## 2 Materials and Methods  
### Cell Cultures and Treatments  
All cells were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA) and were grown in standard conditions of temperature, humidity and CO\({}_{2}\). The mouse 3T3-L1 cell line was used as a simplified in vitro model of adipose tissue to test the effects of metabolic stress on TGF\(\beta\) secretion. Several reasons led to the choice of 3T3-L1 cells; in fact, the cell line is a well-established model of differentiation to adipocytes by a standard protocol [34; 35], and can be stimulated to produce TGF\(\beta\)[34]. Most importantly, 3T3-L1 cells and human fat cells are similar in their response to cytokines and growth factors [36; 37] and they show comparable protein secretion [38; 39]; moreover, the proteins secreted by 3T3-L1 cells are frequently tested on human cell models [40; 41]. 3T3-L1 cells were kept in Dulbecco's modified Eagle medium (DMEM) with 4.5 g/L glucose, with the addition of 1% (\(v/v\)) antibiotics (penicillin/streptomycin solution) and 10% (\(v/v\)) fetal calf serum (FCS, Merck Life Science S.r.l., Milan, Italy). When 3T3-L1 cells reached confluence, to induce differentiation into mature adipocytes, 5 \(\upmu\)g/mL insulin (Merck Life Science S.r.l., Milan, Italy), 1 \(\upmu\)M dexamethasone (Merck Life Science S.r.l., Milan, Italy) and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (Merck Life Science S.r.l., Milan, Italy) were added to the culture medium. After 2 days, the medium was changed to DMEM 4.5 g/L glucose, containing 10% fetal bovine serum (FBS) and 1 \(\upmu\)g/mL insulin. Cells were maintained in this medium for 8 days, and we changed it every 3 days [34; 35]. This protocol has been largely validated in many studies by Oil Red-O staining of differentiated adipocytes [34; 36; 42].  
Both differentiated and undifferentiated 3T3-L1 cells were stimulated with Chemically Defined Lipid Concentrate (lipid mix, 11905-031, Gibco, Thermo Fisher Scientific, Monza, Italy) diluted 1:50 for 12 and 48 h, and differentiated adipocytes were kept for 48 h in mild hypoxic condition (5% O\({}_{2}\)). Mild hypoxia approximates the low tissue oxygen levels observed in vivo in obese rodents and humans, and stimulates adipogenesis and lipogenesis, supporting the role of hypoxia in the development of body metabolic impairments [43]. At the end of the 48 h treatments, the supernatant of differentiated adipocytes (conditioned medium, CM) was removed, centrifuged (14,000\(\times\)\(g\), 20 min) to eliminate any cellular debris, and tested for activity. The CM was then subjected to an acidification procedure necessary to activate the latent form of TGF\(\beta\), following a previously tested protocol [34; 44]. Acidification of CM to pH 3.0 was carried out by dropwise addition of 5 N HCI. After incubation on ice for 30 min, the pH of CM was restored to 7.2 by dropwise addition of 5 N NaOH [44]. This protocol allows the activation of the cytokine by a chemical method without requiring the partial enzymatic proteolysis occurring in vivo.  
The human pancreatic cancer cells PANC-1 and CAPAN-2 were grown in DMEM culture medium supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin/streptomycin (Merck Life Science S.r.l., Milan, Italy). Both cellular lines were derived from primary tumors and are representative of different levels of tumor aggressiveness [29]. PANC-1 and CAPAN-2 cells were stimulated with the conditioned medium diluted 1:3 with fresh growth medium or with 0.5 ng/mL TGF\(\beta\)1 (PeproTech, Rocky Hill, NJ, USA) for 8 days.  
The experimental flowchart is summarized in Figure S1 (Supplementary Materials).