Dissecting the Role of Hedgehog Signaling in Tumor-associated Macrophages and Immunosuppression in the Tumor Microenvironment

Macrophages are terminally differentiated cells of the hematopoietic system that are involved in various important cellular processes, including protection against pathogenic infections, inflammation and tissue homeostasis. Additionally, macrophages coinhabit with malignant cells in various stages of tumor development and can be polarized to two distinctive phenotypes – the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype. These M2 polarized tumor-associated macrophages (TAMs) are known to suppress immune responses and promote neoplastic progression. We sought to gain insights into the regulation of TAM functions and mechanisms by which TAMs exert immunosuppressive changes within the tumor microenvironment (TME).

The hedgehog (Hh) signaling pathway is one of the most important signaling pathways in cancer. Its aberrant activation within cancer cells can lead to increased proliferation and metastasis. Hh ligands, including sonic hedgehog (Shh), desert hedgehog (Dhh) and indian hedgehog (Ihh) can also be secreted from tumor cells to modify the TME in order to create a more favorable environment to foster tumorigenesis. However, little is known about how Hh ligands change the TME to promote tumor progression and what cell type(s) are involved in this process. Using a murine model, we found initial evidence that hedgehog signaling is active in TAMs and deletion of the pathway leads to suppressed tumor growth.

In the first aim of this dissertation, we first sough to investigate whether Hh signaling plays a role in TAM functions. Specifically, we asked whether Hh activation could promote TAM M2 polarization. Using several murine tumor models, we confirmed that Hh signaling in myeloid cells is critical for promoting the immunosuppressive M2 phenotype in TAMs, resulting in accelerated tumor growth. We also found that tumor cells secrete Shh and that tumor derived Shh drives M2 polarization. Furthermore, Hh-induced M2 polarization in TAMs can suppress CD8+ T cell recruitment to the TME through the inhibition of C-X-C motif chemokine ligand 9 (CXCL9) and CXCL10 production by TAMs. Lastly, we demonstrated that Krüppel-like factor 4 (Klf4) mediates Hh-dependent TAM M2-polarization and the immunosuppressive function. Collectively, these findings highlight a critical role for tumor derived Shh in promoting TAM M2 polarization and inhibiting CD8+ T cell infiltration in tumor.

In the second aim of this study, we further elucidated whether Hh-induced M2 TAMs can directly subvert CD8+ tumor-infiltrating lymphocyte (TIL) cytotoxic functions. Specifically, we queried whether expression of programmed death ligand 1 (PD-L1) on TAMs, promoted by Hh signaling activation, play a role in suppressing CD8+ TILs. Using several murine models including a novel myeloid-specific Pdl1 knock-out mouse, we showed the tumor-derived Shh ligand promoted PD-L1 expression on TAMs, resulting in decreased interferon- (IFN-) and granzyme B (GzmB) production in CD8+ TILs. Furthermore, TAM-derived PD-L1 was found to be the major source of PDL1-programmed death 1 (PD1)-axis-mediated CD8+ TIL suppression. Lastly, we demonstrated signal transducer and activator of transcription 3 (Stat3) mediates Hh-dependent PD-L1 upregulation in TAMs. Together, these findings demonstrated that hedgehog signaling plays an important role in suppressing CD8+ TIL effector functions through PD-L1-expressing TAMs.

Collectively, this work sought to dissect the role of hedgehog signaling in TAMs and immunosuppression within the TME in order to identify novel cancer therapeutic strategies.