Mouse bone marrow-derived MSC have been previously described (28) and were kindly provided by Dr. Angelika Hausser (IZI, University of Stuttgart, Germany). These cells were cultivated under sterile conditions, at 37°C in a 5% CO₂ humidified atmosphere, in alpha-MEM supplemented with 10% FBS (HyClone) plus 1% penicillin/streptomycin. MSCs were passaged at a confluence of 70% every 3–4 days if not mentioned otherwise. Colo205 and HCT116 cells were obtained from ATCC (Manassas, VA, USA) and cultured, at 37°C and 5% CO₂, in RPMI-1640 medium (Invitrogen) supplemented with 10% FCS (Thermo Fisher Scientific).

Mesenchymal stem cells were transfected with polyethylenimine (PEI) using a ratio 1:3 for DNA and PEI. Briefly, 150 × 10³ cells/well in a six-well plate were grown in 2-ml culture medium for 18 h. Next, cell culture medium was removed, and 1.5 ml of serum-free alpha-MEM was added. Three hundred microliters of Opti-MEM were incubated with 12 μg of PEI for 5 minutes (min) at room temperature (RT). Next, 4 μg of plasmid DNA was added to the mixture, and after 20 min incubation, the mix was carefully added drop-wise to the cells. After 18 h incubation at 37°C cells were transferred into a flask and allowed to grow in cell culture medium for 24 h. Next, in order to select the transfected cells, 250 μg/ml of geneticin (G418) was added to the medium. Subsequently, a single clone selection, making limiting dilutions with a statistical density of 1 cell/well was performed. The best clone, named MSC.TRAIL, was used for further studies. The coding sequence of Db-scTRAIL (EGFR-specific pCR3-Db-scTRAIL) construct was reported by Siegemund et al. (19).

The EGFR targeting Db-scTRAIL fusion protein (see Figure S1 in Supplementary Material) was produced from transfected MSCs and purified from cell culture supernatant by anti-FLAG affinity chromatography as described previously (19). In brief, cell-free supernatant was incubated with anti-FLAG M2 Affinity Gel (0.3 ml bead volume/100 ml supernatant, Sigma-Aldrich) for at least 2 h or alternatively overnight at 4°C on a roller mixer, prior to collecting of beads in an empty column, washing with TBS, and eluting with 100 μg/ml FLAG peptide in TBS. After dialysis in PBS, eluates were concentrated with Vivaspin 20 devices (50 kDa, Sartorius), and the purified protein was analyzed by western blotting.

Colo205 (4 × 10⁴ cells/well), HCT116 (3 × 10⁴ cells/well), or MSCs (2 × 10⁴ cells/well) were grown in 100-μl culture medium in 96-well plates for 18 h, followed by treatment either with serial dilutions of Db-scTRAIL proteins or supernatant from transfected MSCs, in triplicates. For positive control, cells were killed with 0.25% Triton X-100. Cell death assays were performed in the absence or presence of bortezomib (BZB) (250 ng/ml; UBPBio). TRAIL blocking antibody (1 μg/ml; Enzo Life Sciences) was used in the combined treatments. BZB was added 30 min before incubation with the proapoptotic proteins to sensitize cancer cells for cell death induction. After 16 h of incubation, cell viability was determined by crystal violet staining (19).

Colo205 (1 × 10⁴ cells/well) or HCT116 (8 × 10³ cells/well) were seeded in 24-well plates, in 600 μl of MSC medium (alpha-MEM), and allowed to grow at 37°C. After overnight incubation, MSCs were added using different ratios of MSCs:colorectal cancer (CRC) cells, in a final volume of 1 ml/well. Different treatments with BZB (250 ng/ml) and TRAIL blocking antibody (1 μg/ml) were performed and finally analyzed by crystal violet staining as described above.

