Engineered Tumor-Immune Microenvironment On A Chip to Study T Cell-Macrophage Interaction in Breast Cancer Progression.

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.

Inverse-Vaccines for Rheumatoid Arthritis Re-establish Metabolic and Immunological Homeostasis in Joint Tissues.

Rheumatoid arthritis (RA) causes immunological and metabolic imbalances in tissue, exacerbating inflammation in affected joints. Changes in immunological and metabolic tissue homeostasis at different stages of RA are not well understood. Herein, the changes in the immunological and metabolic profiles in different stages in collagen induced arthritis (CIA), namely, early, intermediate, and late stage is examined. Moreover, the efficacy of the inverse-vaccine, paKG(PFK15+bc2) microparticle, to restore tissue homeostasis at different stages is also investigated. Immunological analyses of inverse-vaccine-treated group revealed a significant decrease in the activation of pro-inflammatory immune cells and remarkable increase in regulatory T-cell populations in the intermediate and late stages compared to no treatment. Also, glycolysis in the spleen is normalized in the late stages of CIA in inverse-vaccine-treated mice, which is similar to no-disease tissues. Metabolomics analyses revealed that metabolites UDP-glucuronic acid and L-Glutathione oxidized are significantly altered between treatment groups, and thus might provide new druggable targets for RA treatment. Flux metabolic modeling identified amino acid and carnitine pathways as the central pathways affected in arthritic tissue with CIA progression. Overall, this study shows that the inverse-vaccines initiate early re-establishment of homeostasis, which persists through the disease span.

Immune response differences in degradable and non-degradable alloy implants.

Alloy based implants have made a great impact in the clinic and in preclinical research. Immune responses are one of the major causes of failure of these implants in the clinic. Although the immune responses toward non-degradable alloy implants are well documented, there is a poor understanding of the immune responses against degradable alloy implants. Recently, there have been several reports suggesting that degradable implants may develop substantial immune responses. This phenomenon needs to be further studied in detail to make the case for the degradable implants to be utilized in clinics. Herein, we review these new recent reports suggesting the role of innate and potentially adaptive immune cells in inducing immune responses against degradable implants. First, we discussed immune responses to allergen components of non-degradable implants to give a better overview on differences in the immune response between non-degradable and degradable implants. Furthermore, we also provide potential areas of research that can be undertaken that may shed light on the local and global immune responses that are generated in response to degradable implants.

Vaccines prevent reinduction of rheumatoid arthritis symptoms in collagen-induced arthritis mouse model.

Metabolic reprogramming of immune cells modulates their function and reduces the severity of autoimmune diseases. However, the long-term effects of the metabolically reprogrammed cells, specifically in the case of immune flare-ups, need to be examined. Herein, a re-induction rheumatoid arthritis (RA) mouse model was developed by injecting T-cells from RA mice into drug-treated mice to recapitulate the effects of T-cell-mediated inflammation and mimic immune flare-ups. Immune metabolic modulator paKG(PFK15 + bc2) microparticles (MPs) were shown to reduce clinical symptoms of RA in collagen-induced arthritis (CIA) mice. Upon re-induction, a significant delay in the reappearance of clinical symptoms in the paKG(PFK15 + bc2) microparticle treatment group was observed as compared to equal or higher doses of the clinically utilized U.S. Food and Drug Administration (FDA)-approved drug, Methotrexate (MTX). Furthermore, paKG(PFK15 + bc2) microparticle-treated mice were able to lower activated dendritic cells (DCs) and inflammatory T helper cell 1 (TH1) and increased activated, proliferating regulatory T-cells (Tregs) more effectively than MTX. The paKG(PFK15 + bc2) microparticles also led to a significant reduction in paw inflammation in mice as compared to MTX treatment. This study can pave the way for the development of flare-up mouse models and antigen-specific drug treatments.

Succinate in the tumor microenvironment affects tumor growth and modulates tumor associated macrophages.

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.

Exponentially decreasing exposure of antigen generates anti-inflammatory T-cell responses.

