Infiltrative growth in pancreatic cancer and its liver metastases

Pancreatic ductal adenocarcinoma (PDAC) is the most common cancer form arising from the pancreas. It has one of the worst prognoses among solid tumors, with a five-year overall survival rate of around 11%. PDAC most frequently metastasizes to the liver. As liver metastases from PDAC, but also colorectal cancer expand, they invade into the hepatic parenchyma in either of two ways: either with direct contact with the hepatocytes (replacement growth pattern [GP]), or with a fibrotic rim separating the tumor cells from the hepatocytes (encapsulated GP). Pancreatic neuroendocrine tumors (panNETs) are a smaller subset of pancreatic tumors that are associated with a better prognosis compared to PDACs. Some panNETs stay relatively confined, grow slowly and do not metastasize, while some display more malignant behavior. PDAC and panNETs approach the pancreatic parenchyma in fundamentally different ways. PDAC is recognized for its desmoplastic reaction, where tumor cells are embedded in a thick, characteristic stroma. Hence, the parenchymal atrophy in relation to PDAC is often critical and severe. On the other hand, panNETs are often surrounded by more well-preserved parenchyma, even in areas where the tumor cells are directly adjacent. Similar to liver metastases, but less characterized, panNETs can show two GPs: lobular, and encapsulated. In this thesis, we explored the invasive patterns of PDAC, panNETs, and liver metastases by generating quantitative measures from digitalized whole-slide microscopy images of resection specimens for the respective tumor types. Murine orthotopic injection models and in vitro cultures were included to provide functional insights into the emergence of distinct GPs. In Study I, we found that a higher fraction of encapsulated GP in human liver metastases is associated with a favorable survival. In addition, the dynamic constituents of the capsule were characterized. Firstly, the inner, tumor-facing edge of the capsule was enriched for fibroblast activation protein. Secondly, the outer, hepatocyte-facing edge of the capsule was enriched for the benign fibrosis markers nerve growth factor receptor (NGFR), a smooth muscle actin, platelet-derived growth factor receptor alpha (PDGFRa), collagen IaI, decorin and fibronectin. From the outer to the inner edge of the capsule, we observed the gradual loss of parenchymal remnants, including portal triads and hepatocytes. In a murine liver injection model, the degree of encapsulation increased upon chemotherapy treatment. Collectively, our results indicated that the capsule forms from the outer edge, where early fibrosis occurs and the liver parenchyma decays, when tumor cell invasion into the liver cell plates fails. In Study II, we studied the features of local PDAC invasion into the pancreatic parenchyma. Although most tumor cells were located in archetypical desmoplastic stroma, lobular invasion occurred in the majority of PDACs. Tumor cells in lobules had upregulated markers characteristic of the previously established classical subtype, such as mucin 5AC oligomeric mucus/gel-forming (MUC5AC) and caudal type homeobox 2 (CDX2). Conversely, tumor cells embedded in desmoplastic stroma displayed a more basal-like expression profile, defined by the markers cytokeratin 17 (CK17), carcinoembryonic antigen 125 (CA125) and high mobility group AT-hook 2 (HMGA2). A further characterization of the lobular microenvironment revealed NGFR+ stromal cells in lobules with early tumor invasion and/or atrophy. NGFR+ stromal cells consistently co-expressed PDGFRa, both in our study population and in three publicly available single-cell sequencing datasets, including murine and human PDAC. Moreover, PDAC cells within the pancreatic lobules were observed in proximity to epithelial cells that had lost their acinar differentiation and instead undergone acinar-to-ductal metaplasia (ADM), a canonical pancreatic injury response. Taken together, our results pointed toward the lobules as a microniche with early signs of a fibro-inflammatory microenvironment, where tumor cells upregulated a classical-subtype related protein expression. In Study III, we characterized the invasion patterns of panNETs, with a focus on their GPs and parenchymal attributes. First, we annotated the GP at the interface to the parenchymal invasion front and found that, among n = 40 panNETs, none grew with only a lobular GP. Instead, all had varying degrees of encapsulated GP, and half of the population were encapsulated to over 90%. A clustering analysis of these primarily encapsulated panNETs revealed three subgroups, which we stratified by tumor size. The smallest panNETs were related to the least parenchymal atrophy. Overall, panNETs of intermediate size were characterized by more frequent anomalous vasculature and greater peritumoral atrophy than smaller tumors. Lastly, the largest tumors were associated with the most severe peritumoral atrophy, and for these tumors, the counts of anomalous vasculature were low. We interpret these data, which are part of an ongoing study, as evidence of peritumoral vascular remodeling, implying greater degrees of atrophy as the tumor expands. To conclude, our data highlight reciprocal interactions between normal parenchyma and tumor cells in the liver and pancreas. Given the differences in GPs of panNETs and PDAC tumor invasion, further studies on the molecular drivers of these differences are motivated. These basic tumor biology insights can lay the groundwork for an improved understanding of how future clinical interventions to inhibit tumor cell invasion could be applied. List of scientific papers I. An idiosyncratic zonated stroma encapsulates desmoplastic liver metastases and originates from injured liver. Carlos Fernández Moro, Natalie Geyer, Sara Harrizi, Yousra Hamidi, Sara Söderqvist, Danyil Kuznyecov, Evelina Tidholm Qvist, Media Salmonson Schaad, Laura Hermann, Amanda Lindberg, Rainer L. Heuchel, Alfonso Martín-Bernabé, Soniya Dhanjal, Anna C. Navis, Christina Villard, Andrea C. del Valle, Lorand Bozóky, Ernesto Sparrelid, Luc Dirix, Carina Strell, Arne Östman, Bernhard Schmierer, Peter B. Vermeulen, Jennie Engstrand, Béla Bozóky, Marco Gerling. Nature Communications 14, 5024 (2023). 2023-08-18. https://doi.org/10.1038/s41467-023-40688-x II. An injury-associated lobular microniche is associated with the classical tumor cell phenotype in pancreatic cancer. Sara Söderqvist, Annika Viljamaa, Natalie Geyer, Anna-Lena Keller, Kseniya Ruksha, Carina Strell, Neda Hekmati, Alexandra Niculae, Jennie Engstrand, Ernesto Sparrelid, Caroline Salmén, Tânia D.F. Costa, Miao Zhao, Staffan Strömblad, Argyro Zacharouli, Poya Ghorbani, Sara Harrizi, Yousra Hamidi, Olga Khorosjutina, Stefina Milanova, Bernhard Schmierer, Béla Bozóky, Carlos Fernández Moro, Marco Gerling. Nature Communications 16, 8307 (2025). 2025-09-26. https://doi.org/10.1038/s41467-025-63864-7 III. Invasion patterns in pancreatic neuroendocrine tumors are interconnected to the peritumoral vascular system. Celina Anjali Limbecker, Sara Söderqvist, Manuel Molina-Centelles, Béla Bózoky, Marco Gerling, Carlos Fernández Moro. [Manuscript]