The killing potential and relative contribution of all granzymes in tumor elimination during immunotherapy as well as the extracellular roles of tumor microenvironment-associated granzymes in tumorigenesis and cancer immunity largely remain unclear. Knowledge on these issues may not only unravel novel mechanistic insights, but also may open new cancer (immuno)therapeutic avenues. In this thesis, we aim to investigate the roles of intracellular and extracellular granzymes in tumor elimination, tumor progression, and tumor immunity.
In chapter 2, we employed a systematic granzyme knockout strategy in NK cells to explore the cytotoxic role and relevance of individual granzymes in a cellular setting of breast cancer immunotherapy. Using CRISP-Cas, we obtained clonal perforin, granzyme B (GrB), granzyme H (GrH), and granzyme M (GrM) knockout NK cells. We found that the granule exocytosis pathway plays a pivotal role in killing breast cancer subtypes, with cancer cell type-dependent roles of GrB, GrM, and GrH. Interestingly, the function of one granzyme is not always taken over by another granzyme. Our data suggests that NK cells use only partially overlapping sets of granzymes to kill different subtypes of breast cancer cells. Furthermore, our data suggest cooperation of killer cell granzymes in breast cancer immunotherapy.
In chapter 3, we show that extracellular granzyme K (GrK) inhibits angiogenesis and triggers endothelial cells to release soluble VEGFR1 (sVEGFR1), a decoy receptor that inhibits angiogenesis by sequestering VEGF-A. GrK does not cleave off membrane-bound VEGFR1 from the cell surface, does not release potential sVEGFR1 storage pools from endothelial cells, and does not trigger sVEGFR1 release via protease activating receptor-1 (PAR-1) activation. GrK induces de novo sVEGFR1 mRNA and protein expression and subsequent release of sVEGFR1 from endothelial cells. GrK protein is detectable in human colorectal tumor tissue and its levels positively correlate with sVEGFR1 protein levels and negatively correlate with T4 intratumoral angiogenesis and tumor size. In conclusion, extracellular GrK can inhibit angiogenesis via secretion of sVEGFR1 from endothelial cells, thereby sequestering VEGF-A and impairing VEGFR signaling. Our observation that GrK positively correlates with sVEGFR1 and negatively correlates with angiogenesis in colorectal cancer, suggest that the GrK-sVEGFR1-angiogenesis axis may be a valid target for development of novel anti-angiogenic therapies in cancer.
In chapter 4, we have studied immune checkpoint B7-H3 expression in a tissue cohort of human pediatric medulloblastoma (MB). Expression of B7-H3 was detected by immunohistochemistry and classified via B7-H3 staining intensity and percentage of B7-H3 positive tumor cells. Subsequently, B7-H3 protein expression was distinguished in MB molecular subtypes and correlated to immune cell infiltrates, patient characteristics, and survival.B7-H3 protein expression was found in 23 out of 24 (96%) human pediatric MB cases and in 17 out of 24 (71%) MB cases > 25% of tumor cells had any level of B7-H3 expression. Immune checkpoint B7-H3 is differentially expressed by the large majority of pediatric MB. This further warrants the development of novel B7-H3-directed (immuno)therapeutic methods for children with incurable, metastatic, or chemo-resistant MB.
In chapter 5, we provided an overview of the current consensus on MB classification and the state of in vitro, in vivo, and clinical research concerning immunotherapy in MB. Based on existing evidence, we will especially focus on immune checkpoint inhibition and CAR T-cell therapy. Additionally, we discussed challenges associated with these immunotherapies and relevant strategies to overcome those.
In chapter 6, we hypothesize that the immune inhibitory function of the PD-1/PD-L1/2 axis is directly regulated by extracellular granzymes in the tumor microenvironment. We used purified PD-1 and its ligands as well as living cell experiments using receptor overexpression and primary immune cells. We found that granzyme A (GrA), GrB, GrK and GrM cannot cleave purified (extracellular parts of) PD-1 and its ligands. In addition, these granzymes did not cleave PD-1 or PD-L1 from living cells, although GrB did cleave PD-1 in cell lysate. GrK and inactive GrK-SA mutant, but not GrA, GrB, and GrM, blocked purified PD-1 binding to purified PD-L1. None of the granzymes impaired Fc-PD-L1 binding to cells that overexpress PD-1. GrA, GrB, GrK, and GrM did not inhibit PD-1/PD-L1 signaling, and these granzymes did not affect Fc-PD-L1 binding to cell-surface native PD-1 on primary T cells. We conclude that extracellular granzymes likely do not interfere with the PD-1/PD-L1/2 immune checkpoint axis.
Altogether, this thesis unravel role of granzymes in cancer development, immunoregulation, and immunotherapy. Further exploration of reliable response biomarkers (B7-H3) and application of efficient intracellular or extracellular granzymes may facilitate immunotherapy in cancer treatment.