of both hypoxia and normoxia. The withdrawal of citrate from the tricarboxylic acid (TCA) cycle has been shown to be critical for lipid biosynthesis in both macrophages and DCs. Interference with this process actually abolishes the ability of DCs to activate T cells. Another TCA cycle intermediate, succinate, activates HIF-1α and promotes inflammatory gene expression. These new insights are providing us with a deeper understanding of the role of metabolic reprogramming in innate immunity.Cell Research advance online publication 5 June 2015; doi: 10.1038/cr.2015.68.
Macrophage infiltration has been identified as an independent poor prognostic factor in several cancer types. The major survival factor for these macrophages is macrophage colony-stimulating factor 1 (CSF-1). We generated a monoclonal antibody (RG7155) that inhibits CSF-1 receptor (CSF-1R) activation. In vitro RG7155 treatment results in cell death of CSF-1-differentiated macrophages. In animal models, CSF-1R inhibition strongly reduces F4/80(+) tumor-associated macrophages accompanied by an increase of the CD8(+)/CD4(+) T cell ratio. Administration of RG7155 to patients led to striking reductions of CSF-1R(+)CD163(+) macrophages in tumor tissues, which translated into clinical objective responses in diffuse-type giant cell tumor (Dt-GCT) patients.
T helper 17 (Th17) cells can give rise to interleukin-17A (IL-17A)- and interferon (IFN)-γ-double-producing cells that are implicated in development of autoimmune diseases. However, the molecular mechanisms that govern generation of IFN-γ-producing Th17 cells are unclear. We found that coexpression of the Th1 and Th17 cell master transcription factors, T-bet and retinoid-related orphan receptor gamma-t (RORγt), respectively, did not generate Th cells with robust IL-17 and IFN-γ expression. Instead, development of IFN-γ-producing Th17 cells required T-bet and Runx1 or Runx3. IL-12-stimulated Th17 cells upregulated Runx1, which bound to the Ifng locus in a T-bet-dependent manner. Reciprocally, T-bet or Runx1 deficiency or inhibition of Runx transcriptional activity impaired the development of IFN-γ-producing Th17 cells during experimental autoimmune encephalomyelitis, which correlated with substantially ameliorated disease course. Thus, our studies identify a critical role for T-bet and Runx transcription factors in the generation of pathogenic IFN-γ-producing Th17 cells.
Macrophages activated by the Gram-negative bacterial product lipopolysaccharide switch their core metabolism from oxidative phosphorylation to glycolysis. Here we show that inhibition of glycolysis with 2-deoxyglucose suppresses lipopolysaccharide-induced interleukin-1β but not tumour-necrosis factor-α in mouse macrophages. A comprehensive metabolic map of lipopolysaccharide-activated macrophages shows upregulation of glycolytic and downregulation of mitochondrial genes, which correlates directly with the expression profiles of altered metabolites. Lipopolysaccharide strongly increases the levels of the tricarboxylic-acid cycle intermediate succinate. Glutamine-dependent anerplerosis is the principal source of succinate, although the 'GABA (γ-aminobutyric acid) shunt' pathway also has a role. Lipopolysaccharide-induced succinate stabilizes hypoxia-inducible factor-1α, an effect that is inhibited by 2-deoxyglucose, with interleukin-1β as an important target. Lipopolysaccharide also increases succinylation of several proteins. We therefore identify succinate as a metabolite in innate immune signalling, which enhances interleukin-1β production during inflammation.
Upon stimulation with different cytokines, macrophages can undergo classical or alternative activation to become M1 or M2 macrophages. Alternatively activated (or M2) macrophages are defined by their expression of specific gene products and play an important role in containing inflammation, removing apoptotic cells and repairing tissue damage. Whereas it is well-established that IL-4 can drive alternative activation, if lack of TGFβ signaling at physiological levels affects M2 polarization has not been addressed.
