Mechanistic Insights and Diagnostic Applications for Hypoxia-Induced Vasorin in Pancreatic Cancer

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Project Summary With the advent of cancer immunotherapies, patients have seen dramatic increases in the availability of treatment options for various malignancies. However, many of these modalities lack efficacy in treating solid tumors, namely because of the unique set of environmental factors that exist within these tumors. One of the most ubiquitous features within the tumor microenvironment (TME) is oxygen deprivation, also known as hypoxia. Hypoxia has long been linked to increased metastasis and poorer prognoses for patients. In response to hypoxic stress, cancer cells activate highly regulated cellular pathways and gene programs that promote survival, migration, immune privilege, and increased mortality for patients. Across all solid tumors, pancreatic cancer exhibits the most severe hypoxic phenotype, which may contribute to the high mortality rate associated with this cancer type. These hypoxia-specific downstream effects suggest that tumor hypoxia can be used to elucidate selective biomarkers of solid pancreatic cancer for diagnostic and therapeutic purposes. Here, I utilize cell surface proteomics to identify a novel hypoxia-regulated target in pancreatic cancer called vasorin (VASN), a target previously implicated in the progression of glioblastoma. Furthermore, I have shown that VASN plays an important role in the growth and survival of pancreatic cancer under hypoxic stress. Finally, I have isolated and expressed ten unique antibody clones against the ectodomain of VASN. Using these antibodies, I found that VASN undergoes numerous cleavage events under hypoxia in vitro, which are unique from previously published results. We hypothesize that VASN cleavage is necessary for the survival and proliferation of pancreatic cancer under hypoxia, and that classification of expression levels for cleavage-specific forms of VASN in relevant tumor models will aid the downstream development of antibody-based diagnostics for hypoxic pancreatic cancer. To test this hypothesis, I will employ proteomic characterization and recombinant protein expression to identify the in vitro cellular consequences of VASN proteolysis. In order to isolate clones towards the membrane-retained form of cleaved VASN, I will use an established phage display-based recombinant antibody strategy. A selective antibody-based biotinylation strategy will be implemented to identify novel interacting partners of the membrane- retained form of cleaved VASN that may play an important role in downstream signaling. Finally, I will functionalize my existing and future anti-VASN antibody clones for serum-based ELISA detection and radiotracer-based immunoPET imaging of VASN, respectively, which will validate the presence of proteolyzed VASN in human serum and tumors, as well as in an in vivo nu/nu mouse model of pancreatic cancer. The proposed studies will provide mechanistic insights into the functional consequences of VASN proteolysis in pancreatic cancer, as well as providing evidence for the development of non-invasive, early-detection diagnostic alternatives for a highly metastatic and deadly disease.