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The prognosis for pancreatic ductal adenocarcinoma (PDAC) remains poor despite decades of effort. The abundant extracellular matrix (ECM) in PDAC comprises a major fraction of the tumor mass and plays various roles in promoting resistance to therapies. However, nonselective depletion of ECM has led to poor patient outcomes. Consistent with that observation, we previously showed that individual matrisome proteins derived from stromal cells correlate with either long or short patient survival. In marked contrast, those derived from cancer cells correlate strongly with poor survival. Here, we studied three cancer cell-derived matrisome proteins that are significantly overrepresented during PDAC progression, AGRN (agrin), SER-PINB5 (serine protease inhibitor B5), and CSTB (cystatin B). Using both overexpression and knockdown experiments, we demonstrate that all three are promoters of PDAC metastasis. Furthermore, these proteins operate at different metastatic steps. AGRN promoted epithelial-to-mesenchymal transition in primary tumors, whereas SERPINB5 and CSTB enhanced late steps in the metastatic cascade by elevating invadopodia formation and in vivo extravasation. All three genes were associated with a poor prognosis in human patients and high levels of SERPINB5, secreted by cancer cells and deposited in the ECM, correlated with poor patient prognosis. This study provides strong evidence that cancer cell-derived matrisome proteins can be causal in promoting tumorigenesis and metastasis and lead to poor patient survival. Therefore, compared with the bulk matrix, mostly made by stromal cells, precise interventions targeting cancer cell-derived matrisome proteins, such as AGRN, SERPINB5, and CSTB, may represent preferred potential therapeutic targets. Significance: This study provides insights into the biological roles of cancer cell-derived matrisome proteins in PDAC and supports the notion that these proteins are protumorigenic and better therapeutic targets.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by rich deposits of extracellular matrix (ECM), affecting the pathophysiology of the disease. Here, we identified galectin 4 (gal 4) as a cancer cell produced protein deposited into the ECM of PDAC tumors and detected high circulating levels of gal 4 in PDAC patients. In orthotopic transplantation experiments we observed increased infiltration of T-cells and prolonged survival in immunocompetent mice transplanted with cancer cells with reduced expression of gal 4. Increased survival was not observed in immunodeficient RAG1-/- mice, demonstrating that the effect was mediated by the adaptive immune system. Furthermore, by performing single-cell RNA-sequencing we found that the myeloid compartment and cancer-associated fibroblast (CAF) subtypes were altered in the transplanted tumors. Reduced gal 4 expression was associated with higher proportion of myofibroblastic CAFs and reduced numbers of inflammatory CAFs. We also found higher proportions of M1 macrophages, T-cells and antigen presenting dendritic cells in tumors with reduced gal 4 expression. Using a co-culture system, we observed that extracellular gal 4 induced apoptosis in T-cells by binding N-glycosylation residues on CD3 epsilon/delta. Hence, we show that gal 4 is involved in immune evasion and identify gal 4 as a promising drug target for overcoming immunosuppression in PDAC. 

The ability to evade the immune system is crucial for cancer cells to survive. In pancreatic ductal adenocarcinoma (PDAC), various mechanisms contributing to immunosuppression have been described, including the recruitment of suppressive immune cells like M2 macrophages, the expression of cell membrane attached proteins like PDL-1, or secretion of extracellular proteins inducing immune cell apoptosis. PDAC is characterized by a rich stroma, consisting of large quantities of extracellular matrix (ECM) proteins, immune cells, fibroblasts and blood vessels. Cancer cell-derived proteins deposited in the stroma can inhibit immune cell function and thereby contribute to the progression of the disease. Galectin 4 (gal 4) is highly expressed by PDAC cancer cells and is secreted into the stroma and has recently been shown to have the capacity to induce T-cell apoptosis in PDAC tumor. High levels of gal 4 is also associated to poor prognosis and reduced immune activity in PDAC patients. Here we show that sets of immunosuppressive genes form groups based on correlation of expression levels. Gal 4 gene expression correlates with other galectin family proteins, collectively clustering into a distinct immune evasion group. This cluster has negative correlation to other more classical immunosuppressive factors, such as PDL-1, CXCL12, and TGFBI, indicating that a subset of tumors mainly relies on galectins to achieve immune evasion. Conversely, tumors with low expression of gal 4 have high expression of one or more of the classical immunosuppressive factors. These results indicate that different tumors rely on different mechanisms to achieve immune evasion and emphasize the need for personalized treatment strategies when targeting immunosuppression in PDAC.