Conditional Knockout of Hypoxia-Inducible Factor 1-Alpha in Tumor-Infiltrating Neutrophils Protects against Pancreatic Ductal Adenocarcinoma.

Large numbers of neutrophils infiltrate tumors and comprise a notable component of the inflammatory tumor microenvironment. While it is established that tumor cells exhibit the Warburg effect for energy production, the contribution of the neutrophil metabolic state to tumorigenesis is unknown. Here, we investigated whether neutrophil infiltration and metabolic status promotes tumor progression in an orthotopic mouse model of pancreatic ductal adenocarcinoma (PDAC). We observed a large increase in the proportion of neutrophils in the blood and tumor upon orthotopic transplantation. Intriguingly, these tumor-infiltrating neutrophils up-regulated glycolytic factors and hypoxia-inducible factor 1-alpha (HIF-1α) expression compared to neutrophils from the bone marrow and blood of the same mouse. This enhanced glycolytic signature was also observed in human PDAC tissue samples. Strikingly, neutrophil-specific deletion of HIF-1α (HIF-1αΔNφ) significantly reduced tumor burden and improved overall survival in orthotopic transplanted mice, by converting the pro-tumorigenic neutrophil phenotype to an anti-tumorigenic phenotype. This outcome was associated with elevated reactive oxygen species production and activated natural killer cells and CD8+ cytotoxic T cells compared to littermate control mice. These data suggest a role for HIF-1α in neutrophil metabolism, which could be exploited as a target for metabolic modulation in cancer.

Targeting Glycolysis in Macrophages Confers Protection Against Pancreatic Ductal Adenocarcinoma.

Inflammation in the tumor microenvironment has been shown to promote disease progression in pancreatic ductal adenocarcinoma (PDAC); however, the role of macrophage metabolism in promoting inflammation is unclear. Using an orthotopic mouse model of PDAC, we demonstrate that macrophages from tumor-bearing mice exhibit elevated glycolysis. Macrophage-specific deletion of Glucose Transporter 1 (GLUT1) significantly reduced tumor burden, which was accompanied by increased Natural Killer and CD8+ T cell activity and suppression of the NLRP3-IL1ß inflammasome axis. Administration of mice with a GLUT1-specific inhibitor reduced tumor burden, comparable with gemcitabine, the current standard-of-care. In addition, we observe that intra-tumoral macrophages from human PDAC patients exhibit a pronounced glycolytic signature, which reliably predicts poor survival. Our data support a key role for macrophage metabolism in tumor immunity, which could be exploited to improve patient outcomes.

Integrated in silico and 3D in vitro model of macrophage migration in response to physical and chemical factors in the tumor microenvironment.

Macrophages are abundant in the tumor microenvironment (TME), serving as accomplices to cancer cells for their invasion. Studies have explored the biochemical mechanisms that drive pro-tumor macrophage functions; however the role of TME interstitial flow (IF) is often disregarded. Therefore, we developed a three-dimensional microfluidic-based model with tumor cells and macrophages to study how IF affects macrophage migration and its potential contribution to cancer invasion. The presence of either tumor cells or IF individually increased macrophage migration directedness and speed. Interestingly, there was no additive effect on macrophage migration directedness and speed under the simultaneous presence of tumor cells and IF. Further, we present an in silico model that couples chemokine-mediated signaling with mechanosensing networks to explain our in vitro observations. In our model design, we propose IL-8, CCL2, and ß-integrin as key pathways that commonly regulate various Rho GTPases. In agreement, in vitro macrophage migration remained elevated when exposed to a saturating concentration of recombinant IL-8 or CCL2 or to the co-addition of a sub-saturating concentration of both cytokines. Moreover, antibody blockade against IL-8 and/or CCL2 inhibited migration that could be restored by IF, indicating cytokine-independent mechanisms of migration induction. Importantly, we demonstrate the utility of an integrated in silico and 3D in vitro approach to aid the design of tumor-associated macrophage-based immunotherapeutic strategies.

Modulating Macrophage Phenotype by Sustained MicroRNA Delivery Improves Host-Implant Integration.

