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Abstract

Despite representing an unprecedented gain for clinical cancer immunotherapy, over the recent years it became evident that the therapeutic approach of immune checkpoint blockade only shows responsiveness in a maximum of 20% of patients suffering from also very common tumors like breast, colon, or prostate cancer, melanoma, and brain tumors. The Tumor infiltrating myeloid cells, such as tumor-associated macrophages of the M2-like phenotype and myeloid-derived suppressor cells, have been identified to represent the most abundant immunosuppressive cell compartment in the tumor microenvironment and have been associated with poor patient prognosis for the most common cancer types. Thus, representing the key obstacle for effective cancer immunotherapy. Even though being in the focus of cancer research, specific druggable targets to therapeutically approach tumor-associated macrophages are largely lacking. Dedicated to this issue, this thesis was therefore aiming to improve the understanding of myeloid cell biology in the context of the tumor microenvironment and to identify novel molecular targets to modify macrophage polarization. This work presents a CRISPR/Cas9-based screening approach to identify druggable targets involved in molecular mechanisms to promote the immunosuppressive (M2) or to prevent the immunostimulatory (M1) macrophage phenotype. The inhibition of these targets will reinstruct tumor-infiltrating myeloid cells to stimulate antitumor immune responses in the tumor microenvironment. As recapitulating relevant key signalling pathways and effector functions of immune cell biology most accurately, primary murine monocytes from a Cas9-expressing mouse line were used as cellular system to perform the CRISPR screen. A comprehensive transduction and differentiation protocol was established that enabled CRISPR/Cas9-mediated functional genomic studies in primary cells. A CRISPR/Cas9-based screen was performed with a target gene library focusing on the myeloid cell transcriptome detecting the effects of sgRNA-mediated knockout on MHC II and CD206 expression in differentiated and polarized primary macrophages. Thereby, a set of genes was identified with a potential role in M2 macrophage polarization. Some of the hits, such as CSF1R and CXCR2 have been described before to be involved in macrophage biology and polarization, supporting the validity of the screening approach. To further characterize the impact of the other identified screening hits, an extensive in vitro validation protocol was established and validated with a positive control gene, TNFAIP3, which represents one of the top hits from a whole-genome CRISPR screen performed in human THP-1 cells by the cooperation partner Anna Montebaur. Investigating the impact of TNFAIP3-deficient primary myeloid cells in the setting of the established validation protocol, the induction of a phenotypic shift towards the pro-inflammatory and immunostimulatory M1 phenotype was documented on a descriptive and also on a functional level. Therefore, the validity of the established validation protocol was proven and is now being used as a readout platform to further investigate on the relevance of the top hits from the focused CRISPR screen for macrophage polarization. Furthermore, a protocol was established to allow the CRISPR/Cas9-mediated ex vivo knockout of target genes at will in hematopoietic stem cells. By reimplanting the knockout cells into recipient mice, the investigation of the target gene function for tumor-associated macrophage polarization in vivo, in the context of the multifactorial influences of the tumor microenvironment, is facilitated. Thus, providing a platform to gain a better understanding of the relevance of specific genes as potential targets for cancer immunotherapy. Based on the results generated in this thesis and also in this cooperation project between the DKFZ and Bayer HealthCare, several studies have been started to further study the role of the screening hits in macrophage polarization, on the basis of the established protocol readouts, to evaluate their potential as starting points for a pharmaceutical development program.