Fig. 2

Chemoresistant derivative exhibit limited sensitivity to CD::UPRT-MSC/5-FC regimen. The resistance increases with number of passages. The efficacy of the GDEPT was evaluated by direct co-culture of the tumor cells and therapeutic CD::UPRT-MSC at a ratio of 5:1 for 6 days after the addition of 5-FC concentration gradient, and the relative luminescence corresponding to the viability was evaluated. Luminescence in co-cultures without 5-FC was set to 100% by default (a, d, e). a CD::UPRT-MSC/5-FC did not exert significant cytotoxic effect on chemoresistant cells HT-29/EGFP/FUR when compared to the parental HT-29/EGFP cells. Chemoresistant cells in “low” passages are 43-fold more resistant to CD::UPRT-MSC/5-FC than parental counterparts. The higher passage of cells (stated as middle, high), the higher chemoresistance as shown by counted IC50 values. Values were expressed as means of triplicates ± SD. Data were statistically analyzed by parametric ANOVA test with Bonferroni’s or Tamhanes’s tests for multiple comparisons and non-parametric Kruskal–Wallis test. *p < 0.05; **p < 0.01; ***p < 0.001. b The red fluorescent representative images from the IncuCyte ZOOM™ kinetic imaging system of the tumor cells stained with NucLight™ Red in the direct co-culture with CD::UPRT-MSC in the presence of 500 µg/ml of 5-FC for 6 days documented the capability of chemoresistant cells (here referred to as FUR/NLR) to withstand the bystander cytotoxicity exerted by the therapeutic cells. c Kinetic measurements based on counting of the red nuclei of the tumor cells in direct co-cultures with CD::UPRT-MSC (5:1 ratio) and 5-FC (500 µg/ml) demonstrated 67% reduction in the proportion of chemoresistant tumor cells in comparison to 91% elimination of parental cells. The number of the NLR cells in the co-culture without 5-FC at a given time point was taken as 100%. Values were expressed as means of sextaplicates ± SD. Mann–Whitney U-test was used for statistical analysis. *p < 0.05. d The analysis of the 3D aggregate co-cultures 7 days after the 5-FC addition showed 97% reduction in the HT-29/EGFP viability in comparison to only 30% reduction in the chemoresistant cells at a concentration of 5-FC of 500 µg/ml. Expressed as means of sextaplicates ± SD. Mann–Whitney U-test was used for statistical analysis. **p < 0.01. e In vivo-derived cells of the HT-29/EGFP/FUR xenograft (referred to as FURiv) retained their resistance even after 5-month-long in vivo growth, and subsequent 2-month-long in vitro cultivation without the selection pressure of the 5-FU. The sensitivity to CD::UPRT-MSC/5-FC was exactly the same as for HT-29/EGFP/FUR cells from which the xenotransplant and subsequent FURiv derivative had been established. Values were expressed as means of quadruplicates ± SD. ns not significant