## 3 Results
### MiR-125b-5p and miR-99a-5p are Highly Expressed in Cells within Human Pancreatic Cancer Stroma  
In order to assess the expression of miR-125b-5p and miR-99a-5p in the tumors, we analyzed the paired resected pancreatic cancer tissues by in situ hybridization (ISH) with DIG-labeled locked nucleic acid miR probes. The positive and negative controls for the ISH assays are shown in Figure S2. MiR-125b-5p is expressed at low levels in the cytoplasm of normal pancreatic acinar cells, as well as in a fraction of the PanIN cells (Figure 3a). However, a small number of cells in the fibrotic stroma surrounding the invasive cancer cells express high levels of miR-125b-5p (Figure 3b). The stainings did not show a difference in the miR-125b-5p-high stromal cells in tumors from patients with short versus long PFS. Interestingly, the miR-125b-5p-high cells are localized in inflammatory cell aggregates in the tumor stroma. The miR-125b-5p-high cells are in low abundance, and resemble large immune cells such as macrophages or plasma cells. Immunohistochemistryistochemistry shows that the miR expressing cells do not overlap with CD68 expressing macrophages (Figure S3). Instead, the miR expressing cells are located within clusters of CD79A positive cells (Figure S3, Figure 3b), indicating that miR-125b-5p may play a role in B-lymphocyte/plasma cell infiltration in pancreatic cancer stroma. MiR-99a-5p expressing cells are less abundant than miR-125b-5p positive cells in the pancreatic tumors. MiR-99a-5p positive cells are only present in limited regions of tumor stroma in a few of the tumors, and are located in muscular and connective tissue where pancreatic cancer cells invade the stroma. Figure 4 shows tumor regions with cells that express high levels of miR-99a-5p. In the centers of the pancreatic tumors rich in epithelial cancer cells, there are no cells that express miR-99a-5p. When comparing the location of CD79A positive cells in the adjacent tissue slides, we observed that the miR-99a-5p expressing inflammatory cells are also in close proximity to cells of the B-cell lineage (Figure 4). The abundance of inflammatory cells that express CD79A protein, or high levels of miR-125b-5p or miR-99a-5p in the resected tumor tissues was not different in patients with short \begin{table}
\begin{tabular}{l l} \hline
**Characteristics** & **All Patients (N = 28)** \\ \hline Age (years) median (range) & 71.9 (35.5) \\ Sex (\%) Male Female & 22 (78.6)/6 (21.4) \\ T-stage * (\%)/1/2/3/4 & 0 (0.0)/0 (0.0)/28 (100.0)/0 (0.0) \\ N-stage * (\%)/0/1 & 5 (17.9)/23 (82.1) \\ Resection margin ** (\%)/R0/R1 /unknown & 13 (46.4)/14 (50.0)/1 (3.6) \\ Adjuvant Chemotherapy (\%) & 22 (78.6) \\ PFS (months)/median (IQR) & 10.7 (6.7–15.8) \\ OS (months)/median (IQR) & 16.0 (10.2–31.6) \\ \hline \end{tabular}
\end{table} 
Table 1: Patient characteristics. Patients who underwent pancreaticoduodenectomy for pancreatic cancer and were included for serum miR analysis. \begin{table}
\begin{tabular}{c c c} \hline
**Serum miR Expression** & **HR (95\% CI)** & _P-Value_ \\ \hline Pre surgery miR-122-5p & 0.941 (0.805–1.100) & 0.444 \\ Change in miR-122-5p & 1.151 (0.982–1.349) & 0.082 \\ Post surgery miR-122-5p & 1.107 (0.901–1.361) & 0.334 \\ Pre surgery miR-99a-5p & 0.999 (0.694–1.440) & 0.997 \\ Change in miR-99a-5p & 1.402 (1.003–1.961) & **0.048** \\ Post surgery miR-99a-5p & 1.734 (1.098–2.740) & **0.018** \\ Pre surgery miR-125b-5p & 1.016 (0.821–1.256) & 0.887 \\ Change in miR-125b-5p & 1.246 (0.984–1.577) & 0.068 \\ Post surgery miR-125b-5p & 1.341 (1.043–1.723) & **0.022** \\ \hline \end{tabular}
\begin{tabular}{c} miR = microRNANA; CI = confidence interval. \\ \end{tabular}
\end{table} 
Table 2: Hazard ratios (HR) for PDAC recurrence after resection according to serum miR expression levelsversus long PFS, although a single section may not represent the overall extent of expression across an entire tumor. Figure 3: MiR-125b-5p detection by in situ hybridization in pancreatic cancer tissues.