Biomedical implant failure due to the host's response remains a challenging problem. In particular, the formation of the fibrous capsule is a common barrier for the normal function of implants. Currently, there is mounting evidence indicating that the polarization state of macrophages plays an important role in effecting the foreign body reaction (FBR). This opens up a potential avenue for improving host-implant integration. Here, electrospun poly(caprolactone-co-ethyl ethylene phosphate) nanofiber scaffolds are utilized to deliver microRNAs (miRs) to induce macrophage polarization and modulate FBR. Specifically, C57BL/6 mice that are treated with M2-inducing miRs, Let-7c and miR-124, display relatively thinner fibrous capsule formation around the scaffolds at both Week 2 and 4, as compared to treatment with M1-inducing miR, Anti-Let-7c. Histological analysis shows that the density of blood vessels in the scaffolds are the highest in miR-124 treatment group, followed by Anti-Let-7c and Let-7c treatment groups. Based on immunohistochemical quantifications, these miR-encapsulated nanofiber scaffolds are useful for localized and sustained delivery of functional miRs and are able to modulate macrophage polarization during the first 2 weeks of implantation to result in significant alteration in host-implant integration at longer time points.

Targeting immune cells for cancer therapy.

Recent years have seen a renaissance in the research linking inflammation and cancer with immune cells playing a central role in smouldering inflammation in the tumor microenvironment. Diverse immune cell types infiltrate the tumor microenvironment, and the dynamic tumor-immune cell interplay gives rise to a rich milieu of cytokines and growth factors. Fundamentally, this intricate cross-talk creates the conducive condition for tumor cell proliferation, survival and metastasis. Interestingly, the prominent impact of immune cells is expounded in their contrary pro-tumoral role, as well as their potential anti-cancer cellular weaponry. The latter is known as immunotherapy, a concept born out of evidence that tumors are susceptible to immune defence and that by manipulating the immune system, tumor growth can be successfully restrained. Naturally, a deeper understanding of the multifaceted roles of various immune cell types thus contributes toward developing innovative anti-cancer strategies. Therefore, in this review we first outline the roles played by the major immune cell types, such as macrophages, neutrophils, natural killer cells, T cells and B cells. We then explain the recently-explored strategies of immunomodulation and discuss some important approaches via an immunology perspective.

Developmental Analysis of Bone Marrow Neutrophils Reveals Populations Specialized in Expansion, Trafficking, and Effector Functions.

Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses.

The Sweet Surrender: How Myeloid Cell Metabolic Plasticity Shapes the Tumor Microenvironment.

Immune cells are one of the most versatile cell types, as they can tailor their metabolic activity according to their required function. In response to diverse environmental cues, immune cells undergo metabolic reprogramming to support their differentiation, proliferation and pro-inflammatory effector functions. To meet a dramatic surge in energetic demand, immune cells rewire their metabolism to utilize aerobic glycolysis. This preferential use of glycolysis even under aerobic conditions is well established in tumor cells, and is known as the "Warburg effect." Tumor cells avidly use glucose for aerobic glycolysis, thereby creating a nutrient-starved microenvironment, outcompeting T cells for glucose, and directly inhibiting T-cell anti-tumoral effector function. Given that both immune and tumor cells use similar modes of metabolism in the tumor stroma, it is imperative to identify a therapeutic window in which immune-cell and tumor-cell glycolysis can be specifically targeted. In this review, we focus on the Warburg metabolism as well as other metabolic pathways of myeloid cells, which comprise a notable niche in the tumor environment and promote the growth and metastasis of malignant tumors. We examine how differential immune-cell activation triggers metabolic fate, and detail how this forbidding microenvironment succeeds in shutting down the vigorous anti-tumoral response. Finally, we highlight emerging therapeutic concepts that aim to target immune-cell metabolism. Improving our understanding of immunometabolism and immune-cell commitment to specific metabolic fates will help identify alternative therapeutic approaches to battle this intractable disease.

Synthetic Nanoparticles That Promote Tumor Necrosis Factor Receptor 2 Expressing Regulatory T Cells in the Lung and Resistance to Allergic Airways Inflammation.

