## 2 Materials and Methods
### Patient Serum miR Quantittation  
Cell-free circulating miRs from the patients were isolated from 200 \(\upmu\)L serum using the miRNeasy serum/plasma miRNA Isolation Kit (Qiagen) and eluted in 15 \(\upmu\)L nuclease free water. Two proprietary pre-mixed spike-in \(\sim\)20 nt control RNAs from MiRXES, Singapore, with sequences distinct from annotated mature human miRNAs (miRNA8 base version21) were added into the lysis buffer prior to sample miR isolation, in order to evaluate RNA isolation efficiency. MiRs were reverse transcribed using IDEAL miR-specific oligos in a multiplex reaction per manufacturer's instruction (MiRXES). In brief, up to 2 \(\upmu\)L sample RNA were mixed with 1 \(\upmu\)L RT Spike-in RNA, RT Buffer, nuclease free water, reverse transcriptase enzyme and a maximum of 10 different miR-specific RT oligos into 20 \(\upmu\)L reactions and incubated at 42 \({}^{\circ}\)C for 30 min followed by heat inactivation at 95 \({}^{\circ}\)C for 5 min in a SimpliAmp thermal cycler (Applied Biosystems). cDNA was stored at \(-20\)\({}^{\circ}\)C (up to 4 weeks) and thawed only once. Before miR quantitation, the cDNA was diluted 1:10 in nuclease free water.  
For the Reverse Transcription quantitative PCR (RT-qPCR), 5 \(\upmu\)L of sample cDNAs were mixed with the individual miRNA qPCR Assays (MiRXES), nuclease free water and the IDEAL miRNA qPCR Master Mix containing the passive reference dye ROX, into reactions of 20 \(\upmu\)L volume. The IDEAL assay from MiRXES utilizes miR-specific reverse transcription, as well as a combination of a miR-specific forward and reverse primer to quantitate miR expression, described by [PERSON] et al. [24], allowing for miR-specific and sensitive PCR. The following protocol in the 7500 Fast Real-Time PCR System (Applied Biosystems) was used: 10 min at 95 \({}^{\circ}\)C, 5 min at 40 \({}^{\circ}\)C, followed by 40 cycles of: 10 s at 95 \({}^{\circ}\)C and 30 s at 60 \({}^{\circ}\)C with FAM fluorescence reading at the end of this step. The Ct values were determined using the 7500 Software (Applied Biosystems). Technical variations introduced during RNA isolation and the process of RT-qPCR were normalized using the measurements of the spike-in control RNAs.  
In the first screen, 250 miRns were measured in the serum before and after surgery of \(n=2\) patients: one with early progressive disease after 7 months, and one patient with late disease progression after 18 months. The Ct value cutoff was 33 cycles to ensure reliability of the miR measurements. MiR levels were normalized using interplate calibrators, two spike-in control RNAs and the average expression value of two stably expressed reference miRns (miR-29c-5p and miR-421). The reference miRns were selected based on their stability factor, previously described by others [25; 26]. MiRs that were altered \(\geq\)3-fold after tumor resection, in opposite direction between the patients with short versus long progression free survival (PFS) were compared to the differentially expressed metastases-related miRns of the KPC mice. The miRns that were upregulated in progressive PDAC in mice as well as in patients, plus the two reference miRns were measured in the serum of \(n=26\) additional patients with resectable PDAC before and after pancreaticoduodenectomy.