## 4 Discussion
The median survival of KPC mice is 5 months and all mice succumb to the disease before the age of one year [23]. We performed a cross-species comparison of serum miR expression: the prognostic miRs we identified in the patients shortly after surgery were also correlated to miRs in KPC mice with PDAC metastases, confirming their significance in pancreatic cancer progression. Most murine miRs are homologues to human miRs and share the same sequence. We made this comparison across species given the notion that patients who develop radiographically visible cancer lesions within 8 months after tumor resection are considered to have occult metastatic disease at the time of surgery. Circulating miRs that are associated with cancer presence often do not originate from cancer cells themselves. For example, miR-125b expression in colorectal liver and lung metastases is \(\sim\)3 fold and \(\sim\)7-fold higher in the stroma than in the cancer cells [34]. Indeed, the majority of circulating miRs are derived from blood cells and the endothelium [35; 36]. PDAC progression goes hand in hand with alterations in systemic immune cell profiles [37] and this link between altered circulating miRs during cancer progression and immune cells was confirmed in our study. We found that miR-125b-5p and miR-99a-5p levels are high in cells that are closely associated with B-lymphocytes in the tumor stroma. Others have shown that miR-125b-5p is upregulated in B-lymphocytes [38] and can cause leukemia in mice [39; 40]. In pancreatic tumors, high levels of infiltrating plasma cells are significantly correlated with worse prognosis in terms of overall survival in patients after surgery [41]. We found that after surgical tumor removal, patients with high levels of serum miR-125b-5p or miR-99a-5p have shorter PFS. MiR-125b-5p and miR-99a-5p belong to the 20 most abundant miRs in human plasma exosomes [42], indicating that these miRs are actively packaged and released into the bloodstream. Circulating miRs are transferred from cell to cell and can elicit immune modulation [43]. Serum miRs interact more with immune-related mRNA genes that with non-immune related genes [44]. MiR-containing T-regulatory-cell-derived exosomes suppress pathogenic T helper 1 cells [45]. A recent study showed that miR-125b-5p and miR-99a-5p both downregulate the activation of \(\gamma\)8 T-lymphocytes and their cytotoxicity towards lymphoma cells [46]. In humans and rats, treatment with methylprednisolone leads to increased plasma miR-99a-5p levels, suggesting miR-99a-5p is involved in systemic immune suppression [47]. Others have shown that miR-99a-5p inhibits the mammalian target of rapamycin (mTOR) signaling in bladder cancer cells [48], which was also described in gastric cancer tissues by [PERSON] et al. [49]. mTOR is not only an important cancer-related pathway, but also crucial for hematopoietic cell fate [50; 51], since the differentiation of naive T-cells into distinct effector T-cells is promoted by mTOR [50]. In the absence of mTOR activity myeloid differentiation is impaired due to a block in glucose uptake and lipid metabolism [52]. Cell-free miR-99a-5p levels are high in the blood of patients with progressive PDAC, suggesting that the miR is produced at high levels by a subset of leukocytes, and can potentially be taken up by other cells, leading to the abrogation of mTOR among other pathways. On the other hand, expression of miR-122-5p is liver specific and absent in most other tissues [53; 54; 55]. In the KPC mice with metastatic PDAC, miR-122-5p was upregulated 22-fold in comparison to mice with pre-invasive lesions. In the patients who underwent surgical tumor resection, serum miR-122-5p levels went up after surgery in patients with early disease recurrence, whereas post-surgery serum miR-122-5p levels did not distinguish patients based on PFS, or based on the presence of liver metastases. This may suggest that liver damage is present at some level in all patients who undergo pancreaticoduodenectomy for PDAC.