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

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

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

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

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 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

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

Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines

<div><p>Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44^high) and a main population which was either weakly positive or negative for CD44 (CD44^low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44^highCD90⁺ sub-population. Moreover, these CD44^highCD90⁺ cells revealed mesenchymal morphology, increased expression of mesenchymal markers <i>N-Cadherin</i> and <i>Vimentin</i>, increased mRNA levels of the embryonic stem cell related genes <i>Nanog</i> and <i>Oct4</i> and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44^highCD90⁺ population a good candidate for the lung CSCs. Both CD44^highCD90⁺ and CD44^highCD90⁻ cells in the PLCCL derived from SCC formed spheroids, whereas the CD44^low/− cells were lacking this potential. These results indicate that CD44^highCD90⁺ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44^high sub-population.</p> </div