HIF-1α and CA-9 protein expression and metastatic potential are highest in tumours derived from follicular (WRO) and anaplastic (8505c) cell lines. Briefly, BcPAP, FTC133, WRO and 8505c cells were implanted sub-cutaneously in female CBA nu/nu nude mice. Once tumours reached 450 mm3, tumour tissue and fresh lung tissue were excised. Tumour sections were immuno-stained for HIF-1α. Freshly excised lung tissue was enzymatically digested, plated out in serial dilution and left until visible colonies formed. Colonies were stained, counted and number of metastatic colonies per mg lung tissue calculated. (For full methods, see supplementary materials and methods which is located at doi: 10.406/2011/762905). (a) Immunohistochemical staining for HIF-1α (brown staining). One example of HIF-1α staining for each tumour type: BcPAP, FTC133, WRO and 8505c are displayed. (b) Semiquantitative analysis of HIF-1α and CA-9 immunostaining quantified according to Allred et al. [109]. The total score is representative of intensity and proportion of staining. HIF-1α was highly expressed in all tumour types with highest expression observed in the 8505c tumours. CA-9 was also expressed in all tumour types with significantly higher expression observed in 8505c tumours (, one way Anova, with Tukey post test). (c) The number of spontaneous metastatic colonies cultured from enzymatically digested lung tissue that was excised from mice bearing WRO, 8505c, FTC133, and BcPAP tumours. The highest number of colonies counted were from the lungs of mice bearing FTC (WRO) and ATC (8505c) tumours. Data are representative of the mean ± S.D. of 5 independent mice for each tumour type.
