br Introduction br Although mesenchymal stem cells
Although mesenchymal stem cells (MSCs) are utilized in re-generative medicine, the development of tumor tropism and promotion of tumorigenesis in pre-existing tumors limit their applicability in cancer patients [1,2]. MSCs support tumor growth through diﬀer-entiation to tumor-associated fibroblasts [3–6]; suppression of immune responses ; promotion of angiogenesis [4,8,9]; stimulation of epi-thelial-mesenchymal transition [10,11]; interaction with cancer stem cells [12,13]; promotion of tumor metastasis [1,14–17]; and inhibition
of tumor cell apoptosis [18–20]. MSCs have also been reported to prevent tumor growth through apoptosis induction, Elafibranor (GFT505) and cel-lular signaling regulation, and immune cell infiltration induction .
Interferons (IFNs) are pleiotropic cytokines that regulate the innate and acquired immunity, activate host defenses against viral and bac-terial infections, and function in tumor surveillance. IFNs comprise two main families: type I (IFN-α, -β, -ε, -κ, and -ω) and type II (IFN-γ) . IFN-α and IFN-β are secreted by many cell types, including lymphocytes (natural killer [NK] cells, B-cells, and T-cells), macrophages, fibroblasts, endothelial cells, and osteoblasts . They stimulate both
Abbreviations: 40K-ASC-CM, CM recovered from 40K-ASCs; 7-AAD, 7-aminoactinomycin D; APC, allophycocyanin; ASCs, adipose tissue-derived MSCs; Cas, caspase; CFSE, carboxyfluorescein diacetate succinimidyl ester; CM, condition medium; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; H460*, CFSE-labeled H460; HBSS, Hank's Balanced Salt solution; IFNs, interferons; mbTRAIL, membrane-bound TRAIL; MSCs, mesenchymal stem cells; MTT, methylthiazolyldiphenyl-tetrazolium bromide; PBS, phosphate-buﬀered saline; RT-PCR, reverse transcription-polymerase chain re-action; SD, standard deviation; sTRAIL, secreted TRAIL; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand
∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (K.S. Bae), [email protected] (Y.W. Eom).
macrophages and NK cells to elicit potent anti-tumor, anti-viral, and immunomodulatory functions [24,25]. Moreover, MSCs that are primed or genetically modified with type I and II IFNs exert antitumor eﬀects via nuclear factor-κB (NF-κB), signal transducer and activator of tran-scription (Stat), or tumor necrosis factor-related apoptosis-inducing li-gand (TRAIL) pathways [26–30].
Previously, we reported that adipose tissue-derived MSCs (ASCs) cultured at a high density expressed type I IFNs and suppressed tumor growth in MCF-7 breast cancer cells . Herein, we investigated TRAIL expression in ASCs cultured at a high density, analyzed the mechanisms controlling IFN-β and TRAIL expression, and evaluated the tumor suppression potential of ASCs against H460 lung cancer cells in vitro and in vivo.
2. Materials and methods
In accordance with the procedures approved by the Institutional Review Board of the Yonsei University Wonju College of Medicine, human adipose tissues from three healthy donors (24–38 years of age) were obtained through elective liposuction procedures under anesthesia at the Wonju Severance Christian Hospital (Wonju, Korea). Informed consent was obtained from all donors. Mononuclear cells were isolated using a modified protocol as described by Zuk et al. . Briefly, li-poaspirates were thoroughly washed with phosphate-buﬀered saline (PBS) to remove contaminated blood cells and local anesthetics. Mononuclear cells were then obtained by digestion with 0.075% type IA collagenase (Sigma-Aldrich, St. Louis, MO, USA) in PBS, cen-trifugation at 1200×g for 5 min, and resuspension in low-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco, Rockville, MD, USA) containing 10% fetal bovine serum (FBS) and penicillin/strepto-mycin. Debris was removed by filtration through a 100-μm mesh filter (Cell Strainer; Becton Dickinson, Franklin Lakes, NJ, USA). Mono-nuclear cells (5 × 106) were seeded in a 100-mm culture dish with low-glucose DMEM containing 10% FBS and penicillin/streptomycin. After 2 days, the medium was replaced to remove non-adherent cells. Thereafter, the cell culture medium was changed twice weekly, and the cells were passaged with 0.25% trypsin/0.1% EDTA (Gibco) until they reached 90% confluence. For experiments, ASCs were seeded at 5,000, 10,000, 20,000, or 40,000 cells/cm2 (5K-ASCs, 10K-ASCs, 20K-ASCs, and 40K-ASCs, respectively) and cultured for the indicated times (1–5 days).
The human lung cancer cell line H460 was purchased from the Korean Cell Line Bank (Seoul, Korea). H460 cells were maintained in low-glucose DMEM (Gibco) supplemented with 10% FBS and peni-cillin/streptomycin. H460 and ASCs were indirectly co-cultured using a Transwell plate. 5K-ASCs or 40K-ASCs were cultured for 3 days (upper chamber) and then co-cultured with H460 cells (lower chamber) for a further 2 days to observe changes in cell morphology or for 1 day to analyze apoptosis rates through annexin-V staining. For direct co-cul-ture, H460 cells were labeled with carboxyfluorescein diacetate succi-nimidyl ester (CFSE, Sigma-Aldrich). Briefly, H460 cells were sus-pended in PBS (1 × 107 cells/ml) and incubated with 5 μM CFSE in the dark at room temperature for 15 min. An equal volume of FBS was added, incubated for an additional 5 min at room temperature in the dark, and washed three times with cold PBS. The same number of 40K-ASCs cultured for 3 days before co-culture with CFSE-labeled H460 (H460∗) cells was seeded and then cultured for an additional 2 days.