Rheumatoid Arthritis (RA) is a chronic debilitating disease characterized by auto-immune reaction towards self-antigen such as collagen type II. In this study, we investigated the impact of exponentially decreasing levels of antigen exposure on pro-inflammatory T cell responses in the collagen-induced arthritis (CIA) mouse model. Using a controlled delivery experimental approach, we manipulated the collagen type II (CII) antigen concentration presented to the immune system. We observed that exponentially decreasing levels of antigen generated reduced pro-inflammatory T cell responses in secondary lymphoid organs in mice suffering from RA. Specifically, untreated mice exhibited robust pro-inflammatory T cell activation and increased paw inflammation, whereas, mice exposed to exponentially decreasing concentrations of CII demonstrated significantly reduced pro-inflammatory T cell responses, exhibited lower levels of paw inflammation, and decreased arthritis scores in right rear paw. The data also demonstrate that the decreasing antigen levels promoted the induction of regulatory T cells (Tregs), which play a crucial role in maintaining immune tolerance and suppressing excessive inflammatory responses. Our findings highlight the importance of antigen concentration in modulating pro-inflammatory T cell responses in the CIA model. These results provide valuable insights into the potential therapeutic strategies that target antigen presentation to regulate immune responses and mitigate inflammation in rheumatoid arthritis and other autoimmune diseases. Further investigations are warranted to elucidate the specific mechanisms underlying the antigen concentration-dependent modulation of T cell responses and to explore the translational potential of this approach for the development of novel therapeutic interventions in autoimmune disorders.

Succinate based polymers drive immunometabolism in dendritic cells to generate cancer immunotherapy.

Boosting the metabolism of immune cells while restricting cancer cell metabolism is challenging. Herein, we report that using biomaterials for the controlled delivery of succinate metabolite to phagocytic immune cells activates them and modulates their metabolism in the presence of metabolic inhibitors. In young immunocompetent mice, polymeric microparticles, with succinate incorporated in the backbone, induced strong pro-inflammatory anti-melanoma responses. Administration of poly(ethylene succinate) (PES MP)-based vaccines and glutaminase inhibitor to young immunocompetent mice with aggressive and large, established B16F10 melanoma tumors increased their survival three-fold, a result of increased cytotoxic T cells expressing RORγT (Tc17). Mechanistically, PES MPs directly modulate glutamine and glutamate metabolism, upregulate succinate receptor SUCNR1, activate antigen presenting cells through and HIF-1alpha, TNFa and TSLP-signaling pathways, and are dependent on alpha-ketoglutarate dehydrogenase for their activity, which demonstrates correlation of succinate delivery and these pathways. Overall, our findings suggest that immunometabolism-modifying PES MP strategies provide an approach for developing robust cancer immunotherapies.

Rescue of dendritic cells from glycolysis inhibition improves cancer immunotherapy in mice.

Inhibition of glycolysis in immune cells and cancer cells diminishes their activity, and thus combining immunotherapies with glycolytic inhibitors is challenging. Herein, a strategy is presented where glycolysis is inhibited in cancer cells using PFK15 (inhibitor of PFKFB3, rate-limiting step in glycolysis), while simultaneously glycolysis and function is rescued in DCs by delivery of fructose-1,6-biphosphate (F16BP, one-step downstream of PFKFB3). To demonstrate the feasibility of this strategy, vaccine formulations are generated using calcium-phosphate chemistry, that incorporate F16BP, poly(IC) as adjuvant, and phosphorylated-TRP2 peptide antigen and tested in challenging and established YUMM1.1 tumours in immunocompetent female mice. Furthermore, to test the versatility of this strategy, adoptive DC therapy is developed with formulations that incorporate F16BP, poly(IC) as adjuvant and mRNA derived from B16F10 cells as antigens in established B16F10 tumours in immunocompetent female mice. F16BP vaccine formulations rescue DCs in vitro and in vivo, significantly improve the survival of mice, and generate cytotoxic T cell (Tc) responses by elevating Tc1 and Tc17 cells within the tumour. Overall, these results demonstrate that rescuing glycolysis of DCs using metabolite-based formulations can be utilized to generate immunotherapy even in the presence of glycolytic inhibitor.

Biomaterial mediated simultaneous delivery of spermine and alpha ketoglutarate modulate metabolism and innate immune cell phenotype in sepsis mouse models.

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.

Drug delivery for metabolism targeted cancer immunotherapy.

Drug delivery vehicles have made a great impact on cancer immunotherapies in clinics and pre-clinical research. Notably, the science of delivery of cancer vaccines and immunotherapeutics, modulating immune cell functions has inspired development of several successful companies and clinical products. Interestingly, these drug delivery modalities not only modulate the function of immune cells (often quantified at the mRNA and protein levels), but also modulate the metabolism of these cells. Specifically, cancer immunotherapy often leads to activation of different immune cells such as dendritic cells, macrophages and T cells, which is driven by energy metabolism of these cells. Recently, there has been a great excitement about interventions that can directly modulate the energy metabolism of these immune cells and thus affect their function and in turn lead to a robust cancer immune response. Here we review few strategies that have been tested in clinic and pre-clinical research for generating effective metabolism-associated cancer therapies and immunotherapies.

Short term, low dose alpha-ketoglutarate based polymeric nanoparticles with methotrexate reverse rheumatoid arthritis symptoms in mice and modulate T helper cell responses.

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.