Project Summary - Overall The mortality rates of breast and pancreatic cancers are intrinsically tied to metastasis. In pancreatic cancer, the 5-year survival rate is only 9% and in ~70% pathological evaluations of the resected tumor, instances of venous invasion are found. Metastasis is a complex multi-step process involving cancer cells, local vasculature, and the surrounding microenvironment at multiple sites. Venous invasion in pancreatic cancer, in which cancer cells gain often invade the portal vein, is an early step in this process and provides the cells a direct path to the liver, the most common site of pancreatic cancer metastasis. As in pancreatic cancer, invasion past normal breast tissue barriers is critically tied to breast cancer outcomes. Most breast tumors can be surgically removed, and so mortality is closely tied to the extent of distant metastasis through lymphovascular invasion. The detection of lymphovascular invasion in a breast tumor correlates with poor prognosis and is not captured in current molecular analyses. The spatial organization of cancer cells, and cellular and stromal components of the tumor microenvironment near and far away from blood vessels is intrinsically three-dimensional, non-symmetric, and highly heterogeneous. In the TECH units of the Johns Hopkins Center for 3D Multiscale Cancer Imaging, we will develop a versatile 3D multiscale imaging method, CODA, which will allow us to probe the phenotypic heterogeneity of tumors from the multi-cm to the micron scale via multiplexing serial imaging. CODA can readily incorporate other imaging modalities to extract high cellular/molecular content from 3D samples. These include immunocytochemistry (CODA+IHC), immunofluorescence (CODA+IF), imaging mass cytometry (CODA+IMC) and spatial transcriptomics/proteomics (CODA+DBiT-seq). These proposed expanded versions of CODA offer a unique opportunity to produce new 3D multi-omic maps of human PDAC and breast tumors near and far from blood vessels. CODA and its integrated versions CODA+X will be tested in the RTB units of the Center in both human/mouse tissue samples and organoids, in breast and pancreatic cancer. Results from these test beds will provide novel mechanistic insights into venous invasion in breast and pancreatic cancer. Exploiting our extensive experience in data dissemination, we will make the large datasets and software produced by our CODA+X platforms and software widely available to the larger cancer research community. The units of the Center will be co-led by Johns Hopkins/Yale engineers, scientists and physicians. Substantial additional support will be provided by the Johns Hopkins U., the JH School of Medicine, the Institute for Convergence, the Department of Pathology, and the Whiting School of Engineering.

Project Narrative We propose to create a Center that will focus on developing new 3D imaging tools to visualize breast and pancreatic tumors at high spatial resolution. Our multidisciplinary team of clinicians, computational biologists, and bioengineers will develop computer algorithms combined with advanced molecular tools to recognize key types of cells that can contribute to the spread of cancer cells from the primary tumor site to distant sites in 3D.