Cancer Letters br Cells and culture conditions br Human
Cancer Letters 444 (2019) 70–81
2.3. Cells and culture conditions
Human pancreatic stellate cell (PSC) lines were established from fresh surgical specimens of pancreatic cancer using the outgrowth method [21–23]. Pancreatic cancer cell (PCC) lines from the primary tumors of KPC mice were also established using an outgrowth method as described previously . In addition, eleven other PCC lines were used in this study: SUIT-2, MIAPaCa-2 (Japanese Cancer Resource Bank, Osaka, Japan), Panc-1 (RIKEN BRC, Tsukuba, Japan), AsPC-1, SW1990, Capan-2, CFPAC-1, BxPC-3 (American Type Culture Collec-tion, VA, USA), KP-3, H48N, and KP-2 (Dr. H. Iguchi, National Shikoku Cancer Center, Matsuyama, Japan). All PCC lines were obtained be-tween 2011 and 2016, and propagated and frozen immediately after arrival. Cell lines were regularly authenticated by matched short tandem repeat DNA profiling. All 170277-31-3 were maintained as described previously .
2.4. Transgenic mouse models and in vivo experiments
The KPC and LSL-KrasG12D/+;Pdx-1-Cre (KC) mouse models have been described previously [6,9]. Mice were genotyped by PCR using primers specific for transgenic alleles. KPC-derived PCCs (1 × 106) were orthotopically implanted into the tail of the pancreas of 7-week-
old KC mice, which is before the formation of ADM in the host pan-creatic parenchyma that results from the influence of KrasG12D muta-
tion. The mice were sacrificed on day 14 to exclude the possibility that acinar cells of the host pancreas change into cancerous lesions via multistep carcinogenesis. All orthotopic tumors, along with sur-rounding tissue, were excised and weighed. Tumor volume was calcu-lated by the formula π/6 × (L × W2), where L is the largest tumor diameter and W is the smallest tumor diameter. All animal experiments were approved by the Ethics Committee of Kyushu University.
2.5. 3D culture of pancreatic acinar cells
For 3D explant cultures [25,26], cell culture plates were coated with collagen I in Waymouth's media. Isolated pancreatic acinar cells (PACs) were resuspended in a mixture of collagen I/Waymouth's media and added on the top of this layer. Waymouth's complete media was then added on top of the cell/gel mixture. The media was replaced the fol-lowing day and then every other day during the course of explant culture. Ductal area was measured using ImageJ software at day four of explant culture.
2.6. Gene expression analysis
Gene expression was analyzed using the Mouse PanCancer Pathways Panel (XTeCSOeMPATH1-12) on the nCounter system (NanoString Technologies, Seattle, WA)  according to manufac-turer's instructions. Raw data were normalized to the stable house-keeping gene, which was selected automatically by the system. The gene expression heatmap was generated in MultiExperiment Viewer version 4.9. Venn diagram analysis was performed using the VENNY tool (Bioinformatics for Genomics and Proteomics). Functional anno-tation clustering analysis was performed using the DAVID database (National Institute of Allergy and Infectious Diseases). Gene set en-richment analysis (GSEA) was performed for the three types of ADM by comparing expression data with that of normal acini. Comparisons were made by entering fold-change expression data from the nCounter ana-lysis into the GSEA software (Broad Institute, UC San Diego) .
2.7. Statistical analysis
Data are represented as the mean ± standard deviation (SD). Comparisons between two groups were carried out using the Student's t-test, with a P value < 0.05 considered to be statistically significant.
Fig. 1. ADM-like lesions are formed within the invasive front of pancreatic cancer. (A) Human pancreatic cancer samples stained with H&E. The border between the tumor and the acini can be divided into two histopathologically distinct regions, the invasive front and the non-invasive front. The invasive front is associated with CAA. In the non-invasive front, the tumor is encapsulated by fibrosis. (B) Immunohistochemical analysis of amylase and CK19 expression in serial sections of pancreatic cancer containing regions of CAA. Red arrowheads indicate the duct-like features of ADM stained with amylase and CK19. Scale bars, 100 μm. r> The Kaplan–Meier method was used to analyze survival, with curves compared using the log-rank test. All statistical analyses were per-formed using JMP Pro 12 software (SAS Institute, Cary, NC, USA).
3.1. ADM-like lesions are formed within the invasive front of pancreatic cancer
Histopathologically, the border between the tumor and the acini can be divided into two visibly distinct zones, the invasive front and the non-invasive front, as we have reported previously by collagen fiber analysis . In the invasive front, cancer cells and/or cancer-