Lab OHSU Center for Spatial Systems Biomedicine Platelet-Derived Soluble and Membrane-Bound Proteins Differentially Influence C-MYC Expression in Pancreatic Cancer Cells

Despite the development of promising targeted cancer therapeutics, many treatments have limited efficacy in patients over time. Often this is due to the ability of aberrant signaling pathways to circumvent druginduced roadblocks. In most cases, this complex reorganization remains unresolved at the single-molecule level because of the inherent difficulty in visualizing individual proteins in situ. Multispectral superresolution microscopy (MSSRM), an extension of single-molecule superresolution microscopy (SRM) where up to 20 proteins in a single pathway may be tagged with distinct fluorophores and imaged at the nanoscale, provides a unique opportunity to obtain both structural and temporal protein information while preserving spatial integrity in cells and tissues. However, MSSRM relies on the ability to site-specifically tag individual protein units with a fluorophore in a controlled manner. Few studies have quantified the efficiency and utility of different protein affinity tags (e.g. whole IgG, antibody fragments, nanobodies, affibodies, aptamers), particularly when multiple targets must be labeled within close proximity. In the current work, HER2, a transmembrane protein overexpressed in approximately 25% of all breast cancer, is used as a model system for quantitative protein labeling studies in fixed cells. By engineering HER2 monomer units with genetically encoded fluorescent protein tags, we are conducting co-localization studies to screen potential affinity tags for use in quantitative MSSRM. These techniques will also be applied to live cell imaging, and offer the possibility for quantitative protein labeling in high-resolution microscopy including SRM and electron microscopy. Andrew Gunderson Coussens Lab Department of Cell, Developmental & Cancer Biology *ePoster Presenter* BTK Signaling Regulates Immune Cell Crosstalk to Promote Pancreatic Carcinogenesis Andrew J. Gunderson1,14, Megan M. Kaneda5,14, Takahiro Tsujikawa1,2, Abraham V. Nguyen5, Nesrine I. Affara7, Brian Ruffell1, Sara Gorjestani5, Shannon M. Liudahl1, Morgan Truitt9, Peter Olson9, Grace Kim7,10, Douglas Hanahan6, Margaret Tempero8,10, Brett Sheppard3,4, Bryan Irving11, Betty Y. Chang12, Judith A. Varner5,13,14, Lisa M. Coussens1,4,14, Abstract: Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive cancers, has a high rate of Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive cancers, has a high rate of chemo-resistance, and has few treatment options. Recently, immunotherapies for patients with late-stage metastatic disease have increased PDA survival rates, giving promise to a once hopeless malignancy. Despite efficacy of these therapies, survival rates remain very low. We recently established a functional role for B cells and humoral immunity as promoting forces in PDA using in mouse models of late-stage PDA growth. Our studies revealed that inhibiting pro-tumorigenic B cells, or the pathways they activate in myeloid cells, improve responses to chemotherapy and mobilize anti-tumor CD8+ T cell responses, as well as reducing desmoplasia, a characteristic feature of PDA. B cells accomplish this through chronic activation of Bruton’s tyrosine kinase (BTK) both directly downstream of the B cell receptor and through paracrine communication with FcR + my Accordingly, the BTK antagonist, Ibrutinib, ameliorated PDA growth in tumor-bearing mice that was associated with attenuated Th2 immunity, enhanced CD8+ T cell activation, and reduced fibrosis when administered in combination with standard of care chemotherapy. These pre-clinical discoveries can be rapidly translated into PDAC patients with chronic pro-tumoral B cell activity.