Enzyme-linked immunosorbent assay assays were performed using the kit OptEIA™ human TRAIL ELISA Set (BD), according to the manufacturer’s instruction. Briefly, the ELISA plate was coated with 100 μl/well of capture antibody and incubated overnight at 4°C, and the remaining binding sites were blocked with 2% (w/v) dry milk/PBS. Next, a titration (1:3) of standard TRAIL protein and either serial dilutions of MSC supernatant or serum blood (dilution 1:20) were added and incubated for 2 h at RT, in duplicate. After five washing steps, working detector solution was incubated for 1 h at RT. Bound proteins were detected using ELISA developing solution (0.1 mg/ml TMB, 100 mmol/l sodium acetate buffer, pH 6.0, 0.006% H₂O₂). The reaction was stopped with H₂SO₄ (1 mol/l). Absorbance was measured at 450 nm in an ELISA reader.

TNF-related apoptosis-inducing ligand secreted by transfected MSCs in culture medium was purified by an anti-FLAG affinity chromatography as described above. The purified proteins were separated on SDS-PAGE (12%) and then blotted on PVDF membrane. After incubation with primary antibody (anti-TRAIL MAB687, R&D), the secondary anti-mouse HRP-conjugated antibody (Sigma-Aldrich) was added. Finally, the membrane was treated with a peroxidase substrate (enhanced chemiluminescence detection system from Pierce) according to the manufacturer’s instructions to visualize the signals and exposed to an X-ray film.

To analyze the expression of surface markers, MSCs (10 × 10⁴ cells/well) were seeded in a 96-well plate and incubated with the directly conjugated antibody (CD9, CD44, CD71, CD105, CD14, and CD34). Signals from respective isotype control antibodies were subtracted from all samples to compensate unspecific antibody binding.

Propidium iodide (PI, Sigma-Aldrich) staining of cells was done after 18 h of treatment. The cells were collected, incubated with 10 μg/ml of PI, and immediately analyzed by flow cytometry.

In order to test cleaved caspase-3 activation, Colo205 (8 × 10⁴ cells/well) were seeded in the bottom of a transwell plate (Costar) and incubated at 37°C overnight. Then, BZB (250 ng/ml) was added into the culture medium and incubated for 30 min. Next, into the upper chamber of the transwell, 16 × 10³ cells for each MSC line were seeded. After 18 h of coculture, Colo205 cells from the lower chamber were collected, fixed in PFA (4%), and permeabilized with 0.1% Triton X-100. Then, primary antibody (Asp 175, Cell Signaling Technology) was incubated for 1 h at RT. After two washing steps and secondary antibody incubation, the cells were resuspended and analyzed.

Mesenchymal stem cells were seeded on glass coverslips and incubated for 3 h at 37°C. Then, coverslips were washed with PBS and cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. The blocking step was performed by incubating the cells with 5% FBS in PBS for 30 min at RT. Next, cells were washed and incubated with primary antibody and DAPI (1 μg/ml, Sigma) diluted in blocking buffer for 2 h. When required, incubation with secondary antibody, diluted in blocking buffer, was performed for 1 h. Coverslips were mounted in Fluoromount G and analyzed with the Spinning Disc (Zeiss) using 488, 543, and 633 nm excitation and a 20×/0.8 DIC objective lens. Images were processed with ZEN software.

For the adipogenic differentiation, MSCs were grown to confluence on Permanox 4-well chamber slides (Thermo Scientific). Then, the culture medium was replaced with adipogenic medium (α-MEM supplemented with 10% FBS, 1% penicillin/streptomycin, 1 μM dexamethasone, 500 μM IBMX, 10 μg/ml human insulin, and 100 μM indomethacin) and changed every 2–3 days. Twelve days after initial adipogenic induction, cells were washed with PBS and fixed for 10 min in 4% Histofix (Roth). Next, cells were rinsed once with H₂O, incubated in 60% isopropanol for 5 min, and then with Oil Red O for 10 min. Finally, the cells were washed once with 60% isopropanol followed by H₂O. Nuclei were counterstained with hemalaun. As a negative control, cells grown in culture medium for 12 days were used. In order to analyze the osteogenic differentiation, MSCs were grown to 90–100% confluence in 24-well plates and then cultured in osteogenic medium (α-MEM supplemented with 15% FBS, 1% penicillin/streptomycin, 100 nM dexamethasone, 50 μg/ml ascorbate-2- phosphate, and 10 mM beta-glycerol phosphate) for 21 days. At this time point, the differentiation was assessed by Alizarin Red staining. In brief, cells were washed with PBS and allowed to dry for 5–10 min. Next, cells were fixed with 50% ethanol for 20 min. The fixed cells were then stained with 1% Alizarin red (Roth) at pH 6.4 for 30 min under continuous shaking. Finally, cells were rinsed three times with H₂O, and transmitted light pictures were taken. As a negative control, cells grown in culture medium for 21 days were used.