Vav1-Cre x TβRIIfx/fx mice, lacking TβRII function in hematopoietic cells, exhibited uncontrolled pulmonary inflammation and developed a lethal autoimmune syndrome at young age. This was accompanied by significantly increased numbers of splenic neutrophils and T cells as well as elevated hepatic macrophage infiltration and bone marrow monocyte counts. TβRII-/- CD4+ and CD8+ T-cells in the lymph nodes and spleen expressed increased cell surface CD44, and CD69 was also higher on CD4+ lymph node T-cells. Loss of TβRII in bone marrow-derived macrophages (BMDMs) did not affect the ability of these cells to perform efferocytosis. However, these cells were defective in basal and IL-4-induced arg1 mRNA and Arginase-1 protein production. Moreover, the transcription of genes that are typically upregulated in M2-polarized macrophages, such as ym1, mcr2 and mgl2, was also decreased in peritoneal macrophages and IL-4-stimulated TβRII-/- BMDMs. We found that cell surface and mRNA expression of Galectin-3, which also regulates M2 macrophage polarization, was lower in TβRII-/- BMDMs. Very interestingly, the impaired ability of these null mutant BMDMs to differentiate into IL-4 polarized macrophages was Stat6- and Smad3-independent, but correlated with reduced levels of phospho-Akt and β-catenin.
Our results establish a novel biological role for TGFβ signaling in controlling expression of genes characteristic for alternatively activated macrophages. We speculate that lack of TβRII signaling reduces the anti-inflammatory M2 phenotype of macrophages because of reduced expression of these products. This would cause defects in the ability of the M2 macrophages to negatively regulate other immune cells such as T-cells in the lung, possibly explaining the systemic inflammation observed in Vav1-Cre x TβRIIfx/fx mice.
While it is well-known that adsorbed proteins on implanted biomaterials modulate inflammatory responses, modulation of dendritic cells (DCs) via adhesion-dependent signaling has only been begun to be characterized. In this work, we demonstrate that adhesive substrates elicit differential DC maturation and adaptive immune responses. We find that adhesive substrates support similar levels of DC adhesion and expression of stimulatory and co-stimulatory molecules. Conversely, DC morphology and differential production of pro- and anti-inflammatory cytokines (IL-12p40 and IL-10, respectively) is adhesive substrate-dependent. For example, DCs cultured on collagen and vitronectin substrates generate higher levels of IL-12p40, whereas DCs cultured on albumin and serum-coated tissue culture-treated substrates produce the higher levels of IL-10 compared to other substrates. Additionally, our results suggest substrate-dependent trends in DC-mediated allogeneic CD4+ T-cell proliferation and T-helper cell type responses. Specifically, we show that substrate-dependent modulation of DC IL-12p40 cytokine production correlates with CD4+ T-cell proliferation and Th1 type response in terms of IFN-γ producing T-helper cells. Furthermore, our results suggest substrate-dependent trends in DC-mediated stimulation of IL-4 producing T-cells, but this Th2 type response is not dependent on DC production of IL-10 cytokine. This work has impact in the rational design of biomaterials for diverse applications such as tissue-engineered constructs, synthetic particle-based vaccines and the ex vivo culture of DCs for immunotherapies.
Myeloid cells are the most abundant nucleated haematopoietic cells in the human body and are a collection of distinct cell populations with many diverse functions. The three groups of terminally differentiated myeloid cells - macrophages, dendritic cells and granulocytes - are essential for the normal function of both the innate and adaptive immune systems. Mounting evidence indicates that the tumour microenvironment alters myeloid cells and can convert them into potent immunosuppressive cells. Here, we consider myeloid cells as an intricately connected, complex, single system and we focus on how tumours manipulate the myeloid system to evade the host immune response.
Aging affects many aspects of the cellular function of macrophages. Macrophages play a critical role in innate immunity, acting as sentinels to fight pathogens, promoting wound healing, and orchestrating the development of the specific acquired immune response. However, little is known about how age influences the ability of macrophage to change phenotypes in response to environmental factors. This study examined the age-associated defects on macrophage polarization toward a pro-inflammatory (M1) or an anti-inflammatory (M2) phenotype. Adherent splenocytes enriched for macrophages were cultured with or without lipopolysaccharide (LPS), a combination of interferon (IFN)-γ and tumor necrosis factor (TNF)-α or interleukin (IL)-4. A panel of M1 markers, inducible nitric oxide synthase (iNOS), IL-6, IL-1β, and TNF-α, and M2 markers, including arginase-1 (Arg1), Ym1, and Found In Inflammatory Zone 1 (FIZZ1), were analyzed. IL-6 mRNA in cells from aged mice was decreased by 78% and 58% compared with young after stimulation with LPS or IFN-γ and TNF-α (P<0.05), respectively. Also, there was a marked reduction in the induced levels of iNOS, IL-1β, and TNF-α in cells from aged mice relative to young controls. Similarly, IL-4 exposure resulted in a reduction of M2 markers in adherent splenocytes from aged mice compared with younger animals. This was consistent with a 28% decrease in splenic F4/80(+)IL-4R(+) cells in aged mice relative to controls, although IL-4R expression on these cells did not vary between age groups. In contrast, levels of M1 and most M2 markers, save for FIZZ1, in bone marrow-derived macrophages were similar between the age groups, irrespective of stimuli. These data imply that impaired macrophage polarization in the elderly may dysregulate the development of the host response, making them more susceptible to infectious diseases and that the aging microenvironment may be a key modulator of these macrophage-elicited responses.
