Glioblastoma (GB) is a highly invasive type of brain cancer exhibiting poor prognosis. As such, its microenvironment plays a crucial role in its progression. Among the brain stromal cells, the microglia were shown to facilitate GB invasion and immunosuppression. However, the reciprocal mechanisms by which GB cells alter microglia/macrophages behavior are not fully understood. We propose that these mechanisms involve adhesion molecules such as the Selectins family. These proteins are involved in immune modulation and cancer immunity. We show that P-selectin mediates microglia-enhanced GB proliferation and invasion by altering microglia/macrophages activation state. We demonstrate these findings by pharmacological and molecular inhibition of P-selectin which leads to reduced tumor growth and increased survival in GB mouse models. Our work sheds light on tumor-associated microglia/macrophage function and the mechanisms by which GB cells suppress the immune system and invade the brain, paving the way to exploit P-selectin as a target for GB therapy.

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Cancer immunotherapy critically depends on fitness of cytotoxic and helper T cell responses. Dysfunctional cytotoxic T cell states in the tumor microenvironment (TME) are a major cause of resistance to immunotherapy. Intratumoral myeloid cells, particularly blood-borne myeloids (bbm), are key drivers of T cell dysfunction in the TME. We show here that major histocompatibility complex (MHC) class II (MHCII)-restricted antigen presentation on bbm is essential to control the growth of brain tumors. Loss of MHCII on bbm drives dysfunctional intratumoral tumor-reactive CD8+ T cell states through increased chromatin accessibility and expression of Tox, a critical regulator of T cell exhaustion. Mechanistically, MHCII-dependent activation of CD4+ T cells restricts myeloid-derived osteopontin that triggers a chronic activation of nuclear factor of activated T cells (Nfat)2 in tumor-reactive CD8+ T cells. In summary, we provide evidence that MHCII-restricted antigen presentation on bbm is a key mechanism to directly maintain functional cytotoxic T cell states in brain tumors.

MotivationCell-cell crosstalk involves simultaneous interactions of multiple receptors and ligands, followed by downstream signaling cascades working through receptors converging at dominant transcription factors which then integrate and propagate multiple signals into a cellular response. Single-cell RNAseq of multiple cell subsets isolated from a defined microenvironment provides us with a unique opportunity to learn about such interactions reflected in their gene expression levels.ResultsWe developed the FLOW framework with the intention of mapping the potential ligand-receptor interactions between different cell subsets based on a maximum flow computation in a network of protein-protein interactions (PPIs). The maximum flow approach further allows characterizing the intracellular downstream signal transduction from differentially expressed receptors towards dominant transcription factors. This, therefore enables the association between a set of receptors and their downstream activated pathways. Importantly, we were able to identify key transcription factors toward which the convergence of signaling of multiple receptors occurs. These identified factors have a unique role in the integration and propagation of signaling following cell-cell interactions.

Glioblastoma (GB) is an aggressive type of brain cancer with high mortality rate. It is a highly angiogenic tumor exhibiting an extremely invasive nature. As such, its brain microenvironment plays a crucial role in its progression. Microglia are the brain resident immune cells which have been shown to facilitate GB cell invasion and immune suppression. The mechanism by which GB cells alter microglia behavior is yet to be fully understood. One proposed mechanism involves adhesion molecules such as the Selectins family of proteins which are expressed on the surface of endothelial and immune cells and are involved in immune modulation and cancer immunity. We have previously shown that P-Selectin (SELP) is expressed by GB cells. Here, we investigated the factional role of SELP in GB-microglia interactions. First, we found that microglia cells facilitate the expression and secretion of SELP by GB cells, and that GB cells facilitate the expression of P-Selectin ligand by microglia. We then showed that SELP mediates microglia-enhanced GB invasion and proliferation in 2D and 3D in vitro models and has a role in microglia activation state. These findings were validated in vivo, showing that inhibition or downregulation of SELP leads to reduced tumor growth, increased overall survival and improved immune response. Single-Cells RNA-seq analysis of the tumors revealed an increase in pro-inflammatory microglia signature, reduction in cancer cell tumorigenesis potential and improved T cell activation. Our results indicated that SELP has an important role in GB progression and microenvironment activation. This work can improve our understanding of tumor-associated microglia function and the mechanisms by which GB cells suppress the immune system and invade the brain tissue. Citation Format: Eilam Yeini, Paula Ofek, Sabina Pozzi, Nitzan Albeck, Dikla Ben-Shushan, Galia Tiram, Sapir Golan, Ron Kleiner, Ron Sheinin, Shlomit Reich-Zeliger, Rachel Grossman, Zvi Ram, Henry Brem, Thomas Hyde, Prerna Magod, Dinorah Friedmann-Morvinski, Asaf Madi, Ronit Satchi-Fainaro. P-selectin axis plays a key role in microglia immunophenotype and glioblastoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2716.