Nanoengineered DNA origami with repurposed TOP1 inhibitors targeting myeloid cells for the mitigation of neuroinflammation

Targeting myeloid cells, especially microglia, for the treatment of neuroinflammatory diseases such as multiple sclerosis (MS), is underappreciated. Here, we screened a library of compounds and identified the topoisomerase 1 (TOP1) inhibitor camptothecin (CPT) as a promising drug candidate for microglial modulation. CPT and its FDA-approved analog topotecan (TPT) inhibited inflammatory responses in microglia and macrophages, and ameliorated neuroinflammation in mice. Transcriptomic analysis of sorted microglia revealed an altered transcriptional phenotype following TPT treatment, with Ikzf1 identified as a potential target. Importantly, TOP1 expression was found elevated in several neuroinflammatory conditions, including human MS brains. To achieve targeted delivery to myeloid cells we designed a nanosystem using DNA origami and loaded TPT into it (TopoGami). TopoGami also significantly suppressed the inflammatory response in microglia and mitigated disease progression in MS-like mice. Our findings suggest that TOP1 inhibition represents a therapeutic strategy for neuroinflammatory diseases, and the proposed nanosystem may foster future research and drug development with a demand to target myeloid cells.

TAMI-53. THE ONCOMETABOLITE D-2HG INDUCE INFLAMMATORY ASTROGLIOSIS CAUSING NEUROTOXICITY AND SEIZURES IN IDH MUTATED GLIOMA

The role of tumor-associated astrocytes in the microenvironment of glioma has long been underestimated but is moving into the focus of current research. We explored the role of reactive astrocytes in IDH-mutated glioma using RNA-sequencing of purified astrocytes and microglia and single-nucleus RNA-sequencing of infiltrating tumor regions. Mapping of the transcriptional phenotype of astrocytes along developmental and reactive trajectories revealed an inflammatory transformation of IDH-mutated associated astrocytes. The major proportion of astrocytes is marked by complement-activation similar to findings in neuroinflammatory diseases. A human neocortical slices model with injected IDH-mutated patient-derived cells or D-2HG treatment (+/- microglia depletion) was used to map shared and unique transcriptional adaptation in astrocytes promoted by either tumor cells or metabolic alteration. High-dimensional electrophysiological profiling was used to investigate alterations in neural response to tumor-induced microenvironmental transformation. We showed that 2HG alone promote the inflammatory pattern of astrocytes, which causes neurotoxicity and seizures in our neocortical slice model. Depletion of microglia rescued the neurotoxicity suggesting that microglia predominantly drive inflammatory astrogliosis as a response to metabolic alteration the tumor environment. We showed that neurotoxic astrogliosis induced by the oncometabolite D-2HG via distinct microglia activation promote the evolution of frequently observed seizures in IDH-mutated glioma patients.

Transcriptional subtype-specific microenvironmental crosstalk and tumor cell plasticity in metastatic pancreatic cancer

ABSTRACTIn pancreatic ductal adenocarcinoma (PDAC), the basal-like and classical transcriptional subtypes are associated with differential chemotherapy sensitivity and patient survival. These phenotypes have been defined using bulk transcriptional profiling, which can mask underlying cellular heterogeneity and the biologic mechanisms that distinguish these subtypes. Furthermore, few studies have interrogated metastases, which are the cause of mortality in most patients with this highly lethal disease. Using single-cell RNA-sequencing of metastatic needle biopsies and matched organoid models, we demonstrate intra-tumoral subtype heterogeneity at the single-cell level and define a continuum for the basal-like and classical phenotypes that includes hybrid cells that co-express features of both states. Basal-like tumors show enrichment of mesenchymal and stem-like programs, and demonstrate immune exclusion and tumor cell crosstalk with specific macrophage subsets. Conversely, classical tumors harbor greater immune infiltration and a relatively pro-angiogenic microenvironment. Matched organoid models exhibit a strong bias against the growth of basal-like cells in standard organoid media, but modification of culture conditions can rescue the basal-like phenotype. This study reframes the transcriptional taxonomy of PDAC, demonstrates how divergent transcriptional subtypes associate with unique tumor microenvironments, and highlights the importance of evaluating both genotype and transcriptional phenotype to establish high-fidelity patient-derived cancer models.

