Find tumor immune evasion strategies by cellular barcoding

Cancer is a leading cause of death worldwide, accounting for 8.8 million deaths in 2015. One of the most recent and highly successful advances in anti-cancer treatment is cancer immunotherapy. As cancer patients are frequently suffering from immunosuppression, immunotherapy aims at re-activating the immune system. Natural killer (NK) cells are at the forefront of the bodys anti-tumor defense capable to directly kill tumor cells and to activate other immune cells. Novel therapies including the infusion of pre-activated NK cells into tumor patients have shown extraordinary performance. However, certain limitations curtail the success story. Malignant cells are highly adaptable to their environment and capable to efficiently evade from immune surveillance. The concept of immunoediting describes the phenomenon that tumors change their appearance and become immune-resistant upon constant pressure imposed by the immune system. This concept has been shown in experimental models such as chemically-induced tumors, but whether and how it applies in other malignancies is still a matter of debate. In the present project we hypothesize that immunoediting takes place in natural tumors. There is unequivocal evidence for the importance of NK cells in the fight against leukemia. We thus choose leukemia as a model system to address whether NK cell-mediated immunoediting is taking place. The design of our study will allow two potential scenarios to be distinguished: (i) NK cells are able to kill most tumor cell clones but spare the few pre-existing resistant sub- clones (clonal selection) or (ii) NK cells shape tumors by classical immunoediting. We further aim to determine the underlying molecular mechanisms for tumor evasion from NK cell- mediated eradication. This is made possible by using a cellular barcoding system that allows tracking of single leukemic cells in mice. The combination of cellular barcoding and next generation sequencing enables us to determine changes causing immune-resistance and tumor outgrowth. A better understanding of the molecular mechanisms underlying tumor immune evasion is the first step to utilize the full potential of NK cells in anti-tumor therapy. Ultimately, this study will pave the way for new therapeutic approaches to treat tumors that have successfully evaded the immune system.

The term tumor immunoediting describes the dual role by which the immune system can suppress and promote tumor growth and is divided into three phases: elimination, equilibrium and escape. It elucidates the phenomenon whereby constant pressure exerted by the immune system can prompt tumors to alter their appearance to become resistant to immune cells. Natural Killer (NK) cells are part of the innate immune response and are primarily responsible for eliminating virus-infected and transformed tumor cells. In this project, we hypothesized that NK cells play a crucial role not only during the elimination phase but in all three stages of tumor immunoediting. To address this question, we developed an in vitro model in which murine BCR/ABLp185+ acute lymphoblastic leukemia (B-ALL) cells were co-cultivated with NK cells over an extended period. To quantitatively assess NK cell-mediated tumor immunoediting, newly generated B-ALL cell lines were labeled with DNA barcodes. The inheritable sequence of the DNA barcode, akin to commercial products, marks each individual B-ALL cell and allows the simultaneous tracking of individual tumor clones under various experimental conditions over extended periods of time. Our results demonstrated that the majority of tumor cell clones were efficiently eliminated by NK cells, with only a small fraction exhibiting intrinsic (primary) resistance to NK cells. Additionally, the application of DNA barcoding allowed us to prove the existence of tumor cell clones with secondary resistance, which developed resistance against NK cells during the course of co-culture. We found that the production of interferon- (IFN-) rather than direct cytotoxicity by NK cells, led to the emergence of highly resistant tumor cells. Besides well-known regulators of immune evasion, such as increased MHC-I expression, our detailed transcriptomic analysis of NK-resistant tumor cells revealed the upregulation of novel genes, such as lymphocyte antigen 6A (Ly6a), which, according to our functional tests, promoted NK cell resistance in leukemic cells. Our results underscore that during the equilibrium phase, tumor cells are actively edited by NK cells, employing various strategies to evade NK cell-mediated elimination.