NK cell therapy for glioblastoma: Modulating cystatin F to improve efficacy

Glioblastoma (GBM) is a highly aggressive type of brain cancer, characterized by tumor cells that are invasive and resistant to apoptosis. Despite the best available treatment options, such as surgery, radiotherapy, and chemotherapy, GBM patients' survival rate remains low due to cancer stem cells which survive these treatments. Therefore, there is a need to develop new and effective therapies to improve the survival rate and quality of life of GBM patients. Immunotherapy has been explored as a new approach for GBM treatment but has had limited success due to the immunosuppressive tumor microenvironment that inhibits the immune response. Given the challenges associated with GBM treatment, adoptive cell therapy using natural killer (NK) cells represents a promising option, as NK cells can recognize and eliminate cancer stem cells, which are resistant to chemo- and radiotherapy and may overcome the immunosuppressive tumor microenvironment. However, NK cells' function is often compromised in cancer patients, which leads to the inability of NK cells to recognize and eliminate cancerous cells, a phenomenon also described as NK cell anergy. The other problem that halts the success of NK cell therapy is the susceptibility of NK cells to immunosuppressive factors in the tumor microenvironment, among them peptidase inhibitors. The activation of cytotoxic granules, the main step in NK cell cytotoxicity, relies on cysteine cathepsins. Cysteine cathepsins C, H, and L activate granzymes and perforin, the executors of target cell killing. However, their activity is blocked by an endogenous inhibitor called cystatin F, which is typically expressed by immune cells. Cystatin F is transported to endo/lysosomes due to its glycosylation moiety. In the lysosomes, it undergoes an activation from a dimeric to an active monomeric form. Recent studies have shown that cystatin F is also expressed by cancer cells. Increased levels of cystatin F in the tumor microenvironment can therefore negatively impact the function of NK cells. Therefore, reducing the expression or activity of cystatin F may increase the cytotoxicity of NK cells and improve their therapeutic effectiveness. We will address our hypothesis in multiple ways. Aim 1 intends to characterize NK cells in the GBM tumor microenvironment and peripheral blood of GBM compared to healthy individuals. Aim 2 will determine the best strategy to modulate cystatin F expression in NK cells from healthy individuals, by either decreasing its expression, modulation of glycosylation status, or preventing its activation to monomeric form, in order to increase NK cell cytotoxicity. In Aim 3 we will evaluate the ex vivo modulated NK cells on organoid GBM models in a microfluidics chip device, enabling us to test NK cells in a clinically relevant environment. The project will be a collaborative effort between several partners who will combine their expertise to successfully manage the project research and administration tasks and to effectively disseminate the results. The project's risk assessment has been conducted, and three different strategies to modulate cystatin F are proposed, to reduce the risk of not achieving one of the primary project goals. Overall, the impact of this project lies in the generation of novel ex vivo modulated NK cells with increased cytotoxicity profile and increased ability to overcome the immunosuppressive tumor microenvironment as a candidate for a preclinical study. The project's use of cystatin F modulation to enhance NK cell function could be applied to other types of cancer as well, as NK cell anergy is a common feature of many types of cancer.