Patients and sample collection
Blood samples were collected in heparinized tubes from patients with MPC (n = 54), RPC (n = 12), and BPC (n = 52) between April 2019 and January 2022. All patients with MPC and RPC were histologically verified; patients with MPC were treatment-naïve and had no other cancers. Blood samples were collected from patients with MPC before the introduction and 2 months after. Blood samples from patients with RPC were collected immediately before surgery, and tissue samples were obtained from the resected specimens. Patients with MPC received S-IROX, modified FOLFIRINOX (mFFX), or GnP. S-IROX was administered in a clinical trial. After receiving S-IROX or mFFX, patients were treated every 2 weeks: S− 1 40 mg/m2 was administered orally twice daily on days 1 to 7 in S-IROX, and 5-fluorouracil 2400 mg/m2 was intravenously administered for 46 h without bolus infusion in mFFX, in addition to intravenous oxaliplatin (85 mg/m2) and irinotecan (150 mg/m2) on day 1. Patients in GnP therapy received gemcitabine 1000 mg/m2 plus nab-paclitaxel 125 mg/m2 intravenously on days 1, 8, and 15 of each 28-day cycle. Computed tomography (CT) or magnetic resonance imaging (MRI) was used to evaluate the response to chemotherapy every 2–3 months according to the guidelines of the Response Evaluation Criteria in Solid Tumors (RECIST) 1.0. Patients with BPC had serous cyst neoplasm (SCN), simple cyst, or intraductal papillary mucinous neoplasm (IPMN), but no malignant findings in the pancreas, as evaluated by image inspection such as CT, MRI, and endoscopic ultrasonography. The study protocol was approved by the Ethics Committee of Kyushu University, and informed consent was obtained in writing from all patients (approval number, 2020–620).
Processing tissue samples
Tissue samples were washed with DMEM (high glucose) (FUJIFILM Wako Pure Chemical Corporation, Japan) and minced into pieces < 0.75 μm in length. BD HorizonTM Dri Tumor and Tissue Dissociation Reagent (TTDR) (BD Biosciences, Tokyo, Japan) was used to extract TILs from tissues. According to the protocol presented, minced tissues were suspended in 1x TTDR and incubated at 37 °C with frequent agitation for 75 min. Three volumes of phosphate-buffered saline (PBS) were added to the TTDR, including tissues, and filtered through a 70-μm filter (Falcon Catalog number 352350). The filtrate was centrifuged at 300×g for 8 min. The pellets, including TILs, were suspended in cell-staining buffer (BioLegend, Tokyo, Japan) and used for flow cytometry.
Flow cytometry
Peripheral blood mononuclear cells (PBMCs) were purified from each venous blood sample by Lymphoprep™ (Serunwerk Bernburg, Oslo, Norway) and washed twice with PBS. PBMCs and TILs, collected as described above, were suspended in cell-staining buffer and FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to block non-specific binding for 10 min. PBMCs and TILs were stained for cell surface molecules, while PBMCs were intracellularly stained for Foxp3 and cytotoxic T-lymphocyte-associated protein 4 (CTLA4). The used antibodies (Abs) are follows: Brilliant Violet 421™ anti-CD122 (clone TU27, BioLegend, Tokyo, Japan), Brilliant Violet 605™ anti-CD45R (clone RA3-6B2, BioLegend, Tokyo, Japan), Brilliant Violet 785™ anti-CD4 (clone RPA-T4, BioLegend, Tokyo, Japan), Brilliant Violet 785™ anti-CD8 (clone SK1), fluorescein isothiocyanate (FITC) anti-CD3 (clone OKT3, BioLegend, Tokyo, Japan), FITC anti-CD4(clone RPA-T4, BioLegend, Tokyo, Japan), Pacific Blue™ anti-CD3 (clone OKT3, BioLegend, Tokyo, Japan), phycoerythrin (PE) anti-FOXP3 (clone 150D, BioLegend, Tokyo, Japan), PE anti-CD107a (clone H4A3, BioLegend, Tokyo, Japan), phcoerythrin-cyanine™ 7 (PE-Cy™7) anti-CD28 (clone CD28.2, BioLegend, Tokyo, Japan), Allophycocyanin (APC) anti-CD25 (clone BC96, BioLegend, Tokyo, Japan), APC anti-CD19 (clone HIB19, BioLegend, Tokyo, Japan), Alexa Fluor647™ anti-CTLA4 (clone L3D10, BioLegend, Tokyo, Japan), Brilliant Violet 421™ mouse IgG1κ (clone MOPC-21, BioLegend, Tokyo, Japan), Brilliant Violet 605™ mouse IgG1κ (clone MOPC-21, BioLegend, Tokyo, Japan), Brilliant Violet 785™ mouse IgG1κ (clone MOPC-21, BioLegend, Tokyo, Japan), FITC mouse IgG1κ (clone MOPC-21, BioLegend, Tokyo, Japan), FITC mouse IgG2aκ (clone MOPC-173, BioLegend, Tokyo, Japan), Pacific Blue™ mouse IgG2aκ (clone MOPC-173, BioLegend, Tokyo, Japan), PE mouse IgG1κ(clone MOPC-21, BioLegend, Tokyo, Japan), PE-Cy™ 7 mouse IgG1κ (clone MOPC-21, BioLegend, Tokyo, Japan), APC mouse IgG1κ (clone MOPC-21, BioLegend, Tokyo, Japan), Alexa FluorR 647 mouse IgG1κ (clone MOPC-21, BioLegend, Tokyo, Japan). We used Zombie NIR™ dye (BioLegend, Tokyo, Japan) to determine cell viability. PBMCs were stained with Ab and Zombie dye on the surface at room temperature in the dark for 20 min. Next, using the Fixation/Permeabilization Buffer Set (eBioscience, Tokyo, Japan), we stained intracellular FOXP3 or CTLA4 using the manufacturer’s instructions. Briefly, each sample was incubated with a fixation/permeabilization buffer at room temperature in the dark for 30 min. Each sample was washed twice with permeabilization buffer, anti-FOXP3 Ab or anti-CTLA4 Ab was added, and the samples were incubated at room temperature in the dark for 30 minutes. Each sample was washed twice with permeabilization buffer, diluted in cell-staining buffer, and analyzed by flow cytometry. All the prepared samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, California, USA). The gating strategy is illustrated in Online Resources 1–3.
Statistical analysis
Statistical analysis was conducted using the JMP v. 16 software (SAS and JMP, Institute Inc., Cary, NY, USA). We analyzed the differences in each PBL subtype between patients with MPC and BPC using Student’s t-test. To evaluate the correlation between two continuous variables, we performed correlation analysis using Spearman’s rank correlation coefficient. The Kaplan–Meier method was employed with the log-rank test to compare survival rates between the two groups. The chi-square test was used to compare categorical groups. Differences between two values of the same groups were analyzed using paired t-test with Wilcoxon signed-rank test. Multivariate analysis was performed using a multiple logistic regression model or a Cox multivariate proportional hazard regression model. Statistical analysis was performed by statistical experts from the Academic Research Organization at Kyushu University.