Project 3 Summary (Coffey) The Coffey lab has identified important links between WNT and EGFR signaling in cetuximab (CTX) resistance and intestinal crypt homeostasis. We recently reported a new mode of CTX resistance due to increased WNT signaling mediated by miR-100/-125b. These two miRs are upregulated in extracellular vesicles (EVs) released by CTX-resistant cells and these EVs can transfer CTX resistance. In a unique EGFR and WNT reporter mouse model, we show that activation of WNT signaling in an EGFR-sensitized background dramatically increases both EGFR and non-cell autonomous WNT activity. Based on these findings, we propose a model of opposing gradients of EGFR and WNT activity in the colonic stem cell niche (SCN) that contribute to homeostasis and disruption of the gradient is a feature of neoplastic transformation. We hypothesize that in the normal crypt niche EVs and exomeres released by the EGFR-active and WNT-active compartments reinforce the EGFR-WNT gradient and in CRC these nanoparticles serve to drive tumor growth and define cancer progression due to their oncogenically altered constituents. The model also provides a framework to further examine the role of EVs and exomeres in conferring CTX resistance, at least in part, via increased WNT signaling. To examine this model and to determine how EVs participate in CTX resistance, with the ultimate goal of devising strategies to overcome CTX resistance, we propose three Aims. Aim 1 is to determine the effect of EVs and exomeres isolated from highly informative paired cell lines on EGFR and WNT activity in reporter cell lines. Aim 2 is to test the hypothesis that these EVs and exomeres regulate normal stem cell patterning and tumor progression using our unique EGFR and WNT reporter mouse models and their derived organoids. Aim 3 is to elucidate mechanistic underpinnings of EV participation in resistance to EGFR blockade. This work has the potential to alter our fundamental understanding of normal stem cell function, regulation of tumor growth and processes regulating drug resistance.