Animal care and all experiments performed were in accordance with federal guidelines and had been approved by university and state authorities. Female NMRI nu/nu mice (Janvier), 8 weeks old, were injected subcutaneously (s.c.) with 3 × 10⁶ Colo205 cells in 100 μl PBS at left and right dorsal sides. Treatment started 10 days after tumor cell inoculation when tumors reached ~100 mm³. In particular, 4 × 10⁶ MSCs were resuspended in 100 μl PBS mixed with 100 IU/ml of heparin (29) and then peritumorally injected (p.t.). During the injections of all cell lines, mice were anesthetized with isoflurane. The Colo205-bearing mice received three p.t. injections of MSCs at day 10, 17, and 27. In addition, 5 μg of BZB in 100 μl PBS were injected i.p. every second day, starting from day 11 until day 31. Mice in the control groups received either 100 μl PBS i.p. injected or MSCs.Mock. Tumor growth was monitored as described (30). Blood samples were taken from the tail at day 31, centrifuged (10,000 × g, 10 min, 4°C) and then stored at −80°C. Activity of ALT was determined by an enzymatic assay accordingly to the manufacturer’s instructions (BIOO Scientific, Austin, TX, USA).

All values are expressed as means ± SD, while for the analysis of the in vivo studies the 95% confidence interval (95% CI) was used. Significances, for each experiment, were calculated with GraphPad prism one-way ANOVA with Tukey’s post-test. In particular: * represents a p-value < 0.05, ** represents a p-value < 0.01, and *** represents a p-value < 0.001.

As a prerequisite to study the application of MSCs as cell-based therapy for Db-scTRAIL fusion protein expression, we first investigated the TRAIL sensitivity of these cells in comparison to a CRC cell line Colo205. In accordance with our previous study (19, 30), combinatorial activity of the Db-scTRAIL fusion protein with the apoptosis sensitizer BZB exerts a potential superior apoptotic effect on CRC cells. In fact, we observed a strong enhancement of cell death induction on Colo205 cells upon combined treatment resulting in a ~9-fold increase of TRAIL-mediated apoptosis induction with BZB (EC₅₀ values: Db-scTRAIL 19 pM; Db-scTRAIL + BZB 2.2 pM) (Figure 1A). Conversely, under the same conditions, MSCs were fully resistant to Db-scTRAIL activity even in the presence of the sensitizer (Figure 1B), confirming that MSCs are a well-suited cell delivery system of highly active TRAIL variants. Next, we tested Db-scTRAIL expression after transient transfection by ELISA and immunoblotting as well as in vitro bioactivity. MSCs were able to secrete Db-scTRAIL, revealing an accumulation of intact product over the observed time period of 5 days (Figures 1C,D). With the applied transient transfection protocol, Db-scTRAIL production, as revealed by specific ELISA and bioassay (induction of Colo205 cell death) was highest 1 day after transfection (~0.44 pg/cell) and declined thereafter, with still significant protein and bioactivity detectable after 5 days of culture and four subsequent media changes (Figure 2). Additionally, we tested cell death induction with PI staining after coculture of MSCs and Colo205 at ratios of 1:5 and 1:50, showing a significant increase of PI levels in a cell-dose and BZB-dependent manner (Figure S2 in Supplementary Material). These data collectively demonstrate that MSCs are a suitable system for the expression of bioactive Db-scTRAIL proteins.