It is known that adsorbed adhesive proteins on implanted biomaterials modulate inflammatory responses; however, modulation of dendritic cell (DC) responses upon interaction with adhesive proteins has only begun to be characterized. DCs are specialized antigen-presenting cells that modulate both innate and adaptive immune responses. Previously we have shown that the activation and stimulatory capacity of DCs derived from C57BL6/j mice is differentially modulated by adhesive substrates. Here we extend our investigation of adhesive substrate modulation of DC responses to consider the case where the DCs had maturational defects associated with diabetes. Understanding the adhesive responses of DCs in diabetics is potentially important for immunotherapy and tissue engineering applications. In this work we use the non-obese diabetic (NOD) mouse, an established animal model for type 1 diabetes, to generate DCs (NOD-DCs). We demonstrate that NOD-DCs cultured on different adhesive substrates (collagen, fibrinogen, fibronectin, laminin, vitronectin, albumin and serum) respond with substrate-dependent modulation of the surface expression of the stimulatory molecule MHC-II and the co-stimulatory molecules CD80 and CD86 and production of the cytokines IL-12p40 and IL-10. Furthermore, the capacity of NOD-DCs to stimulate CD4(+) T-cell proliferation and cytokine production (IL-4 and IFN-γ) showed substrate-dependent modulation. Specifically, NOD-DCs cultured on vitronectin induced the highest IL-12p40 production, whereas collagen induced the highest IL-10 production. Dendritic cells cultured on collagen, fibrinogen and serum-coated substrates stimulated the highest CD4(+) T-cell proliferation. It was further determined that DCs cultured on vitronectin induced the highest percent population of IL-4-producing T-cells and DCs cultured on a fibronectin-coated substrate induced the highest expression of IFN-γ in T-cells. Pearson's correlation analysis revealed high correlations between T-cell proliferation and DC expression level of CD80 and T-cell production of IL-4 and DC production of IL-10. This demonstration of substrate-based control of NOD-DC activatory and stimulatory capacity, distinct from non-diabetic B6-DC responses, establishes the field of adhesive modulation of immune cell responses and informs the rational design of biomaterials for patients with type 1 diabetes.
Myelin-specific effector Th1 cells are able to perpetuate CNS inflammation in experimental autoimmune encephalomyelitis, an animal model representative of multiple sclerosis. Although the effects of cytokines in the CNS microenvironment on naive CD4(+) T cells have been well described, much less is known about their ability to influence Ag-experienced effector cells. TGF-beta is a multifunctioning cytokine present in the healthy and inflamed CNS with well-characterized suppressive effects on naive T cell functions. However, the effects of TGF-beta on effector Th1 cells are not well defined. Using myelin-specific TCR transgenic mice, we demonstrate that TGF-beta elicits differential effects on naive versus effector Th1 cells. TGF-beta enhances cellular activation, proliferation, and cytokine production of effector Th1 cells; however, adoptive transfer of these cells into naive mice showed a reduction in encephalitogenicity. We subsequently demonstrate that the reduced encephalitogenic capacity is due to the ability of TGF-beta to promote the self-regulation of Th1 effector cells via IL-10 production. These data demonstrate a mechanism by which TGF-beta is able to suppress the encephalitogenicity of myelin-specific Th1 effector cells that is unique from its suppression of naive T cells.
We describe a quantitative histological study of 34 breast biopsies using a marker for human macrophages, the monoclonal antibody EBM/11. Seventeen of the biopsies were of malignant tumours. Both benign and malignant breast tissue contained large numbers of macrophages with significantly higher numbers occurring in the malignant group. An analysis was made of macrophage counts according to stage, grade and prognostic index of the malignant tumours. There was no correlation between macrophage numbers and any of these parameters in malignant breast tumours. We discuss the possible reasons why some earlier studies (using other markers such as lysozyme), have shown an apparently insignificant number of intratumoral macrophages.
