br Fig TRAIL expression in ASCs cultured
Fig. 1. TRAIL expression in ASCs cultured at high density. A–B) Expression of IFN-β and TRAIL in ASCs at diﬀerent cell seeding densities. ASCs were cultured at doses of 5000 (5K), 10,000 (10K), 20,000 (20K), or 40,000 (40K) cells/cm2 for 5 days, and the expression of IFN-β and TRAIL was evaluated by RT-PCR (A) and immunoblotting (B). C–D) TRAIL expression in 40K-ASCs after culture for the indicated time periods using RT-PCR (C) and immunoblotting (D). E) TRAIL expression in 5K-ASCs treated with IFN-β. After treatment with IFN-β for 48 h, TRAIL expression was analyzed by immunoblotting. F) Expression of membrane-bound TRAIL (mbTRAIL) in ASCs. 5K-ASCs or 40K-ASCs were cultured for 1 or 5 days, and the mean fluorescence intensity (MFI) of mbTRAIL was analyzed by flow cytometry after staining with Fluor 488-conjugated mbTRAIL antibody and APC-conjugated DR5 antibody. G) Expression of secreted TRAIL (sTRAIL). ASCs were cultured for 5 days, and conditioned media were harvested by centrifugation. Data represent the mean ± SD from three independent experiments. *P ≤ 0.05; **P ≤ 0.01. ns: non-specific band.
was observed in media when ASCs were cultured for 5 days at a cell concentration of > 10K or when cultured at 40K for more than 3 days . Indeed, in the absence of both serum and glucose, TRAIL induced necrotic cell death in H460 cells, but not apoptosis (Fig. 3A). These results suggest that both IFN-β and TRAIL played key roles in inducing necrotic cell death in H460 cells, which depended on nutrient depletion (i.e., serum and glucose) during co-culture with 40K-ASCs.
3.4. Tumor suppression by ASCs
40K-ASCs induced H460 cell death in vitro by expressing IFN-β and TRAIL. Therefore, we investigated whether 40K-ASCs could control tumor growth in an in vivo tumor model. H460 Benazepril were sub-cutaneously injected with or without 5K-ASCs or 40K-ASCs into nude mice. When H460 cells were co-injected with 5K-ASCs or 40K-ASCs, tumor volumes were 2.5-fold and 1.5-fold greater than those in the control group after 1 week, respectively. However, from week 2, the tumor volumes of mice in the control group were greater than those of mice in the ASC co-injected groups (Fig. 4A and B). Furthermore, tumor
weight was reduced by 15% and 23% at the end of 3 weeks in the 5K-ASC and 40K-ASC co-injected groups, respectively (Fig. 4C). These re-sults show that 5K-ASCs and 40K-ASCs could suppress tumor growth in vivo, suggesting that IFN-β and TRAIL expression in 40K-ASCs may more eﬀectively inhibit tumor growth.
3.5. Mechanism of TRAIL expression in ASCs
Since serum depletion is known to induce the expression of type I IFNs in macrophages , we investigated whether serum depletion would induce IFN-β and TRAIL expression in ASCs. Interestingly, when 40K-ASCs were cultured in serum-free medium, IFN-β expression was undetectable at 3, 24, and 72 h but was detectable at 6, 12, 96, and 120 h. TRAIL mRNA expression was detectable from 3 h and was highly expressed from 24 h (Fig. 5C). TRAIL protein expression in 40K-ASCs was detectable from 48 h and increased gradually in a time-dependent manner (Fig. 5D). However, 5K-ASCs cultured in serum-free media did not express IFN-β or TRAIL (Fig. 5A and B). Moreover, when 40K-ASCs expressing IFN-β and TRAIL were re-seeded at a low density (5K), IFN-β
Fig. 2. Cell death of H460 cells after treatment with 40K-ASCs. A) Growth inhibition of H460 cells in conditioned media obtained from cultured 40K-ASCs (40K-ASC-CM) after 1–5 days. H460 cells were treated with 40K-ASC-CM for 24 h, and their viability was assessed using an MTT assay. Error bars represent the mean ± SD of triplicate wells. Data are from one of three independent experiments. *P ≤0.05. B) Morphology of H460 cells indirectly co-cultured with 40K-ASCs. H460 cells were co-cultured with 40K-ASCs for 2 days, and then the morphology of H460 cells was observed using a light microscope (100 × ). C) Annexin-V/7-AAD staining of H460 cells indirectly co-cultured with 40K-ASCs for 2 days. Data represent the mean ± SD of three independent experiments. D) Growth suppression of CFSE-labeled H460 cells (H460∗) directly co-cultured with 40K-ASCs. Cell cycle analysis of H460∗ cells co-cultured with 40K-ASCs or alone for the indicated time points by Vybrant DyeCycle Ruby staining. Approximate H460∗ cell populations are marked with red squares (upper panel); only the cell cycle stages of H460∗ cells were analyzed, represented by a bar graph (lower panel). Error bars represent the mean ± SD from triplicate analyses. Data were obtained from one of three independent ex-periments. TW: Transwell. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)