Toll-like receptors (TLRs) are transmembrane proteins mainly involved in innate immune response, which differ depending on the ligand they bind and their localization in the cell. However, they can also be expressed on cancer cells where their role is still unresolved. TLR3 activation is triggered by dsRNA originating from viral infection or its synthetic analogues, poly(I:C) or poly(A:U). TLR3 has a dual role in cancer; its activation can trigger apoptosis as well as stimulate cancer cell survival, proliferation and progression. In this project we will further explore the role of TLR3 activation in cancer survival and progression by focusing especially on the role of its endogenous ligands. These ligands originate from necrotic cancer cells and may stimulate cancer progression. We will study this on several models: a) TLR3 reporter cell line HEK-Blue to validate the hypothesis of in vivo and in vitro existence of TLR3 endogenous ligands derived from necrotic cells and tissue, b) head and neck cancer cell lines stably transfected with shRNA for TLR3 in order to confirm that the obtained results are TLR3-dependant and c) cancer tissue specimens isolated from head and neck cancer patients which will either be stained with TLR3, CD133, aldehyde dehydrogenase (ALDH) and antibodies for endogenous ligands or checked for the expression of genes involved in tumorigenesis. We will produce endogenous ligands in vitro by stressing the cancer cell lines by serum starvation, irradiation, hypoxia and oxidative stress. In vivo we will obtain endogenous ligands by aspiration from xenografted tumors in mice or from head and neck cancer patients. Second approach will include studying the role of TLR3 and its endogenous ligands in cancer stem cells (CSC) development and self-renewal. In CSC, we will determine the role of TLR3 in tumorosphere formation, ”stemness” gene induction, ALDH activity and endogenous ligands release in the microenvironment. We will also study CSC on proteomic level with the aim to reveal novel CSC markers in cancer tissue. Lastly, we will seek to develop a novel anti-cancer therapy against CSC. These cells show resistance to classical therapy and may trigger recurrence thus targeting them is a novel and reasonable strategy in cancer therapy development. Our approach will include combining proton or γ-rays irradiation with the treatment with poly(I:C) and pharmacological inhibitors of endogenous ligands. TLR3 agonists are currently being investigated as potential novel cancer therapy adjuvants and apoptosis inducers, alone or in combination with other therapeutic options. This project will focus on the delivery of new knowledge on the detrimental role of TLR3 in cancer and it is of great importance to elucidate it before the introduction of TLR3 agonists into clinical practice. Moreover, the results of our novel therapeutic approach might also initiate the development of innovative anti-cancer therapy.