Stem cell behaviour is controlled both by intrinsic mechanisms and by external signals from the local microenvironment or niche. Interactions with the niche are reciprocal, since stem cells are capable of remodelling their environment. Using adult epidermis as an experimental model, I propose to discover the relative importance of specific intrinsic and extrinsic signals in regulating stem cell fate (aim 1) and reveal how altered intrinsic signalling in stem cells impacts on neighbouring cells of the epidermis and dermis (aim 2). I will apply these findings to squamous cell carcinomas of the oral cavity (HNSCC) (aim 3). In aim 1 I will develop live cell imaging tools to visualise commitment and differentiation of single human epidermal stem cells on micro-patterned extracellular matrix (ECM)-coated substrates. I will examine whether engagement of specific intercellular receptors or attachment of stem or differentiated cells affects the decision of an adherent stem cell to differentiate. I will also investigate whether intrinsic activation of Wnt, Notch/Delta-like1 (Dll1), EGF receptor (EGFR) or beta1 integrin signalling, interconnected pathways known to affect the epidermal stem cell compartment, influences microenvironmental responses. In aim 2 I will extend the studies in vivo by examining reconstituted human epidermis in immunocompromised mice and mice in which the epidermal stem cell compartment is genetically modified. I will discover the functional significance of heterogeneous Dll1 expression by epidermal stem cells and evaluate how intrinsic epidermal alteration of Dll1, Lrig1 and beta1 integrins impacts on cells of the dermis, thereby identifying specific stromal signatures of genetic alterations in the epidermis. In aim 3 I will study the microenvironmental responsiveness of human HNSCC stem cells in vitro using tools developed in aim 1, correlating the data with HNSCC growth in xenografts. I will correlate genetic alterations with altered niche responses in vitro and with the composition of the tumour stroma in vivo, relating the data to the stromal signatures defined in aim 2. I will use this information to devise strategies to block tumour growth in xenografts. The research has broad translational applications. The micro-patterned substrates can be used to probe niche interactions in any adherent stem cell population and as a platform for drug discovery; identification of stromal signatures that reflect specific genetic alterations in epithelia will provide improved diagnostic and prognostic indicators in a wide variety of tumours; and modifying niche interactions offers an exciting strategy for treating cancer.