Characterisation of Mesothelioma-Initiating Cells and Their Susceptibility to Anti-Cancer Agents
Fig 4
Mesospheres form tumours in serial transplantations.
(A) Ist-Mes-2 sphere cells (generation 1, G1) were subcutaneously grafted into Balb-c/nude mice at 106 cells per animal. When tumours reached about 2,000 mm3, the mice were sacrificed, tumours excised and malignant cells grew in vitro as a cell line. The adherent cells were converted into spheres (G1) and these were grafted into Balb-c/nude mice to form tumours that were used for generation 2 (G2) spheres. This procedure was repeated two more times to derive G3 and G4 spheres. The inset in panel A shows the lag to tumour appearance following cell grafting for individual sphere cell generations. Cells of individual generations were evaluated for stemness markers as shown using qPCR (B) and WB (panel C shows the blots, panel D their densitometric evaluations) and for CS activity (E). (F) Tumours derived from adherent cells and spheres of individual generations were excised, paraffin-embedded and stained for the MM marker mesothelin (upper images show staining with the exclusion of the primary IgG) and with H&E. Data shown in panel A are derived from 5 animals and are mean values ± S.E.M, data in panels B and E are mean values from three independent experiments ± S.D. Images in panel C are representative of two individual experiments and their densitometric evaluations in panel D represent mean values with differences lower than 10%. Images in panel F were obtained using one tumour for each condition (generation). The symbol ‘*’ denotes statistically significant differences with p<0.05. Images are representative of three different tumours.