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A. Cruz-Bermúdez et al.
Fig. 4. The role of Fibroblast OXPHOS function in metabolic re-
programming of co-cultured cells. (A) OXPHOS function character-ization of fibroblast cell lines. (CF1; Control Fibroblast 1, CF1 +EtBr; Control Fibroblast 1 + Ethidium Bromide, AF; Aﬀected Fibroblast). Note the profound diﬀerences both in their galactose to glucose growth rate ratios as well as in their MIMP and LDHA mRNA levels. (B) Left panel, immunofluorescence confocal images of MCT4 induction in fi-broblasts after 4-day co-culture with H1299 cells. (MCT4 antibody in green and TO-PRO-3 staining the nucleus in blue). Right panel, MCT4 mRNA levels induced by co-culture with H1299 Durvalumab (C) MIMP changes produced by co-culture with diﬀerent fibroblast. (D) PGC-1α mRNA levels on H1299 cell line when co-cultured with control and Aﬀected Fibroblasts and on both fibroblasts co-cultured with H1299 cell line. Data are means from at least three diﬀerent experiments. Error bars indicate standard deviation. Student's t-test p-value was considered to be statistically significant when was < 0.05 (*=p ≤0.05).
function (Fig. 4C). On the other hand, the AF and the CF1 treated with EtBr do not behave the same way as the CF1 when co-cultured with H1299. Whereas the CF1 decrease its MIMP the AF or the CF1 treated with EtBr did not show any statistical diﬀerences (Fig. 4C). It is possible
that the latter cell lines are not able to diminish their OXPHOS function more than it is already reduced, which would also explain why they do not increase their expression of MCT4. Moreover, we measured PGC-1α mRNA levels as indirect
A. Cruz-Bermúdez et al.
Fig. 5. Reactive Oxygen Species and TGF-β in meta-
bolic reprogramming. ROS levels were evaluated using
the H2DCFH-DA fluorescent probe by flow cytometry. (A)
Basal ROS levels in monocultured A549 and H1299 cells.
Note the fourfold increase in ROS levels for the H1299 cells (B) ROS levels on A549 cells and control fibroblasts, ROS levels are not modified after co-culture. (C) H1299
cells increase their ROS levels after co-culture with two diﬀerent fibroblasts cell lines. (D) TGF-β mRNA levels measured by RT-qPCR in the cells were increased in all
the cell lines studied. Note that the co-culture with af- fected fibroblasts augments the eﬀect on H1299 cells compared to control fibroblasts. (E) Eﬀect on MIMP and ROS levels of TGF-β addition to monocultured cells. Data are means relative to monocultured tumor cells from at least three diﬀerent experiments. Error bars indicate
standard deviation. Student's t-test p-value was considered to be statistically significant when was < 0.05
measurement of the mitochondrial biogenesis, which, as we had seen previously, was altered by the co-culture with CF1. Our results show a greater increase of the mitochondrial biogenesis when the H1299 line is co-cultivated with the AF. In turn, the decrease of PGC-1α expression is
higher for these fibroblasts than for the CF1 when co-cultured with H1299 (Fig. 4D). These results would indicate that this process occurs even on an OXPHOS-aﬀected fibroblast. In fact, this seems to facilitate the MIMP and PGC-1α increase of the tumor cells.
Fibroblasts with an aﬀected OXPHOS function seems to facilitate the reverse Warburg eﬀect probably through their higher supply of lactate due to their impaired OXPHOS function . Recently, lactate has been proposed as a potential carbon source for lung tumors in vivo  and metabolic diﬀerences between CAFs from high and low glycolytic tu-mors have been found .
All this data combined with the variability of mitochondrial func-tion among healthy individuals (a common drawback in the study of mitochondrial diseases ) would place the OXPHOS performance of fibroblasts as a modifier element of tumor metabolism.
In relation to the previous sections, these results confirm that the H1299 cell line presents the ability to modify the metabolic micro-environment in which is present, since it occurs in the presence of two fibroblasts from diﬀerent origins. Furthermore, since the AF do not become significantly activated in the presence of the H1299 cell line (Fig. 3A), but nevertheless they produce a greater eﬀect on the MIMP of the tumor line, this result confirms that this process of metabolic re-programming is independent of fibroblast activation measured as α-SMA induction.
3.5. Reactive oxygen species levels in co-cultured NSCLC cells
One of the main factors responsible for metabolism reprogramming is ROS . We measured the basal ROS, in both tumor cell lines, using the fluorescent probe H2DCFH-DA. The results showed a significant fourfold increase of ROS for H1299 cells compared to A549 cells (Fig. 5A).
When we analyzed the levels of ROS in the co-cultures, we observed a change of them for H1299 cells, but not for A549 cells, that maintain the same ROS levels (Fig. 5B-C). Interestingly in H1299 co-culture, ROS levels increase in both cell types, suggesting some kind of reciprocal signal.