Dulbecco modified eagle medium (dmem)

Primary human lung fibroblasts were isolated by explant outgrowth from fresh or cryopreserved tumor-free tissue derived from distal airway-free lung tissue (parenchymal fibroblasts) or small airways (peribronchial fibroblasts) following published protocols (31 (link), 73 (link)–75 (link)). Briefly, 6–7 microdissected lung parenchyma or airway pieces were placed per well into 6-well plates, left for 30 minutes at room temperature without medium to improve explant attachment, and carefully covered with 1 mL of DMEM (Thermo Fisher Scientific) supplemented with 2% FBS (Gibco, Thermo Fisher Scientific) (DMEM⁺⁺⁺, Supplemental Table 3). Explants were left undisturbed for 3–4 days, and afterward the medium was exchanged every 2 days and the outgrowth of fibroblasts from the explants was followed daily. Cells were collected from several explant pieces when reaching 70% confluence to preserve the fibroblast heterogeneity. Possible epithelial contamination was prevented by trypsinizing for shorter periods (3 minutes, 0.05% trypsin with EDTA, Thermo Fisher Scientific) and keeping the cells in DMEM⁺⁺ (Supplemental Table 3). Fibroblasts were used within passages 2–4.

MB cells were brought to a single cell suspension in serum free media. For neurosphere formation cultures, cells were seeded at 0.5 x 10⁶ cells/mL on low-attachment plates in their control or experimental media, in the presence of 20 ng/mL bFGF and 20 ng/mL EGF. Serum free ACM was collected by culturing astrocytes in DMEM (high glucose, no glutamine, no phenol red, Thermo Fisher Scientific) and collecting the supernatant after 48 h. Control media is DMEM alone, and as a positive control condition for sphere formation, DMEM was mixed 1:1 with Ham’s F12 nutrient mix (Thermo Fisher Scientific). At 48 h, cells were imaged using a Keyence BZ-X microscope. In a six-well plate, 20 pre-programmed brightfield images per well were taken at 4X magnification with 1.7X digital zoom applied. The images were analyzed using Keyence analysis software, where sphere diameter was measured. To quantify, all spheres greater than 75 μm in diameter were measured and counted. Representative images used for visualization of the neurospheres were taken at 10X magnification and 1.8X digital zoom was applied. Measurement of neurosphere size was also done previously using a rough calibration of pixels to micrometer (μm), and then used ImageJ for further data processing. Both methods (Keyence software analysis and ImageJ analysis) yielded similar data.

Breast cancer cell lines (MDA-MB-231, SKBR-3, and MCF-7) were obtained from the Shanghai Institute of Life Science, Chinese Academy of Sciences (Shanghai, China) and maintained in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS (PAN-Biotech, Adenbach, Bavaria, Germany), 50 units/mL penicillin and 50 units/mL streptomycin. All these cells have been authenticated by short tandem repeats analysis (Genetic Testing Biotechnology Corporation, Suzhou, China). The 3T3-L1 preadipocyte cell line was obtained from American Type Culture Collection (Manassas, VA, USA) and the growth and differentiation of these cells were performed as previously described [15 (link)]. Cells were cultivated in DMEM containing 10% fetal calf serum (FCS) (Thermo Fisher Scientific) and reached confluency after 3 days. For further differentiation, these cells were maintained in DMEM supplemented with 10% FBS (Gibco, Thermo Fisher Scientific), 0.5 mM IBMX (Sigma, St. Louis, MO, USA), 1 μM dexamethasone (Sigma), and 10 μg/mL insulin (Sigma) for 3 days, subsequently, cultured in DMEM containing 10% FBS and 10 μg/mL insulin for 6 days until they reached full differentiation, and then exposed to DMEM containing 10% FBS for 3 days. oil red O staining and BODIPY staining were performed to confirm differentiation efficiency.

Dermal fibroblasts were isolated from skin biopsies from eight NHS. Under local anesthesia with 1% xylocaine, 5-mm punch skin biopsies were performed in the distal forearm. Samples were minced into small pieces, and digested by collagenase type I (ThermoFisher Scientific Inc., Waltham, MA, USA) for 2 h at 37 °C with 5% CO₂. After centrifugation at 300 × g for 10 min, pellets were resuspended in 1 ml D-MEM (Gibco-Life Technologies, Groningen, the Netherlands) supplemented with 20% fetal bovine serum (FBS; PAA-GE Healthcare, Buckinghamshire, UK), 2 mM glutamine (Sigma-Aldrich, Saint Louis, MO, USA), penicillin (100 U/ml)–streptomycin (100 μg/ml) (Sigma-Aldrich) and transferred into a T25 plate (Corning Incorporated, NY, USA). Cultures were maintained at 37 °C in 5% CO₂-humidified incubator until confluence. Nonadherent cells and dermal tissue were removed by washing, and established fibroblasts were passaged after trypsin/EDTA (ThermoFisher Scientific) release up to the eight passage. Cells were maintained in D-MEM with 10% FBS, 2 mM glutamine, penicillin (100 U/ml)–streptomycin (100 μg/ml) (ThermoFisher Scientific) or incubated overnight in D-MEM with 1% FBS before functional studies.
The purity of fibroblast culture was 98% as detected by flow cytometry using a mouse anti-human CD90 and a mouse anti-human CD45 antibodies–PE conjugated (BD Biosciences, San Jose, CA, USA).