## 4 Discussion  
Our initial analyses provided a surprising and interesting observation that ASA can modulate the impact of pro-cancerous Fas signaling on HCT116 and HT29 cancer cell lines. Our results suggested the synergistic relationship between both active compounds: ASA and anti-Fas Ab. Currently, the shortage of reports combining the activity of ASA and anti-Fas Ab is highlighted by the increasing number of data depicting these agents' independent activity in different contexts and conditions, including anti-cancer effect. In our experimental panel, ASA when added alone for cancer cells' treatment, reduced sphere sizes but at the same time the number of CD133+ CSCs was the same as in control. Original pro-cancerous role of Fas signaling (previously presented in [20]) was ceased after ASA was included into samples. We found that simultaneous treatment induced the reduction of sphere sizes, the number of CD133+, CD44+ and CD29+ cancer cells in colonospheres, along with higher apoptosis rate. Incubation with ASA + anti-Fas Ab elevated the number of Fas+ cancer cells (probably more vulnerable to apoptosis) what is confirmed by cytometric apoptosis assay. Moreover, in samples with higher apoptosis, the higher caspase-2 and-3 protein relative levels were also found. Moreover, the level of caspases remains at higher level than in control. Our combined treatment modified the caspases level what seemed to influence other measured parameters. Our results highlighted the potential crucial role of caspases in CSCs function in both cancer cell lines we used. To establish the type of cell death and/or pro-tumorigenic activity resulting from the combined treatment of CRC CSCs with anti-Fas Ab and ASA, we assessed the levels of caspase-2 and caspase-3, the latter known as an executioner type of a cysteine-aspartic protease involved in the apoptotic process. Recently [PERSON] et al., have shown that caspase-3 inhibitor didn't enhance STAT1 activation and the lack of caspase-3 expression resulted in the Fas signaling activation even without its stimulation [31]. Caspase-3 is known to be associated with stemness of CSCs and [PERSON] et al., revealed that a subgroup of CRC patients with low levels of an active form of caspase-3 was characterized by increased disease-free survival [32]. Moreover, [PERSON] et al., in in vitro and in vivo experiments proved that dying breast cancer cells following radiotherapy produced caspase-3 and other paracrine factors that stimulated the growth of the remaining cancer cell population [33]. Our observations seem to confirm these results. Although we measured the non-cleaved form of caspase-3, the elevated relative level of this protein was clearly visible in samples with the most advanced apoptosis. It is commonly believed that the active form of caspase-3 is directly engaged in apoptosis since not the whole pool of proteins after translation can be a trigger for the executioner phase of programmed cell death. Since we discovered a similar phenomenon in both studied CRC cell lines, the elevated caspase-3 level seems to have a biologically relevant meaning and require further analyzes. In these samples the low proportion of CD133+ cells is probably associated with the silencing of CSCs metabolism for cancer evasion, protecting mechanism from anti-cancerous agents. It is well known that caspases may participate in different cell death types, i.e., apoptosis, necrotosis and DICE (death induced by CD95 or CD95L elimination) [31; 34]. However, it has to be stressed that their function is not limited to the regulation of cell death mechanisms [35]. Caspase-2 plays multiple roles in normal cells, including DNA-damage-induced apoptosis, cell cycle regulation and genomic stability maintenance. Moreover, cumulative evidence also implicates caspase-2 as an important driver of cell maturation and differentiation [34]. Caspase-2 was suggested to be a negative regulator of the Fas/STAT1 axis supporting stemness of cancer cells, demonstrated on the MCF-7 breast cancer cell line [31].