Fig. 4

The chemoresistant HT-29/EGFP/FUR cells spontaneously form lung metastases whilst retaining chemoresistance. a The lung inspection unraveled multiple tumor metastasis foci in the lung parenchyma of the animals subcutaneously injected with HT-29/EGFP/FUR cells in comparison to the lung of mice injected with chemonaïve HT-29/EGFP cells (b). c The histological and immunohistochemical analysis of mouse tissues revealed the lung metastases development five months after subcutaneous administration of HT-29/EGFP/FUR cells. a Subcutaneous tumor. Hematoxylin/eosin staining, original magnification × 100. b, c Detection of EGFP-expressing tumor cells in subcutaneous tumor. Immunohistochemical reaction with anti-EGFP polyclonal antibody, original magnification × 200, × 400, visualization with 3,3′-diaminobenzidine. d Lung tumor. Hematoxylin/eosin staining, original magnification × 100. e, f Detection of EGFP-expressing tumor cells in lung tumor. Immunohistochemical reaction with anti-EGFP polyclonal antibody. Original magnification × 100, × 400, visualization with 3,3′-diaminobenzidine. d The molecular analysis of lungs of mice (n = 7) injected with HT-29/EGFP/FUR cells with detected metastases. DNA was PCR amplified for human and mouse gene detection. 1. Molecular weight standard (50 bp). 2. – 8. Product of duplex PCR - seven mouse lungs. Both human HBB (146 bp) and mouse Rapsn (116 bp) are present. 9. Positive control for human DNA. 10. Positive control for mouse DNA. 11. Positive control for both human and mouse DNA. On the contrary, we did not detect the presence of human sequences in lungs of mice (n = 7) injected with chemonaïve HT-29/EGFP cells (e; lines 2. – 8.). f The representative images taken at 12, 24 and 36 h after the monolayer wounding exhibit higher cell confluence in the wounded area of HT-29/EGFP/FUR cells. Blue lines indicate the initial scratch wound border, scale bar: 400 μm. The chemoresistant cells significantly increased the migration as determined by higher relative wound confluence. Mann-Whitney U test was used for statistical analysis. g The immunocytochemical staining of F-actin showed dramatic morphological difference of in vivo-derived HT-29/EGFP/FUR (referred to as FURiv) cells to fibroblastoid-like shape. Magnification × 630. h FURiv cells retained their resistance even after 5-months-long in vivo growth, and subsequent 2-months-long in vitro cultivation without the selection pressure of 5-FU. The cells were treated with different doses of 5-FU for 6 days, and the viability was evaluated by the luminescence assay. The sensitivity to 5-FU was exactly the same as for HT-29/EGFP/FUR cells from which the xenotransplant and subsequent FURiv-derivative had been established. Kruskal-Wallis test was used for statistical analysis