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.

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.

PP4 inhibition sensitizes ovarian cancer to NK cell-mediated cytotoxicity via STAT1 activation and inflammatory signaling.

BACKGROUND: Increased infiltration of T cells into ovarian tumors has been repeatedly shown to be predictive of enhanced patient survival. However, despite the evidence of an active immune response in ovarian cancer (OC), the frequency of responses to immune checkpoint blockade (ICB) therapy in OC is much lower than other cancer types. Recent studies have highlighted that deficiencies in the DNA damage response (DDR) can drive increased genomic instability and tumor immunogenicity, which leads to enhanced responses to ICB. Protein phosphatase 4 (PP4) is a critical regulator of the DDR; however, its potential role in antitumor immunity is currently unknown. RESULTS: Our results show that the PP4 inhibitor, fostriecin, combined with carboplatin leads to increased carboplatin sensitivity, DNA damage, and micronuclei formation. Using multiple OC cell lines, we show that PP4 inhibition or PPP4C knockdown combined with carboplatin triggers inflammatory signaling via Nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription 1 (STAT1) activation. This resulted in increased expression of the pro-inflammatory cytokines and chemokines: CCL5, CXCL10, and IL-6. In addition, IFNB1 expression was increased suggesting activation of the type I interferon response. Conditioned media from OC cells treated with the combination of PP4 inhibitor and carboplatin significantly increased migration of both CD8 T cell and natural killer (NK) cells over carboplatin treatment alone. Knockdown of stimulator of interferon genes (STING) in OC cells significantly abrogated the increase in CD8 T-cell migration induced by PP4 inhibition. Co-culture of NK-92 cells and OC cells with PPP4C or PPP4R3B knockdown resulted in strong induction of NK cell interferon-γ, increased degranulation, and increased NK cell-mediated cytotoxicity against OC cells. Stable knockdown of PP4C in a syngeneic, immunocompetent mouse model of OC resulted in significantly reduced tumor growth in vivo. Tumors with PP4C knockdown had increased infiltration of NK cells, NK T cells, and CD4+ T cells. Addition of low dose carboplatin treatment led to increased CD8+ T-cell infiltration in PP4C knockdown tumors as compared with the untreated PP4C knockdown tumors. CONCLUSIONS: Our work has identified a role for PP4 inhibition in promoting inflammatory signaling and enhanced immune cell effector function. These findings support the further investigation of PP4 inhibitors to enhance chemo-immunotherapy for OC treatment.

Single cell enhancer activity distinguishes GABAergic and cholinergic lineages in embryonic mouse basal ganglia.

Enhancers integrate transcription factor signaling pathways that drive cell fate specification in the developing brain. We paired enhancer labeling and single-cell RNA-sequencing (scRNA-seq) to delineate and distinguish specification of neuronal lineages in mouse medial, lateral, and caudal ganglionic eminences (MGE, LGE, and CGE) at embryonic day (E)11.5. We show that scRNA-seq clustering using transcription factors improves resolution of regional and developmental populations, and that enhancer activities identify specific and overlapping GE-derived neuronal populations. First, we mapped the activities of seven evolutionarily conserved brain enhancers at single-cell resolution in vivo, finding that the selected enhancers had diverse activities in specific progenitor and neuronal populations across the GEs. We then applied enhancer-based labeling, scRNA-seq, and analysis of in situ hybridization data to distinguish transcriptionally distinct and spatially defined subtypes of MGE-derived GABAergic and cholinergic projection neurons and interneurons. Our results map developmental origins and specification paths underlying neurogenesis in the embryonic basal ganglia and showcase the power of scRNA-seq combined with enhancer-based labeling to resolve the complex paths of neuronal specification underlying mouse brain development.