## 3 Results
### MnTnHex Reduces Collective Cell Migration  
To guarantee that the findings obtained with the wound-healing assay were due to an impairment in cell migration and not a consequence of reduced cell viability, it was necessary to first determine which non-toxic concentrations of MnTnHex and cisplatin should be used. For that, A549 cells were treated with two low concentrations of MnTnHex (1 and 5 uM) and cisplatin (0.25 and 0.5 uM) (Figure S1A), and H1975 cells were treated with MnTnHex (0.25 and 0.5 uM) and cisplatin (0.5 and 1 uM) (Figure S1B). All experiments were performed using a culture medium with 2% FBS, and cytotoxic effects were assessed using the MTS reduction assay. In A549 cells, both cisplatin concentrations displayed 100% cell viability. For MnTnHex, both concentrations tested caused only a minor decrease in cell viability. Therefore, we selected the representative non-toxic concentrations of 0.5 uM for cisplatin and 5 uM for MnTnHex. In H1975 cells, none of the two concentrations of cisplatin tested showed cytotoxic effects. For MnTnHex, the concentrations of 0.25 uM and 0.5 uM had a decrease in cell viability of less than 10%. Hence, we chose the non-toxic concentrations of 1 uM for cisplatin and 0.5 uM for MnTnHex. After selecting the non-cytotoxic concentrations of both compounds, the migration capability of A549 and H1975 cells were assessed using the wound-healing assay (Figure 6), which evaluates the collective cell migration through a horizontal surface. In A549 cells, MnTnHex alone significantly reduced wound closure by approximately 30% (\(p<0.01\)) at 24 h and 32 h (Figure 6A,B). When combined with cisplatin, this compound also tends to reduce cell migration. In H1975 cells, a significant reduction occurred at 24 h, either using MnTnHex alone or combined with cisplatin, of approximately 19% (\(p<0.05\)) and 15% (\(p<0.05\)), respectively (Figure 6D,E). A similar % of reduction was also observed at 32 h, although without statistical significance. MnTnHex Alone and/or Combined with Cisplatin Induced Alterations in the Metabolic Response of NSCLC Cells  
First, the analytical reproducibility of the metabolomics experiment was confirmed by unsupervised analysis (PCA) of GC-MS data (Figure S3), including all samples under study and QCs, which revealed a well-defined QCs cluster. From a multivariate discriminant analysis (PLS-DA) perspective, no clear separation was found between the extracellular media of A549 or H1975 cells exposed to MnTnHex (0.5 and 1 uM) and cisplatin (1 uM) alone or in combination. Hence, altered levels of VCCs were investigated from a univariate analysis perspective using Volcano plots (Figure 7). Among the 21 VCCs detected in extracellular media of both cell lines by our experimental approach (Table S1), statistically significant VCCs were only found in the extracellular medium of H1975 cells exposed to 1 uM of MnTnHex (Figure 7A) and 1 uM of MnTnHex combined with 1 uM cisplatin (Figure 7B) when compared with the control group. Cisplatin alone did not significantly increase levels of VCCs, possibly being an inferior external source of H2O2 relative to MnTnHex. Interestingly, significant alterations were observed for the same VCCs in both exposed groups (Figure 7C), namely isobutanal, 3-methylpentanal and benzaldehyde. The relative comparison with blanks (i.e., culture medium without cells), also represented in Figure 7C, allowed the interpretation of VCCs alterations in terms of release or uptake by cells. Thus, our findings unveiled that H1975 cells exposed to both conditions released significantly higher levels of isobutanal and 3-methylpentanal and uptook lower levels of benzaldehydebenzaldehyde than control cells. Although no statistically significant alterations were found for extracellular media of exposed A549 cells, the same trend was observed for isobutanal and benzaldehyde levels, showing a similar behavior for higher release of isobutanal and lower uptook of benzaldehyde after exposure to MnTnHex alone and combined with cisplatin (Figure S4).