Basic fibroblast growth factor (bfgf)

Endothelial differentiation was performed as described previously with some modifications.[12 (link)] The iPSCs at subconfluency were detached using CTK solution consisting of 0.1 mg/ml collagenase IV (Invitrogen), 0.25% trypsin (Invitrogen), 0.1 mM CaCl2 (Nacalai tesque) and 20% KSR and seeded onto Matrigel-coated dishes at a ratio of 1:5 to 1:10. We treated iPSCs with 50 ng/mL bone morphogenetic protein 4 (BMP4; R & D Systems, Minneapolis, MN) and 50 ng/mL basic fibroblast growth factor (bFGF; Wako, Osaka, Japan) for the first 24 h; 40 ng/mL vascular endothelial growth factor (VEGF; Invitrogen, Waltham, MA) and 50 ng/mL bFGF for 2 days; and 40 ng/mL VEGF, 50 ng/mL bFGF, and 20 μmol/L SB431542 (Miltenyi Biotec, Teterow, Germany) for 3–4 days. On days 6–7, ECs were purified using FITC-conjugated anti-CD31 antibody (1 μg/1 × 10⁶ cells, WM59; BioLegend, San Diego, CA) and APC-conjugated anti-CD144 antibody (0.5 μg/1.0 × 10⁶cells; 16B1, eBioscience) with a FACSAria III (BD Biosciences, San Jose, CA).

CRC cell lines were trypsinized in 0.25% trypsin-EDTA (Gibco) and suspended at a density of 5 × 10⁴ cells/mL in flasks pre-coated with 0.24% polyhydroxyethylmethacrylate (polyHEMA) (Sigma-Aldrich, St. Louis, MO, USA) in DMEM-F12 supplemented with 0.24% (v/v) methylcellulose (Sigma-Aldrich), 1× B27 supplement (Gibco), 20 ng/mL of epidermal growth factor (EGF, Miltenyi Biotec, Germany), 10 ng/mL of basic fibroblast growth factor (bFGF) (Miltenyi Biotec), 0.4% (v/v) of bovine serum albumin (BSA) (Nacalai Tesque, Japan) and 1 mg/mL of insulin (Gibco). The medium was topped up on day 7. The spheroids formed were collected by centrifugation on day 14, dissociated using Accutase (Gibco) and seeded in new flasks under the same culture conditions for propagation.

All cell lines listed in Supplementary Table 1 were obtained from American Type Culture Collection (ATCC). Cells were regularly (once per month) tested for mycoplasma contamination. No contaminations have been observed within the performed experiments. Cells were cultured in DMEM 5030 supplemented with 17.5 mM glucose, 2 mM glutamine and 10% FBS at 5% CO₂ and 37 C. HAP1 cells were obtained from Patel’s laboratory^{8 (link)} and were cultured in IMDM medium supplemented with 10% FBS. NCH601 cells (derived from the lab of Christel Herold-Mende, Heidelberg) were cultured as non-adherent spheres in DMEM-F12 medium (Lonza) containing 1xBIT100 (Provitro), 2 mM glutamine, 30 U/ml Pen/Strep, 1 U/ml heparin (Sigma), 20 ng/ml bFGF (Miltenyi) and 20 ng/ml EGF (Provitro) as previously described in^{15 (link), 23 (link)}.
For metabolic profiling we deployed previously established protocols for absolute quantification of exchange fluxes and intracellular fluxes of one-carbon metabolism^{10 (link), 12 (link), 24 (link)} to a panel of cancer cell lines. To determine the growth rate, we assumed exponential growth and recorded start count and end count in every experiment and calculated the growth rate as described in^{10 (link)}. For the determination of the SCI we calculated the product of serine level multiplied with the NAD⁺/NADH ratio.