PROJECT SUMMARY/ABSTRACT Glioblastoma multiforme (GB) or grade IV glioma, is one of the most lethal human malignancies and the most common malignant primary brain tumor in adults, with a current median survival of only 14 months. Despite aggressive standard-of-care treatments including surgical resection, radiation, and chemotherapy, local recurrence of GB is essentially universal, and recurrent tumors are highly resistant to conventional cytotoxic treatments. New treatment strategies based on an improved understanding of recurrence mechanisms are desperately needed to improve overall survival for these patients. It has been highly suggested that treatment-resistant glioma cells, particularly glioma stem cells (GSCs), i.e., tumor-initiating cells or tumor-propagating cells, contribute to GB recurrence via translocation from parenchymal GSC niches. We propose an intriguing new mechanism whereby glioma cells in circulation can similarly contribute to tumor development/regrowth. Utilizing human specimen and orthotopic, genetic mouse tumor models our preliminary data demonstrate that these circulating glioma cells (CGCs) acquire a cancer stem cell-like phenotype: activated in stemness, resistant to genotoxic treatments, and more importantly, capable of homing to a primary tumor site to repopulate locally and contribute to new tumor formation. This suggests a previously unidentified role of CGCs in tumor micrometastases and local relapse in GB and possibly other solid tumors. We are uniquely positioned (as the first group to report on the identification of circulating glioma cells - CGCs) to extend our investigations to: i) decipher the key molecular features underlying CGC development and the potential contribution of CGCs to tumor bed recurrences, ii) discover novel therapeutic interventions against CGCs to overcome the universal local recurrence patterns seen in GB. Our proposal seeks to accomplish these translationally-relevant objectives through an innovative set of complementary strategies. Based on our preliminary results and expertise of the assembled team, we propose to test the hypothesis that CGCs recapitulate the features of CSCs, contribute to primary tumor reseeding and that molecular targeting of CGCs provide a novel strategy to overcome GB therapy resistance. To test this hypothesis, we propose the following specific aims: AIM 1. Define the potential stem cell features and transcriptional landscape of CGC by performing single cell RNA-seq. AIM 2. Determine the WNT- dependent mechanisms for CGC-mediated GB tumorigenesis. AIM 3. Test the therapeutic efficacy of WNT inhibition in GB tumorigenesis and therapy resistance. By accomplishing these aims via our combined interdisciplinary expertise, infrastructure, and discovery of a novel GB recurrence paradigm, we seek to build the foundation for an improved therapeutic approach for GB.