Generation and Characterisation of Novel Pancreatic Adenocarcinoma Xenograft Models and Corresponding Primary Cell Lines

<div><p>Pancreatic adenocarcinoma is one of the most lethal cancer types, currently lacking efficient treatment. The heterogeneous nature of these tumours are poorly represented by the classical pancreatic cell lines, which have been through strong clonal selection <i>in vitro</i>, and are often derived from metastases. Here, we describe the establishment of novel pancreatic adenocarcinoma models, xenografts and corresponding <i>in vitro</i> cell lines, from primary pancreatic tumours. The morphology, differentiation grade and gene expression pattern of the xenografts resemble the original tumours well. The cell lines were analysed for colony forming capacity, tumourigenicity and expression of known cancer cell surface markers and cancer stem-like characteristics. These primary cell models will be valuable tools for biological and preclinical studies for this devastating disease.</p></div

Morphology of the cell lines.

<p>A: Phase contrast pictures of the generated cell lines at the following passages: PpaC1 p2, PpaC2 p4, PpaC6 p8 and PpaC8 p10, at 10× magnification. B: Disseminating cell clusters in cultures with cobblestone growth pattern. The regular smooth colony border is indicated with a white arrow and the protruding group of cells with a black arrow. 20× magnification. Picture is from p4.</p

Global mRNA expression pattern.

<p>Heat map showing the hierarchical clustering of normal pancreatic tissue, original tumour material and the corresponding cell lines. The data set consists of 10 948 genes after filtering genes commonly regulated in fresh samples or cell lines. The RNA is isolated from the cell lines at the following passages: PpaC1 p16, PpaC6 p11 and PpaC8 p16.</p

Colony forming capacity of PpaC1.

<p>The table shows the % of cells able to generate colonies in the indicated semi-solid media in different <i>in vitro</i> passages, n.d. = not done.</p

Characterisation of tumour mutation status and xenograft tumours.

1<p>: p53 staining is scored as hot spots where 5 = 30–60% and 6 = 60–100%, and the staining intensity is given in brackets (1 = weak, 2 = medium and 3 = strong).</p>2<p>: S100A4 is scored as % positive cells where 1 = 1–4%, 2 = 5–9%, 3 = 10–14%, 4 = 15–49%, 5≥50%, and the staining intensities is given in brackets (1 = weak, 2 = medium and 3 = strong).</p>3<p>: Time in weeks required for the implanted patient material to reach 10 mm.</p>4<p>:Time in weeks required for the xenograft passage to reach 10 mm for F3 and forward.</p

Cell surface expression of known cancer and stem cell markers.

<p>Shown here is the average % positive cells out of single, live cells, n = at least 3, n.d = not done.</p><p>Cells were analysed in the following passage spans: PpaC1 p1–p10, PpaC2 p3–p14, PpaC6 p5–p9 and PpaC8 p4–p20.</p

<i>In vivo</i> tumourigenicity.

<p>Shown is the number of tumours growing/the number of injected sites in NSG mice for each cell lines, harvested at the following passages: PpaC1 p6, PpaC2 p12, PpaC6 p6, PpaC8 p13.</p

Characterisation of patient material.

1<p>: PDAC = Pancreatic ductal adenocarcinoma, P = pancreatobiliary subtype, I = intestinal subtype.</p><p>IPMN = Intraductal papillary mucinous neoplasia.</p>2<p>: The degree of differentiation for each tumour is according to the pTNM Classification of Malignant Tumours <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103873#pone.0103873-Sobin1" target="_blank">[14]</a>.</p>3<p>: T3 = Tumour extends beyond pancreas, but without involvement of celiac axis or superior mesenteric artery, Tis = Carcinoma <i>in situ</i>.</p>4<p>: 0 = resection margin free and 1 = resection margin not free.</p>#<p>: Patient only followed up the first year after operation, due to non-malignant disease.</p><p>*: Patient is alive, the number of days given refers to the latest follow-up date.</p

Colony forming capacity of the cell lines.

<p>Each diamond represent the fraction of cells able to generate colonies >50 µm in methylcellulose/stem cell medium II in one experiment (n = 3–9). The red line shows the average and the yellow line shows the median colony forming capacity for cells in the following passage span: PpaC1 p1–p31, PpaC2 p5–p13, PpaC6 p2–p7 and PpaC8 p4–p14.</p

Circulating microRNAs associated with prolonged overall survival in lung cancer patients treated with nivolumab

<p><b>Background:</b> The introduction of immune check-point inhibition in non-small cell lung cancer (NSCLC) therapy represents improved prospects for the patients. The response rates to check-point inhibitors are approximately 20% in unselected NSCLC patients. Increasing levels of tumor PD-L1 expression are associated with higher response rates. However, patients with low PD-L1 levels may also have durable responses, and improved strategies for patient stratification are needed.</p> <p><b>Material and methods:</b> In this study, we investigated circulating microRNAs aiming to identify circulating predictive biomarkers associated with increased overall survival after immune check-point treatment. Using next generation sequencing, we performed microRNA profiling in serum from NSCLC patients (<i>n</i> = 20) treated with nivolumab. Serum samples from 31 patients were used for validation using qPCR assays. Serum samples were collected prior to immune therapy initiation.</p> <p><b>Results:</b> Based on multivariate regression analysis, we identified a signature of seven microRNAs (miR-215-5p, miR-411-3p, miR-493-5p, miR-494-3p, miR-495-3p, miR-548j-5p and miR-93-3p) significantly associated with overall survival (OS) > 6 months in discovery cohort (<i>p</i> = .0003). We further validated this in another similar set of samples (<i>n</i> = 31) and the model was significantly associated with overall survival (OS) > 6 months (<i>p</i> = .001) with sensitivity and specificity of 71% and 90%, respectively.</p> <p><b>Conclusions:</b> In this study of circulating microRNAs, we have identified a 7-miR signature associated with survival in nivolumab-treated NSCLC patients. This signature may lead to better treatment options for patients with NSCLC, but a validation in an independent cohort is needed to confirm the predicted potential.</p