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Although different metabolic pathways have been associated with distinct macrophage phenotypes, the field of utilizing metabolites to modulate macrophage phenotype is in a nascent stage. In this report, we developed microparticles based on polymerization of alpha-ketoglutarate (a Krebs cycle metabolite), with or without encapsulation of spermine (a polyamine metabolite), to modulate cell phenotype that are critical for resolution of inflammation. Poly (alpha-ketoglutarate) microparticles encapsulated and released spermine (spermine (encap)paKG MPs) in vitro, which was accelerated in an acidic environment. When delivered to bone marrow-derived-macrophages, spermine (encap)paKG MPs induced a complex phenotypic profile outside of the typical M1/M2 paradigm, with distinct effects in the presence or absence of the pro-inflammatory stimulus lipopolysaccharide. Of particular interest was the increase in expression of CD163, which has been linked to anti-inflammatory responses in sepsis. Therefore, we systemically administered spermine (encap)paKG MPs to two different murine models of sepsis using acute or chronic injection of LPS. Macrophages and neutrophils in the liver and spleen of animals treated with spermine (encap)paKG MPs increased expression of CD163, concomitant with normalizing of glycolysis and oxidative phosphorylation, in both models. Overall, these results show that spermine (encap)paKG MPs modulate macrophage phenotype in vitro and in vivo, with potential applications in inflammation-associated diseases.
Activated effector T cells induce pro-inflammatory responses in rheumatoid arthritis (RA) which then lead to inflammation of the joints. In this report, we demonstrate that polymeric nanoparticles with alpha keto-glutarate (aKG) in their polymer backbone (termed as paKG NPs) modulate T cell responses in vitro and in vivo. Impressively, a low dose of only three administrations of methotrexate, a clinically and chronically administered drug for RA, in conjunction with two doses of paKG NPs, reversed arthritis symptoms in collagen-induced arthritis (CIA) mice. This was further followed by significant decreases in pro-inflammatory antigen-specific T helper type 17 (TH17) responses and a significant increase in anti-inflammatory regulatory T cell (TREG) responses when CIA treated splenic cells were isolated and re-exposed to the CIA self-antigen. Overall, this study supports the concurrent and short term, low dose of paKG NPs and methotrexate for the reversal of RA symptoms.
Dendritic cells (DCs) rely on glycolysis for their energy needs to induce pro-inflammatory antigen-specific immune responses. Therefore, inhibiting DC glycolysis, while presenting the self-antigen, may prevent pro-inflammatory antigen-specific immune responses. Previously we demonstrated that microparticles with alpha-ketoglutarate (aKG) in the polymer backbone (paKG MPs) were able to generate anti-inflammatory DCs by sustained delivery of the aKG metabolite, and by modulating energy metabolism of DCs. Herein, we demonstrate that paKG MPs-based delivery of a glycolytic inhibitor, PFK15, using paKG MPs induces anti-inflammatory DCs (CD86LoMHCII⁺) by down-regulating glycolysis, CD86, tnf and IL-6 genes, while upregulating oxidative phosphorylation (OXPHOS) and mitochondrial genes. Furthermore, paKG MPs delivering PFK15 and a self-antigen, collagen type II (bc2), in vivo, in a collagen-induced autoimmune arthritis (CIA) mouse model, normalized paw inflammation and arthritis score, by generating antigen-specific immune responses. Specifically, these formulations were able to reduce activation of DCs in draining lymph nodes and impressively generated proliferating bc2-specific anti-inflammatory regulatory T cells in joint-associated popliteal lymph nodes. These data strongly suggest that sustained glycolytic inhibition of DCs in the presence of an antigen can induce antigen-specific immunosuppressive responses, therefore, generating a technology that can be applicable for treating autoimmune diseases.
