Phenotypes and karyotypes of human malignant mesothelioma cell lines

Abstract

Background: Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma.

Methods: Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM) and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs.

Results: Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30-72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5-17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions.

Conclusion: These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of mesothelioma during maintenance in artificial culture systems. These characteristics support their potential as in vitro model systems for studying cellular, molecular and genetic aspects of mesothelioma.

Figure 1. Light microscopy of cell lines.

Figures showing morphology using light microscopy MM04 (A), MM05 (B), MM12 (C), MM13 (D), MM1081 (E) and PF1038 (F).

Figure 2. Transmission electron micrograph of cell lines.

Cell lines showing microvilli (dark arrows) and tight intracellular junctions (dotted arrows). MM05 (A), MM04 (B), MM12 (C), MM13 (D), MM1081 (E) and PF1038 (F).

Figure 3. Anchorage independent growth assay.

The assay showing the colony formation for all the cell lines (A549, NFF, MM04, MM05, MM12, MM13, MM1081 and PF1038). 10,000 cells were grown on 96 well plates on soft agar for 10 days before adding Wst-1. Optical Density (OD) was measured at 450 nm and referenced at 620 nm. A549 (Human lung adenocarcinoma epithelial cell line) cell line was used as a positive control and NFF (Neonatal foreskin fibroblasts) as a negative control.