## 4 Discussion
There has been very little to no research that compares the levels of miR after surgical removal of primary pancreatic tumors. In our study, we compared serum miRs pre- and post-resection in treatment-naive patients that were treated at the Erasmus Medical Center in the Netherlands between 2013 and 2017. Recent studies show that patients with (borderline) resectable PDAC that undergo preoperative chemo/radiotherapy may have improved survival [56; 57; 58; 59]. From now on, all patients with (borderline) resectable PDAC in the Netherlands will be offered to receive preoperative chemotherapy (with or without radiotherapy) in the context of a clinical trial (PREOPANC-2 trial), if the performance status of the patient permits and the patient is willing to undergo systemic treatment. Chemotherapy has a vast impact on the immune landscape [60; 61]. Whether serum miR-125b-5p and miR-99a-5p are still predictive of disease progression after surgery in pre-treated patients remains to be evaluated. Therefore, we are currently collecting blood samples to evaluate the changes in the serum miRs of patients with (borderline) resectable tumors who undergo pre-operative FOLFIRINOX chemotherapy or gemcitabine-based chemoradiation. In summary, we found that serum miR-125b-5p and miR-99a-5p levels in patients with PDAC are potential indicators of early disease recurrence after surgery and are likely originating from immune cells. This is a pilot study, the reliability and validity of these circulating biomarkers to accurately detect early PDAC recurrence needs to be validated in a larger independent cohort. Moreover, performing dual stainings for miR expression by ISH and immune related protein expression by immunohistochemistry is one of our future directions. Further dissection of the dynamics and functions of miR-125b-5p and miR-99a-5p in the immune response to pancreatic cancer will provide fundamental information to assist in the development of biomarkers and better immune therapies. **Supplementary Materials:** The following are available online at [[http://www.mdpi.com/2076-3417/9/22/4784/s1](http://www.mdpi.com/2076-3417/9/22/4784/s1)]([http://www.mdpi.com/2076-3417/9/22/4784/s1](http://www.mdpi.com/2076-3417/9/22/4784/s1)). **Figure S1. ** Different stages of pancreatic neoplasia in KPC mice at the age of 5 months, (a) Images of formalin fixed, paraffin embedded (FFFE), H&E stained pancreatic tissues obtained from _LSL-Kras_S12D/+, _LSL-Trp53_R12 ZH/+, _P48-Cre_ (KPC) mice at the age of 5 months. PanIN-3 lesions are indicated by the green arrows. 20X magnification, scale bar = 100 \(\upmu\)m. (b) Images of FFPE, H&E stained liver and lung tissues from KPC mice at the age of 5 months. Metastatic pancreatic cancer lesions are indicated by the black arrows. 10X magnification, scale bar = 100 \(\upmu\)m; **Figure S2. ** MiR in situ hybridization (ISH) controls. Images of FFPE tissues that are stained with silver after in situ hybridization of dual-DIG labeled miR probes leading to brown/black staining. Nuclei are stained with Hematoxylin in blue. (a) U6 expression in human colon and testis tissue, serve as positive controls for the ISH assay. Scale bars = 250 \(\upmu\)m. (b) MiR-125b-5p expression in Her2 Neu positive human breast cancer and human testis tissues. Note the distinct staining patterns in the breast cancer cells and Sertoli cells that show low miR-125b-5p expression (green arrows) versus the single cells in the stroma that have high miR-125b-5p expression (red arrows). Scale bars = 250 \(\upmu\)m. (c) MiR-99a-5p expression in human pancreatic cancer and liver. Cells in the stroma with high levels of miR-99a-5p are indicated by the red arrows. (d) Scramble miR expression in human colon and testis tissues, serves as a negative control for the ISH assay; **Figure S3. ** MiR-125b-5p, CD79A and CD68 expression in human PDAC tumor stroma.