Metabolites control immune cell functions, and delivery of these metabolites in a sustained manner may be able to modulate function of the immune cells . In this study, alpha-ketoglutarate (aKG) and diol based polymeric-microparticles (termed paKG MPs) were synthesized to provide sustained release of aKG and promote an immunosuppressive cellular phenotype. Notably, after association with dendritic cells (DCs), paKG MPs modulated the intracellular metabolic-profile/pathways, and decreased glycolysis and mitochondrial respiration in vitro. These metabolic changes resulted in modulation of MHC-II, CD86 expression in DCs, and altered the frequency of regulatory T cells (Tregs), and T-helper type-1/2/17 cells in vitro. This unique strategy of intracellular delivery of key-metabolites in a sustaine manner provide a paradigm-shift in the immunometabolism field-based immunotherapy with applications in different diseases associated with immune disorders.
Significance
Vascularized composite allotransplantation (VCA) is an emerging field that is particularly beneficial for select amputees and patients with devastating soft tissue loss that is not amenable to conventional reconstructive surgeries. As with solid organ transplantation, VCA recipients are subjected to a lifelong regimen of antirejection drugs with a well-established sequela. Herein, we report a synthetic, controlled-release microparticle system (referred to as TRI-MP) that aims to locally enrich naturally occurring, suppressive lymphocytes to prevent allograft rejection and promote tolerance. While this study exclusively focuses on VCA, this technology has implications for other conditions characterized by aberrant inflammation.
Biomaterials in regenerative medicine are designed to mimic and modulate tissue environments to promote repair. Biologic scaffolds (derived from decellularized tissue extracellular matrix) promote a wound-healing (proregenerative) immune phenotype and are used clinically to treat tissue loss, including in the context of tumor resection. It is unknown whether a biomaterial microenvironment that encourages tissue formation may also promote tumor development. We implanted a urinary bladder matrix (UBM) scaffold, which is used clinically for wound management, with syngeneic cancer cell lines in mice to study how wound-healing immune responses affect tumor formation and sensitivity to immune checkpoint blockade. The UBM scaffold created an immune microenvironment that inhibited B16-F10 melanoma tumor formation in a CD4 ⁺ T cell–dependent and macrophage-dependent manner. In-depth immune characterization revealed an activated type 2–like immune response that was distinct from the classical tumor microenvironment, including activated type 2 T helper T cells, a unique macrophage phenotype, eosinophil infiltration, angiogenic factors, and complement. Tumor growth inhibition by PD-1 and PD-L1 checkpoint blockade was potentiated in the UBM scaffold immune microenvironment. Engineering the local tumor microenvironment to promote a type 2 wound-healing immune signature may serve as a therapeutic target to improve immunotherapy efficacy.
Purpose: We have shown that the aged microenvironment increases melanoma metastasis, and decreases response to targeted therapy, and here we queried response to anti-PD1.
Experimental Design: We analyzed the relationship between age, response to anti-PD1, and prior therapy in 538 patients. We used mouse models of melanoma, to analyze the intratumoral immune microenvironment in young versus aged mice and confirmed our findings in human melanoma biopsies.
Results: Patients over the age of 60 responded more efficiently to anti-PD-1, and likelihood of response to anti-PD-1 increased with age, even when we controlled for prior MAPKi therapy. Placing genetically identical tumors in aged mice (52 weeks) significantly increased their response to anti-PD1 as compared with the same tumors in young mice (8 weeks). These data suggest that this increased response in aged patients occurs even in the absence of a more complex mutational landscape. Next, we found that young mice had a significantly higher population of regulatory T cells (Tregs), skewing the CD8+:Treg ratio. FOXP3 staining of human melanoma biopsies revealed similar increases in Tregs in young patients. Depletion of Tregs using anti-CD25 increased the response to anti-PD1 in young mice.
Conclusions: While there are obvious limitations to our study, including our inability to conduct a meta-analysis due to a lack of available data, and our inability to control for mutational burden, there is a remarkable consistency in these data from over 500 patients across 8 different institutes worldwide. These results stress the importance of considering age as a factor for immunotherapy response. Clin Cancer Res; 24(21); 5347–56. ©2018 AACR.
See related commentary by Pawelec, p. 5193
The clinical successes in immunotherapy have been both astounding and at the same time unsatisfactory. Countless patients with varied tumor types have seen pronounced clinical response with immunotherapeutic intervention; however, many more patients have experienced minimal or no clinical benefit when provided the same treatment. As technology has advanced, so has the understanding of the complexity and diversity of the immune context of the tumor microenvironment and its influence on response to therapy. It has been possible to identify different subclasses of immune environment that have an influence on tumor initiation and response and therapy; by parsing the unique classes and subclasses of tumor immune microenvironment (TIME) that exist within a patient's tumor, the ability to predict and guide immunotherapeutic responsiveness will improve, and new therapeutic targets will be revealed.
