PROJECT SUMMARY Hepatoblastoma (HB) is the most common liver cancer seen in children and has a poor overall survival in patients that present with vascular invasion (VI). Standard of care treatments with additional chemotherapy and surgical treatment have been developed, but despite this aggressive treatment, the survival rate remains <50%. Furthermore, our group recently reported that HB patients with microvascular invasion (micro-VI) had worse overall survival rates than those without micro-VI. Despite the understanding that VI is the precursor to multifocal disease and metastasis, little is known about the mechanisms that enable VI in HB, nor are there any targeted therapies to address this mechanism of disease. Therefore, to create effective therapies, further understanding of the biomarkers and biology of VI in HB is critically needed. Our prior work identified the JAK/STAT3 pathway as being significantly upregulated in the tumor thrombus of patients with high-risk HB and VI. In addition, downstream targets of the JAK/STAT3 pathway were upregulated in a patient-derived xenograft model of high-risk HB, and STAT3 inhibition in our patient-derived cell lines countered HB cell growth and invasion. Based on the rigor of our and other’s prior research, we hypothesize that HB tumors with VI exhibit unique expression signatures that involve JAK/STAT3 signal activation, which can inform the development of VI-targeted therapies. To address this, our first aim (Aim 1) will be to investigate changes in protein expression between HB cells in the primary tumor and those involved in VI to identify a VI-specific protein signature using innovative Phenocycler analysis. This signature will then be validated in a cohort of FFPE patient tissues as a novel biomarker panel to identify patients at risk for VI, as well as pathway targets for future drug development. Then in Aim 2, we will study the role of JAK/STAT3 in VI. Our laboratory has shown that JAK/STAT3 plays a role in enabling cancer cells to invade the blood, emphasizing the role that JAK/STAT3 may have in HB metastasis and VI. Using gain- and loss-of-function assays, we will prove the necessity and sufficiency of STAT3 function to drive HB tumor cell invasion. We will then test targeted JAK/STAT3 pathway inhibitors in vitro and in vivo using two of our unique high-risk patient derived xenograft models as a validation tool. Taken together, these aims will result in new prediction models and preclinical data to inform future clinical trials for high-risk HB patients with VI. Completion of this project will also provide me with a foundation in novel research techniques and help develop my scientific acumen so that I can build a successful career as a surgeon- scientist.