Recent studies show that Hedgehog (Hh) signaling correlates with tumor expansion and reduced survival rates in triple negative breast cancer (TNBC) patients, suggesting a potential therapeutic value for Hh inhibitors. Several studies have highlighted mesenchymal cells as potential targets in the tumorigenicity of Hh signaling, yet their diagnostic value and influence in the therapeutic response of Hh inhibitors have not been established. A main limitation is the lack of models that capture distinct contributions of mesenchymal cell sub-types in the growth response to Hh inhibitors. Inhibition of Hh signaling can lead to tumor expansion or suppression; a bi- modal growth mechanism that can negatively impact the therapeutic outcome in tumors treated with Hh inhibitors. Our studies and preliminary data support the influence of mesenchymal cell sub-types in the tumorigenic potential of Hh signaling. Our evaluation of individual contributions of fibroblasts and mesenchymal stem cells suggest that changes in the composition of the mesenchymal niche influence tumorigenic signals of the Hh pathway and response to Hh inhibitors. Our long-term goal is to provide biomimetic and multi-cell type in vitro models that enable the identification of new targets in clinically challenged endocrine tumors. Our overall goal is to validate an in vitro model for prediction of therapeutic efficacy and identify oncogenic cues associated to the composition of the stroma that can be used as oncogenic markers and targets to improve therapeutic response to Hh inhibitors. Our central hypothesis is that the oncogenicity of Hh signaling is regulated by the composition of the adjacent stroma. Our hypothesis has been formulated based on previous studies and our preliminary data supporting distinct contributions of mesenchymal cell sub-types in the response of tumor cells to Hh signaling. Our rationale is that the evaluation of the interplay between the mesenchymal and immune cell niches in the sensitivity of tumors to Hh inhibitors will be valuable towards understanding tumor transitions to therapeutic resistance driven by the stroma. The following aims are proposed: Aim 1- Model paracrine Hh signaling contribution to stemness and resistance to therapy, Aim 2- In vivo validation of a Hh paracrine model through evaluation of response to Hh inhibition, and Aim 3- Impact of immune cells in the oncogenicity of Hh paracrine signaling. These aims will support recapitulation of in vivo observations in our in vitro models and confirm the influence of the mesenchymal cell niche in the tumorigenicity of Hh signaling. This contribution is significant as it will provide new biological insights into the modulation of tumor cell behavior in response to shifts in the stromal niche and establish the therapeutic value of stromal cell components in Hh signaling. The technology is innovative because it provides with a simple and customizable culture model in which to evaluate tumor transitions towards a more aggressive state and the impact of immune cells in Hh paracrine signaling.

Project Narrative Despite increasing evidence supporting the role of mesenchymal cells in the tumorigenicity of tissues with active Hedgehog (Hh) signaling, the value of the mesenchymal cell niche has not yet been established for potentiating therapeutic efficacy of Hh inhibitors; a prime limitation being the lack of models that capture distinct contributions of cell subtypes in tumor expansion and response to Hh inhibitors. The overall goal for this project is to validate an in vitro model for prediction of therapeutic efficacy and identify cues derived from multi-cell type interactions that support the tumorigenicity of Hh signaling in triple negative breast tumors. The results of this study will provide new biological insights into the modulation of tumor cell behavior and therapeutic response via interactions with mesenchymal and immune cells, and support use of multi-cell type models to unravel the complexity of the tumor microenvironment.