Metabolic reprogramming contributes to tumor development and introduces metabolic liabilities that can be exploited to treat cancer. Chemotherapies targeting metabolism have been effective cancer treatments for decades, and the success of these therapies demonstrates that a therapeutic window exists to target malignant metabolism. New insights into the differential metabolic dependencies of tumors have provided novel therapeutic strategies to exploit altered metabolism, some of which are being evaluated in preclinical models or clinical trials. Here, we review our current understanding of cancer metabolism and discuss how this might guide treatments targeting the metabolic requirements of tumor cells.
As tumors employ complementary overlapping and/or independent mechanisms to evade immune surveillance, many emerging cancer immunotherapies attempt to target multiple pathways to eradicate malignant cells. Although modulation of independent pathways by simultaneous administration of multiple immune modulators (e.g., checkpoint inhibitors, cytokines, and growth factors) has shown great promise, the clinical impact remains limited due to severe toxicity associated with high systemic levels of many of these drugs. Therefore, novel platforms for efficient delivery of multi-component therapies at lower effective doses would be enabling. Here, a drug delivery platform called immunomodulatory molecule delivery system (iMods), which provides sustained extracellular delivery of a checkpoint inhibitor (anti-PD-L1) and simultaneously, targeted intracellular delivery of a tumor antigen (OVA) along with adjuvant (poly(I:C)), and the indoleamine deoxygenase inhibitor 1-MT is described. In melanoma tumor-bearing mice, combinatorial delivery of these factors with iMods leads to regression of both treated and untreated (contralateral) melanoma tumors and 100% survival. These promising therapeutic outcomes are attributed to significantly enhanced ratios of anti-tumor CD8 T-cell/tumor-protective regulatory T-cell (Treg) in tumors and tumor draining lymph nodes. Overall, the iMods delivery platform described here represents a promising advance in multi-factor cancer immunotherapy.
Disease phenotypes of pulmonary sarcoidosis are distinguished by clinical rather than immunological criteria. We aimed to characterise patterns of CD4 ⁺ T-cell lineage plasticity underlying the differences in clinical presentation and disease course between the acute form, Löfgren's syndrome, and the heterogeneous, potentially progressive “non-Löfgren” form.
33 pulmonary sarcoidosis patients and nine controls underwent bronchoscopy with bronchoalveolar lavage. CD4 ⁺ T-cell transcription factor, chemokine receptor and T-cell receptor expression, proliferation and cytokine production were assessed in the lavage fluid and peripheral blood using flow cytometry and multicolour FluoroSpot.
CD4 ⁺ T-cells simultaneously expressing the T-helper cell (Th)1 and Th17 transcriptional regulators T-bet and RORγT (T-bet ⁺ RORγT ⁺ ) were identified in the lavage, but not blood, of all subjects, and to a significantly higher degree in Löfgren's patients. T-bet ⁺ RORγT ⁺ cells proliferated actively, produced interferon (IFN)γ and interleukin (IL)-17A, co-expressed the chemokine receptors CXCR3 and CCR6, and correlated with nonchronic disease. T-cell receptor-restricted Vα2.3 ⁺ Vβ22 ⁺ T-cells strongly co-expressed T-bet/RORγT and CXCR3/CCR6. Cytokine production was more heterogeneous in Löfgren's patients, with significantly higher IL-17A, IL-10, IL-22 and IL-2, but lower IFNγ.
Here we demonstrate the presence of lung T-bet ⁺ RORγT ⁺ CXCR3 ⁺ CCR6 ⁺ CD4 ⁺ T-cells and Th17-associated cytokines especially in sarcoidosis patients with a favourable prognosis, suggesting a Th1/Th17-permissive environment in the lung with implications for disease resolution.