P11.58 Astrocytic Environment Aid the Evolution of Immunosuppressive Environment in Glioblastoma

BACKGROUND Glioblastomas are referred to as immunologically “cold” tumors since their cellular environment promotes an anti-inflammatory environment leading to an unresolved barrier to immunotherapy. Although the role of reactive astrocytes in other inflammatory diseases has been investigated in several studies, the immunoregulatory functions of astrocytes in the tumor environment remains poorly understood. MATERIAL AND METHODS We purified reactive astrocytes from de-novo glioblastoma and non-infiltrated cortex specimens by immunoprecipitation and analyzed the transcriptional phenotype by RNA sequencing. In order to investigate the origin of astrocytic transformation, we used a microglia loss-of-function model in human organotypic slices. Microglia was depleted by clodronat stimulation. We injected tumor cells and analyzed gene expression of the astrocytes after 7d incubation by RNA sequencing. Environmental cytokines were analyzed by multi-ELISA. Immunostainings of slices were processed by confocal microscopy and 3D reconstruction. RESULTS Here we address the immunological impact of tumor-associated astrocytes, we were able to identify a novel reactive subtype marked by JAK/STAT pathway activation and CD274 expression. Our results show a distinct astrocytic transcriptional phenotype that mutually arises from both microglia and astrocytes of the tumor environment. This interaction leads to a large release of anti-inflammatory cytokines such as TGFß and IL10. The reactive subtype switch of astrocytes was recovered by inhibition of the JAK pathway, which caused an increase of pro-inflammatory environment. CONCLUSION Our results can form the basis for a novel therapeutic approach by directly targeting tumor-associated astrocytes in order to transformimmunologically “cold” into “warm” tumors.

Abstract 447: Atherosclerosis-driven Treg Plasticity Results in the Formation of a Dysfunctional Subset of Plastic IFNg + Th1/Tregs

Foxp3 + T regulatory cells (Tregs) are key players in maintaining immune homeostasis. Emerging evidence suggests that Tregs respond to environmental cues to permit or suppress inflammation. In atherosclerosis, Th1-driven inflammation affects Treg homeostasis, but the mechanisms governing this phenomenon are unclear. Here, we addressed whether atherosclerosis impacts Treg plasticity or functionality in Apoe -/- mice and what effect Treg plasticity might have on the pathology of atherosclerosis. We demonstrate that atherosclerosis promotes Treg plasticity, resulting in the reduction of CXCR3 + Tregs, and the accumulation of an intermediate Th1-like IFNγ + CCR5 + Treg subset (Th1/Tregs) within the arterial wall. Using Foxp3 Gfp/Gfp and Foxp3 Yfp-cre/Yfp-cre Rosa tdTomato/tdTomato lineage tracing mice, we demonstrate that Th1/Tregs arise in atherosclerosis from bona fide Tregs, rather than T effector cells. Th1/Tregs recovered from atherosclerotic mice are dysfunctional in suppression assays. Importantly, using Mir146a -/- mice, we demonstrate that plasticity-prone Mir146 -/- Tregs fail to reduce atherosclerosis, arterial Th1, or macrophage content in Apoe -/- recipients. Lastly, we utilized single cell RNA-sequencing to gain whole transcriptome information on Th1/Tregs, Treg, and Th1 cells. Th1/Tregs yielded a unique transcriptional phenotype characterized by co-expression of Treg and Th1 lineage genes, and down-regulation of Treg-related genes, including Ikzf2 , Ikzf4, Tigit, and Lilrb4 . Additionally, an ingenuity pathway analysis further implicates IFNγ, IFNα, IL-2, IL-7, CTLA4, T cell receptor, and Csnk2b-related pathways in regulating Treg plasticity. In conclusion, atherosclerosis drives Treg plasticity, resulting in the accumulation of dysfunctional IFNγ + Th1/Tregs that permit further arterial inflammation and atherogenesis.

A Molecular Classification of Human Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSC) are widely used, isolated from a variety of tissues and increasingly adopted for cell therapy, but the identity of these cells is poorly defined and commonalities between MSC from different tissues sources is controversial. Here we undertook a comprehensive review of all public MSC expression studies to assess whether cells derived from different sources shared any common molecular attributes. In doing so, we discovered an over-arching transcriptional phenotype shared by a wide variety of MSC, freshly isolated or cultured cells, and under a variety of growth conditions. We developed a modified variable selection protocol that included cross platform normalisation, and assessment of the selected gene stability and informativeness. A 16-gene signature classified MSC with >97% accuracy, discriminating these from fibroblasts, other adult stem/progenitor cell types and differentiated cells. The genes form part of a protein-interaction network, and mutations in more than 65% of this network were associated with Mendelian disorders of skeletal growth or metabolism. The signature and accompanying datasets are provided as a community resource at www.stemformatics.org resource, and the method is available from the CRAN repository.