## 3 Results
### The Analysis of HCT116 and HT29 Cell Death after Their Treatment with ASA and Anti-Fas Ab  
Since the Fas signaling in its canonical form is pro-apoptotic, we decided to correlate the dying/apoptosis rate with all previously presented parameters. The proportion of apoptotic cells was assessed using flow cytometry and Annexin V-FITC and PI staining. Despite the elevated percentage of nonviable cells found in colonospheres, the overall number of cells within these structures was constant and as we stated in our previous manuscript, it was the result of cell differentiation and weaker cell-cell interactions [23]. The cell death was quantified after 10 days of cells' incubation with ASA and/or anti-Fas Ab. Additional isotype control IgM Ab wasn't found to influence the viability of cells following the introduced treatment. We found that the isotype control IgM Ab exerted no statistically significant impact also on cells phenotype in our experimental conditions (data not shown).  
Our analyses revealed that combined treatment induced more intensive apoptosis of CSCs in our culture conditions (Figure 4). The treatment of HCT116 and HT29 cells with anti-Fas Ab and ASA induced a higher rate of apoptosis than the effect observed in cultures of untreated cells or treated exclusively with anti-Fas Ab. The number of apoptotic cells was consistent with the level of caspase-3 measured in our samples by Western blot (Figure 5).  
Figure 4: The cytometric analysis of cells’ apoptosis during 10 day–expansion of HCT116 and HT29 cells in spherical forms with agonistic anti-Fas antibody (200 ng/mL), isotype control IgM Ab and/or aspirin (ASA) (2.2 mM or 1.8 mM for HCT116 or HT29, respectively). Annexin V–FITC/PI were used for assessing apoptotic cells. Y-axis presents Annexin V + PI cells frequency (%). Statistically significant differences were assessed by Kruskal-Wallis test followed by Dunn’s test as a post hoc procedure. Bars and whiskers represent median \(\pm\) interquartile range (\(\text{\textdagger}\)\(\textdagger}\)\(\textdagger}\)\(\textdagger}\)\(\textdagger}\)\(\textdagger}\)\)\(\textdagger}\)\(\textdagger}\)\)\(\textdagger}\)\(\textdagger}\)\)\(\textdagger}\)\(\textdagger}\  
Since caspases are the essential proteins engaged in diverse death pathways, in the next step of our study, we evaluated the protein level of caspase-2 and caspase-3 with Western blot (Figure 5). Our results demonstrated the variability of caspase-2 relative level. HCT116 spheroids were found to express the highest caspase-2 after combined treatment, while HT29 cells presented the highest content of this protein following incubation only with ASA. These differences were statistically significant in comparison to control, untreated cells. Moreover, we discovered that combined treatment resulted in increased levels of caspase-3 in both cell lines (Figure 5) (\(p<0.05\), U Mann-Whitney test).  
Surprisingly, the elevated level of caspase-3 was observed in the same HCT116 cells, which displayed also elevated caspase-2 (after ASA + anti-Fas Ab incubation) (Figure 5). At the same time HT29 cancer cells presented inverted tendency in caspases expression. The differences visible in the effect induced in HCT116 and HT29 CRC cell lines are probably associated with different cancer progression status (HCT116-TNM3, HT29-TNM2) and distinct gene status of these cells (e.g., KRAS: G13D and WT; BRAF: WT and V600E; PIK3 CA: H1047R and P449T; PTEN: WT and WT; TP53: WT and R273H for HCT116 and HT29 cell line, respectively) [29].