The tumor microenvironment is a complex ecology of cells that evolves with and provides support to tumor cells during the transition to malignancy. Among the innate and adaptive immune cells recruited to the tumor site, macrophages are particularly abundant and are present at all stages of tumor progression. Clinical studies and experimental mouse models indicate that these macrophages generally play a protumoral role. In the primary tumor, macrophages can stimulate angiogenesis and enhance tumor cell invasion, motility, and intravasation. During monocytes and/or metastasis, macrophages prime the premetastatic site and promote tumor cell extravasation, survival, and persistent growth. Macrophages are also immunosuppressive, preventing tumor cell attack by natural killer and T cells during tumor progression and after recovery from chemo- or immunotherapy. Therapeutic success in targeting these protumoral roles in preclinical models and in early clinical trials suggests that macrophages are attractive targets as part of combination therapy in cancer treatment.
Succinate is an intermediate of the tricarboxylic acid (TCA) cycle, and plays a crucial role in adenosine triphosphate (ATP) generation in mitochondria. Recently, new roles for succinate outside metabolism have emerged. Succinate stabilizes the transcription factor hypoxia-inducible factor-1α (HIF-1α) in specific tumors and in activated macrophages, and stimulates dendritic cells via its receptor succinate receptor 1. Furthermore, succinate has been shown to post-translationally modify proteins. This expanding repertoire of functions for succinate suggests a broader role in cellular activation. We review the new roles of succinate and draw parallels to other metabolites such as NAD(+) and citrate whose roles have expanded beyond metabolism and into signaling.
As we age, the innate immune system becomes dysregulated and is characterized by persistent inflammatory responses that involve multiple immune and non-immune cell types and that vary depending on the cell activation state and tissue context. This ageing-associated basal inflammation, particularly in humans, is thought to be induced by several factors, including the reactivation of latent viral infections and the release of endogenous damage-associated ligands of pattern recognition receptors (PRRs). Innate immune cell functions that are required to respond to pathogens or vaccines, such as cell migration and PRR signalling, are also impaired in aged individuals. This immune dysregulation may affect conditions associated with chronic inflammation, such as atherosclerosis and Alzheimer's disease.
Inflammatory responses play decisive roles at different stages of tumor development, including initiation, promotion, malignant conversion, invasion, and metastasis. Inflammation also affects immune surveillance and responses to therapy. Immune cells that infiltrate tumors engage in an extensive and dynamic crosstalk with cancer cells, and some of the molecular events that mediate this dialog have been revealed. This review outlines the principal mechanisms that govern the effects of inflammation and immunity on tumor development and discusses attractive new targets for cancer therapy and prevention.
Metazoan organisms are dependent on a continuous supply of O(2) for survival. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates oxygen homeostasis and plays key roles in development, physiology, and disease. HIF-1 activity is induced in response to continuous hypoxia, intermittent hypoxia, growth factor stimulation, and Ca(2+) signaling. HIF-1 mediates adaptive responses to hypoxia, including erythropoiesis, angiogenesis, and metabolic reprogramming. In each case, HIF-1 regulates the expression of multiple genes encoding key components of the response pathway. HIF-1 also mediates maladaptive responses to chronic continuous and intermittent hypoxia, which underlie the development of pulmonary and systemic hypertension, respectively.
Macrophages within the tumor microenvironment facilitate angiogenesis and extracellular-matrix breakdown and remodeling and promote tumor cell motility. Recent studies reveal that direct communication between macrophages and tumor cells leads to invasion and egress of tumor cells into the blood vessels (intravasation). Thus, macrophages are at the center of the invasion microenvironment and are an important drug target for cancer therapy.
While tumour incidence is known to augment with age, paradoxically tumour growth and metastasis were often found to proceed at a slower rate at late ages. This age-related biological behaviour of tumours actually imposes a differential therapeutic approach to the old cancer patient. Several mechanisms of the age-related reduced tumour progression have been demonstrated: decreased tumour cell proliferation, increased apoptotic cell death, decreased angiogenesis and anti-tumoural immune response changes. We postulated that it might be possible to design age-adjusted treatment modalities based on the mechanisms responsible for the reduced tumour progression rate in the aged. Based on these mechanisms, we compared the effect of different treatments (apoptosis-inducing agents, Hydrocortisone and Adriamycin, anti-angiogenic agent, TNP-470, and immunomodulators-Levamisole and BCG) on two experimental tumours (B16 melanoma and AKR lymphoma) growing in young and old mice. Most treatments showed, in both tumours, a higher inhibitory effect on tumours growing in old mice than on those developing in young ones, to our knowledge, a feature not described before for anti-tumoural agents. We suggest that designing ageing conditions in tumours of young patients might possibly alleviate neoplastic aggressiveness in these patients as well.