Tumor microenvironment-targeted therapies are emerging as promising treatment options for different cancer types. Tumor-associated macrophages and microglia (TAMs) represent an abundant non-malignant cell type in brain metastases and have been proposed to modulate metastatic colonization and outgrowth. We used an inhibitor of colony stimulating factor 1 receptor (CSF1R) to target TAMs at distinct stages of the metastatic cascade in preclinical breast-to-brain metastasis models and found that CSF1R inhibition leads to anti-tumor responses in prevention and intervention trials. However, in established brain metastases, compensatory CSF2Rb-STAT5-mediated pro-inflammatory TAM activation blunted the ultimate efficacy of CSF1R inhibition by inducing neuro-inflammation gene signatures in association with wound repair responses that fostered tumor recurrence. Consequently, combined blockade of CSF1R and STAT5 signaling led to sustained tumor control, a normalization of microglial activation states and amelioration of neuronal damage.

Background: Myelodysplastic syndromes (MDS) are a heterogenous group of stem cell driven disorders primarily affecting the elderly and characterized by inefficient production of mature blood cells and a high risk (30%) of evolution to secondary acute myeloid leukemia. Despite tremendous progress in the past decade, treatment options for MDS patients remain limited, and primarily address disease symptoms, rather than altering disease course. This points to the urgent need to better understand the pathogenesis of this heterogenous group of syndromes to develop new therapies that address disease vulnerabilities. However, this effort has been largely hampered by the limited availability of model systems that allow the exploration of MDS biology in a fully humanized setting. In recent years, studies from our lab and others, have highlighted the crucial role niche cells play in human MDS, hence reinforcing the notion that MDS is a disease of a tissue rather than hematopoietic cells alone. Therefore, exploration of MDS biology requires the further development of fully human MDS models in which both constituents of the disease, namely hematopoietic and niche cells, are present. Methods: To address this issue we successfully isolated endothelial cells (ECs) and mesenchymal stromal cells (MSC) from bone marrow biopsies obtained from MDS patients or healthy age matched controls, and subsequently utilized them to develop fully human 2D and 3D organotypic niche models, which were successfully used to support normal and MDS HSPCs expansion ex-vivo. The 3D system makes use of a collagen scaffold, as this protein makes up for 90% of the matrix proteins in the bone. Importantly, MSC and EC cultures could be successfully established from several independent donors and immortalized to generate primary cell lines that can be used to reproducibly establish these ex-vivo systems in a robust manner. Moreover, we could show that these niche cells were easily amenable to genetic editing using CRISPR-Cas9 technology as well as modified to carry fluorescent reporter proteins for tracking cellular interactions using live cell imaging and confocal microscopy. Results: In this work, we successfully isolated human mesenchymal and endothelial cells, from primary bone marrow biopsies (MDS and healthy) and established fully human 2D and 3D organotypic co-cultures ex-vivo. Of note, although bone marrow ECs represent an essential component of the hematopoietic niche, they have so far been omitted in previously described human bone marrow niche models, owing to the notorious difficulties in isolating and expanding this cell type from primary bone marrow biopsies. Therefore, we established immortalized EC lines (iECs) that faithfully recapitulate the morphological, phenotypic and functional features of primary bone marrow ECs. When cultured at defined ratios and under defined conditions, MSCs instructed ECs and iECs to form of vessel-like structures that mimic the meshwork observed in vivo and are typically escheated by aSMA positive cells that stabilize the structures. Genetic manipulation of the cellular components of the niche also allowed to explore the functional relevance of a specific ECM protein, which we previously identified to be significantly upregulated in MSCs isolated from MDS patients, namely the Secreted Protein Acidic and Rich in Cysteine (SPARC). SPARC ablation triggered enhanced proliferation of MDS derived HSPCs and sensitized them treatment with 5-Azacytidine, a standard of care hypomethylating agent used for the treatment of MDS patients. Additional studies are underway to further understand the underlying molecular mechanisms and define a potential druggable target that could sensitize MDS cells to standard of care treatment. Besides gene targeting studies, these organotypic models are also being used to evaluate the relative fitness of MDS and healthy stem/progenitor cells in healthy versus patient derived niches, to explore the contribution of niche components to the establishment of the progressive clonal dominance observed in MDS. Disclosures Bönig: Terumo BCT: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kiadis: Honoraria; Bayer: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fresenius: Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Uniqure: Research Funding; medac: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Healthineers: Current equity holder in publicly-traded company; Chugai: Honoraria, Research Funding; Erydel: Research Funding; Miltenyi: Honoraria, Research Funding; Polyphor: Research Funding; Sandor-Hexal: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Stage: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Platzbecker:Amgen: Honoraria, Research Funding; Geron: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Götze:Celgene: Research Funding. Medyouf:Bergenbio: Consultancy, Research Funding.

Brain metastases represent the most common intracranial tumor in adults associated with poor prognosis and median survival of only a few months. Despite current success in the development of targeted or immuno-therapies against different cancer entities, those strategies are ineffective against brain metastases. Hence, treatment options for brain metastasis patients largely remain limited to surgical resection and radio- and/or chemotherapy. This paucity can in part be attributed to the immune-privileged status of the brain where the blood brain-barrier restricts the entry of blood-borne immune cells. However, recent insights into the immune landscape of primary brain cancers indicate that tumor progression leads to an infiltration of blood-borne immune cells into the brain. We employ a comprehensive set of experimental brain metastasis models to characterize the immune landscape of brain metastases from different primary cancer entities at distinct disease stages and in response to radiotherapy. Our data indicate that brain metastases induce massive infiltration of myeloid and lymphoid cell populations into the central nervous system. This leads to the establishment of a dynamic and highly complex tumor microenvironment that affects tumor progression and therapy response. Fractionated whole-brain radiotherapy leads to enhanced infiltration of blood-borne myeloid and lymphoid cells. Transcriptome analysis of brain-resident and recruited myeloid cells indicate a switch from a proinflammatory towards an immune-suppressive environment at advanced disease stages. Importantly, radiotherapy was found to induce gene signatures that are associated with proinflammatory innate immune responses that could revert the establishment of an immune-suppressive environment. Consequently, radiotherapy might sensitize brain metastases towards immuno-therapies. Our goal is to identify pathways or molecular targets that are induced by radiotherapy in the tumor microenvironment to overcome resistance against immuno-therapy. In this project, we seek to test strategies to maintain or induce proinflammatory immune responses for improved targeted or immuno-therapies against brain metastasis. Citation Format: Michael Schulz, Katja Niesel, Anna Salamero Boix, Woon Hyung Chae, Birgitta Michels, Alexander Schaeffer, Maja Strecker, Tijna Alekseeva, Stefan Stein, Henner Farin, Franz Roedel, Patrick Harter, Karlheinz Plate, and Lisa Sevenich. Effects of ionizing radiation on brain metastasis-associated inflammation and its implication for immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A111.