## 3 Results
### Differentiated 3T3-L1 Adipocytes Subjected to Metabolic Stress Produce TGF\(\beta\)
Based on these previous observations, we decided to use the conditioned medium obtained from adipocytes exposed to lipid mix to evaluate the metabolic modulation exerted by the inflammatory cytokine on the same pancreatic tumor lines. PANC-1 and CAPAN-2 cell lines were treated with conditioned medium (CM) diluted three times with fresh medium to ensure the nutrients necessary for growth. The final TGF\(\beta\) concentration was therefore about 0,35 ng/mL, close to the lowest doses of the cytokine used in other experimental settings [61; 62]. First, we carried out a flow cytometric analysis with JC-1 dye to measure mitochondrial respiratory activity and this investigation revealed some interesting metabolic changes, as shown in Figure 3A. In fact, when PANC-1 and CAPAN-2 cells were exposed to prolonged (8 days) treatment with the conditioned medium, we observed an increase in mitochondrial membrane potential similar to the one observed following prolonged treatment with commercial TGF\(\beta\). To further investigate the metabolic effect of the cytokine produced by adipocytes subjected to metabolic stress, we evaluated its transcriptional effects on mitochondrial respiration. In PANC-1 cells, the 8-day treatment with the conditioned medium resulted in a strong transcriptional induction of some subunits of respiratory chain and ATP synthase (COX-II, MT-ATP6 and ATP-5B) compared to control, whereas the prolonged treatment with commercial TGF\(\beta\) was less effective (Figure 3B). These results were confirmed by the analysis of the second pancreatic cancer cell line CAPAN-2, as shown in Figure 3C. The results of these experiments demonstrated the metabolic efficacy of TGF\(\beta\) secreted in the conditioned medium; interestingly, the effect on the transcription of respiratory elements is greater than that exerted by similar dosage of synthetic TGF\(\beta\). Figure 2: Hypoxia and lipid mix stimulate the production of TGF\(\beta\) in its latent form. (**A**) After 48 h of treatment, the amount of TGF\(\beta\) secreted by differentiated adipocytes was measured in conditioned growth medium by ELISA method. (**B,C**) The induction of vitamin D receptor (VDR) expression was used as a readout of TGF\(\beta\) activity. The expression of VDR was evaluated by Western blot analysis of total lysates from PANC-1 cells treated for 48 h with adipocyte-conditioned growth medium or with 10 ng/mL TGF\(\beta\). The band corresponding to VDR is indicated by the arrow. Actin expression was evaluated as loading control. Data are shown as means \(\pm\) SEM of three independent experiments. *** \(p<\) 0.001 vs. ctrl. ; SSSS \(p<\) 0.001. The Conditioned Medium from Adipocytes Exposed to Metabolic Stress Favors the Progression of Tumor Cells from a Proliferative to a Migratory State  
The last part of our study investigated two distinctive features of TGF\(\beta\)-induced tumor progression, namely the nuclear effects of the cytokine on epithelial-mesenchymal transition (EMT) and cellular migration. We sought to confirm whether the prolonged exposure to the cytokine secreted from dysmetabolic adipocytes could enhance EMT and invasion of pancreatic cancer, as demonstrated in our previous in vitro study on TGF\(\beta\)[29]. We incubated PANC-1 and CAPAN-2 cells for 8 days with both the conditioned medium and 0.5 ng/mL of synthetic TGF\(\beta\), to equalize the dosage of TGF\(\beta\) used in treatments. The morphological changes indicative of EMT were observed by light microscopy (Figure S5 in Supplementary Materials) and suggested further analysis of EMT markers by RT-PCR. In addition to the stretched appearance of pancreatic cancer cells, we confirmed the ongoing transition by measuring the transcriptional suppression of E-cadherin and the increase of fibronectin mRNA after the treatment of both cell types with conditioned medium. The transcriptional effects exerted by the similar dosage of synthetic TGF\(\beta\) were less intense. These results are shown in the first two panels of Figure 4A (PANC-1 cells) and Figure 4B (CAPAN-2 cells).