Synthetic glycine coated 50 nm polystyrene nanoparticles (NP) (PS50G), unlike ambient NP, do not promote pulmonary inflammation, but instead, render lungs resistant to the development of allergic airway inflammation. In this study, we show that PS50G modulate the frequency and phenotype of regulatory T cells (Treg) in the lung, specifically increasing the proportion of tumor necrosis factor 2 (TNFR2) expressing Treg. Mice pre-exposed to PS50G, which were sensitized and then challenged with an allergen a month later, preferentially expanded TNFR2+Foxp3+ Treg, which further expressed enhanced levels of latency associated peptide and cytotoxic T-lymphocyte associated molecule-4. Moreover, PS50G-induced CD103+ dendritic cell activation in the lung was associated with the proliferative expansion of TNFR2+Foxp3+ Treg. These findings provide the first evidence that engineered NP can promote the selective expansion of maximally suppressing TNFR2+Foxp3+ Treg and further suggest a novel mechanism by which NP may promote healthy lung homeostasis.

Warburg metabolism in tumor-conditioned macrophages promotes metastasis in human pancreatic ductal adenocarcinoma.

Patients with pancreatic ductal adenocarcinoma (PDAC) face a clinically intractable disease with poor survival rates, attributed to exceptionally high levels of metastasis. Epithelial-to-mesenchymal transition (EMT) is pronounced at inflammatory foci within the tumor; however, the immunological mechanisms promoting tumor dissemination remain unclear. It is well established that tumors exhibit the Warburg effect, a preferential use of glycolysis for energy production, even in the presence of oxygen, to support rapid growth. We hypothesized that the metabolic pathways utilized by tumor-infiltrating macrophages are altered in PDAC, conferring a pro-metastatic phenotype. We generated tumor-conditioned macrophages in vitro, in which human peripheral blood monocytes were cultured with conditioned media generated from normal pancreatic or PDAC cell lines to obtain steady-state and tumor-associated macrophages (TAMs), respectively. Compared with steady-state macrophages, TAMs promoted vascular network formation, augmented extravasation of tumor cells out of blood vessels, and induced higher levels of EMT. TAMs exhibited a pronounced glycolytic signature in a metabolic flux assay, corresponding with elevated glycolytic gene transcript levels. Inhibiting glycolysis in TAMs with a competitive inhibitor to Hexokinase II (HK2), 2-deoxyglucose (2DG), was sufficient to disrupt this pro-metastatic phenotype, reversing the observed increases in TAM-supported angiogenesis, extravasation, and EMT. Our results indicate a key role for metabolic reprogramming of tumor-infiltrating macrophages in PDAC metastasis, and highlight the therapeutic potential of using pharmacologics to modulate these metabolic pathways.

PD-1 expression on dendritic cells suppresses CD8+ T cell function and antitumor immunity.

Programmed death one (PD-1) is a well-established co-inhibitory regulator that suppresses proliferation and cytokine production of T cells. Despite remarkable progress in delineating the functional roles of PD-1 on T lymphocytes, little is known about the regulatory role of PD-1 expressed on myeloid cells such as dendritic cells (DCs). Here, we show that CD8+ T cells can be more potently activated to secrete IL-2 and IFNγ by PD-1-deficient DCs compared to wild-type DCs. Adoptive transfer of PD-1-deficient DCs demonstrated their superior capabilities in inducing antigen-specific CD8+ T cell proliferation in vivo. In addition, we provide first evidence demonstrating the existence of peripheral blood DCs and CD11c+ tumor-infiltrating myeloid cells that co-express PD-1 in patients with hepatocellular carcinoma (HCC). The existence of PD-1-expressing HCC-infiltrating DCs (HIDCs) was further supported in a mouse model of HCC. Intratumoral transfer of PD-1-deficient DCs rendered recipient mice resistant to the growth of HCC by promoting tumor-infiltrating CD8+ effector T cells to secrete perforin and granzyme B. This novel finding provides a deeper understanding of the role of PD-1 in immune regulation and has significant implications for cancer immunotherapies targeting PD-1.