Adipored assay

For screening applications, spheroids were fixed with 0.4% (v/v) methanol‐free formaldehyde, washed with PBS, and stained overnight with Hoechst33342 and NileRed. Imaging was performed on a high‐throughput confocal microscope (Opera Phenix, Revvity) with a 10× objective. Twenty‐five image fields across 10 z‐stacks were obtained for each well. Spheroid roundness, area, and location were extracted using the PhenoLOGIC algorithm in Harmony and steatosis was automatically quantified for each spheroid as the ratio of triglyceride to nuclear signal. For validation experiments, lipid accumulation was measured using the AdipoRed Assay (Lonza) following the manufacturer's instructions.

Lipid accumulation was visualized using the AdipoRed assay (Lonza), following the manufacturer’s protocol. Briefly, 15 µL of AdipoRed assay reagent and 10 µL of Hoechst 33258 were mixed with 1 mL of DMEM to prepare AdipoRed staining solution. The cell culture chambers and channels were washed twice with PBS. AdipoRed staining solution (10 µL) was added and incubated at 37 °C for 15 min. The chambers were washed three times with fresh DMEM solution.

The effect of the 18 PFASs on triglyceride levels was determined using the AdipoRed assay essentially according to the instructions of the supplier (Lonza, Basel, Switzerland). We used the approach as applied in the study of Luckert et al. (2018 (link)), in which HepaRG cells were exposed to the steatotic compound cyproconazole. In that study, 72 h was shown to be the optimal time point to assess the effects of cyproconazole on triglyceride accumulation as determined with the AdipoRed assay. We first assessed whether this time point was also the optimal time point for assessing effects of PFASs on triglyceride accumulation, by studying the effects of a 24-h or a 72-h exposure to PFOA, PFNA, PFHxS and PFOS in the AdipoRed assay, also including cyproconazole as positive control. After exposure for 24 or 72 h, the medium was removed and the cells were washed with 200 μL DPBS and subsequently incubated for 10 min at room temperature with 200 μL AdipoRed-DPBS solution. The latter solution was prepared by adding 25 μL AdipoRed to 1 mL DPBS. Subsequently, fluorescence was measured using a 485/20 nm excitation and 590/35 emission filter set on the Synergy HT Microplate Reader. The results from that study indicate that a 24-h exposure was considered better than a 72-h exposure to study effects of PFASs (see Results section). Therefore, all other PFASs were tested upon a 24-h exposure. For each PFAS, three independent biological replicates, with three technical replicates per condition were obtained. Data were used for dose–response analysis using PROAST software (see below).

Colorectal cancer and adipose specimens were collected from CRC patients who underwent surgical resection, in accordance with the ethical policy of the University of Palermo Committee on Human Experimentation. Isolation and propagation of CR-CSphCs and ASCs were performed as previously reported^{43 (link),64 (link)}. CR-CSphC lines #1, #8, #9, and #21 were isolated from lean CRC patients. Adipose stromal cells from VAT and SAT were obtained from the greater omentum and subcutaneous anterior abdominal wall, respectively. The study received ethical approval for the purification and culture of CR-CSphC and ASCs, by Ethics Committee 1 board, University of Palermo - Azienda Ospedaliera Universitaria “Paolo Giaccone” (authorization CE 6/2015). The study complied with all the ethical regulations for work with human participants, including obtaining the informed consent for both CRC patients with healthy weight, and affected by obesity.
Human samples were crosscut into small pieces and grinded using scalpels and surgical scissors and digested at 37 °C for 30 min in DMEM medium supplemented with 0.6 mg/ml of collagenase (Gibco) and 10 µg/ml of hyaluronidase (Sigma). The cell pellet was resuspended: i. for CRC, in serum-free stem cell medium (SCM) supplemented with EGF and b-FGF; ii. for adipose tissue, in mesenchymal stem cell medium (ThermoFisher Scientific), in ultra-low attachment cell culture flasks, leading to cell growth as spheroids. When cancer and adipose spheroids reached approximately 80% of confluence, cells were mechanically and enzymatically disaggregated, using Accutase (ThermoFisher Scientific). ASCs were routinely frozen and stored in liquid nitrogen at early passages to maintain their pluripotency (passage 1–12). Differentiated adipose cells were obtained by exposing ASCs, for up to 28 days, to adipogenesis differentiation medium (ThermoFisher Scientific). Differentiation efficiency was evaluated by AdipoRed assay (Lonza). Colorectal cancer sphere-derived adherent cells (SDACs) were obtained by culturing cells in adherent condition, in presence of 10% FBS, as previously described^{41 (link),65 (link)}. Huvec cells prescreened for angiogenesis were purchased by Lonza (C2519AS) and cultured according to the manufacturer’s instructions.
CR-CSphC and ASC lines were routinely authenticated by short tandem repeat (STR) analysis using a multiplex PCR assay, including a set of 24 loci (GlobalFiler™ STR kit, Applied Biosystem), by comparing them to the parental patient tissues⁶⁶ . Conditioned medium was collected 48 h after cells reached subconfluence in SCM. The viability of CR-CSphC culture, which is routinely estimated by trypan blue and 7-AAD, is 93 ± 7%.
CR-CSphCs were treated with recombinant IL-6 (2 ng/ml; Novus), HGF (10 ng/ml; Peprotech), VEGF (10 ng/ml; Novus) and exposed to neutralizing antibodies against IL-6 (100 ng/ml; R&D), HGF (200 ng/ml; R&D), NGF (0.2 µg/ml; R&D), CD271 (0.5 µg/ml; Merck), and VEGF (10 ng/ml; R&D), tocilizumab (10 µg/ml; Selleckchem) or crizotinib (30 nM; Selleckchem), or to STAT3 inhibitor C188-9 (10 µM; Selleckchem). CM, cytokines, and neutralizing antibodies were added every 48 h to the cell culture.
Cell viability assay was performed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) according to the manufacturer’s instructions and analyzed by using the GDV MPT reader (DV 990 BV6). To monitor the acquisition of epithelial versus a mesenchymal phenotype, 5 × 10³ viable CR-CSphCs were embedded in 1:10 SCM/Matrigel solution and seeded as a single drop in a pre-warmed 24 well plate. Following matrigel polymerization, 500 µl of SCM were overlaid to each well and 3D organoid formation was followed up to 21 days.