## 3 Results
### Influence of ASA and/or Anti-Fas Ab Treatment on DCs Length  
We assume that the simultaneous colonospheres' treatment with ASA and anti-Fas Ab exerted an advantageous effect against CRC cells. Our group has recently analyzed dendritic cells in anti-cancerous context [22]; thus we decided to test whether our experimental conditions used for the treatment of CRC CSCs would have a significant impact on DCs basic features after their in vitro modification. We have previously shown that cancer cells-derived lysates and conditioned medium can improve the efficacy of DCs preparation for potential DC-based immunotherapy, especially when administrated with LPS [22].  
Figure 5: **(A)** Representative western blot analysis images of caspase-2 and caspase-3 proteins in HCT116 and HT29 cells expanded 10 days in spherical forms with agonistic anti-Fas antibody (200 ng/mL) and/or aspirin (ASA) (2.2 mM or 1.8 mM for HCT116 or HT29, respectively). Protein assessment was performed as described in the Materials and methods. Merged image of all proteins as they were analyzed in the same western blot membrane. (**B,C**). Densiometric analysis of the caspase-2 (**B**) and caspase-3 (**C**) expression at the protein level by western blot Bars and whiskers represent median \(\pm\) interquartile range normalized to GAPDH level in each sample (* \(p<0.05\) vs. control unstimulated cells, * \(p<0.05\) ASA vs. ASA/anti-Fas antibody). Statistically significant differences were assessed by Kruskal-Wallis test followed by Dunn’s test as a post hoc procedure. Each experiment was performed in triplicate, except HCT116 caspase-2 analysis which was performed in duplicate.  
The current study design included DC incubation with lysates obtained from HCT116 and HT29 CRC cells treated with ASA and/or anti-Fas Ab. DCs are known to navigate anti-cancerous response, especially during the first, initial phases of cancer progression [30] thus the analysis of their reaction seems to be reasonable.  
Colonospheres after 10-day incubation with anti-Fas Ab and/or ASA, or 5-FU were polled and cellular lysates were prepared according to commonly used method relying on freezing and thawing sequences as described previously [22]. Afterwards, DCs obtained from monocytes of healthy volunteers were stimulated for 24 h with 100 \(\upmu\)L of lysates of colorectal cancer cells or with LPS. LPS, a potent DC activator, was used as the specific internal positive control of stimulatory potential. We assessed the morphological diversity of obtained DCs with a digital camera and measured DCs length to estimate the particular lysates influence (Figure 6). The DC length analysis revealed that the final effect depended on the cell line used for lysates preparation. HCT116-derived lysates exerted a more prominent impact on DCs size in comparison to HT29-derived lysates. For HT29-derived lysates, we noticed a less dynamic effect since the incubation of DC with lysates didn't change the DCs length. For almost all of our experimental options, the size of DCs was significantly smaller than size of DCs stimulated with LPS. The incubation of DCs with lysates of HCT116 and HT29 cells pretreated with 5-FU resulted in shortening of DCs and these cells were smaller than DCs after ASA/anti-Fas stimulation. Comparing the effect of lysates obtained after stimulation of CRC cell lines with ASA \(\pm\) anti-Fas Ab, we observed opposite results for the studied cell lines. Additionally, HCT116 cells stimulated with both ASA and anti-Fas Ab provided lysates which increased the size of DCs more effectively than ASA-stimulated CRC cells.