Based on the positive results with transient transfection of MSCs, we aimed at generation of stable producer clones by standard selection methods and isolation of individual clones by limiting dilution. The two highest expressing clones out of 13 identified positive clones after the first screening round proofed to be stable expressors in vitro and one clone, named MSC.TRAIL was used for further analyses of long-term expression and in vivo activity (Figure S3 in Supplementary Material). MSC.TRAIL showed cumulative secretion of the protein during culture for 5 days and stable expression of the product in vitro for 44 passages (data not shown). Western blot analysis of the purified TRAIL verified secretion of full-length protein (Figure 3A). Bioactivity of the secreted Db-scTRAIL was tested in coculture assays with Colo205 as target cells in the presence or absence of BZB (Figure 3B). A strong reduction of the cell viability in combination with BZB and complete neutralization of cell death in the coculture by a TRAIL blocking antibody proofed secretion of bioactive protein and strictly TRAIL-dependent cell death. The same results were observed after coculture of MSC.TRAIL with a different CRC cell line (HCT116) sensitive to TRAIL (Figures S4A,B in Supplementary Material). In order to confirm that the observed reduction of cell number is due to an apoptotic process, we analyzed cleaved caspase-3 levels as a hallmark of apoptosis induction. For this, we performed coculture using a double chamber system with a membrane allowing free exchange of soluble mediators. Colo205 cells were seeded in the bottom chamber and the MSC.TRAIL or the Mock cells were seeded in the upper chamber. Cleaved caspase-3 levels in Colo205 cells were determined by flow cytometry after 18 h of coculture (Figure 3C). As expected, a strong increase of cleaved caspase-3 levels was found when Colo205 cells were exposed to Db-scTRAIL-producing cells in combination with the sensitizer BZB. MSC.Mock served as negative control and a weak signal was noted upon incubation with sensitizer BZB only. Collectively, the in vitro studies show that stable MSC producer clone can be established exerting long-term expression of a highly bioactive Db-scTRAIL fusion protein.

In order to investigate whether the transfection and isolation of a stable producer cell line affected MSC characteristics in vitro, we analyzed the properties of this cell line at different passages and compared it to mock-transfected and untransfected MSCs. We first tested the phenotype of MSCs by staining the cells with phalloidin to visualize the F-actin. All cell lines (MSCs untransfected, MSC.Mock, and MSC.TRAIL) displayed a typical spindle-shaped phenotype as described for MSCs (31). Remarkably, the phenotype did not change during in vitro cultivation up to passage 42 (Figure 4A). Next, we investigated the expression of stem cell markers. In accordance with the International Society of Cellular Therapy, all MSC lines analyzed were positive for CD9, CD44, CD71, and CD105 and lacked the expression of CD14 and CD34, as show in Figure 4B. No differences between untransfected MSCs and the stably transfected cell lines were observed. Interestingly, the pattern of marker expression was maintained from early passage (p9) up to passage 42 (Figure S5 in Supplementary Material) and is in accordance with murine MSCs lines described by others [reviewed by Boxall and Jones (32)]. Finally, we verified the multilineage differentiation capability of MSCs, which is the most characteristic feature. All MSC lines showed, on one hand, the capability to generate lipid droplets which indicate a successful adipogenic differentiation (Figure 4C). In rare cases, a spontaneous adipogenic differentiation was observed, probably due to a high cell density in the differentiation cultures, without a statistically significant frequency. Further, the cell lines were also able to display mineralization, observed by Alizarin red staining, confirming osteogenic differentiation ability (Figure 4D). The same results were observed at late passages for all cell lines (data not shown). These findings demonstrate that the stable non-viral transfection and the constitutive Db-scTRAIL secretion do not alter the typical properties of MSCs, even during long-term in vitro culture.

Based on our in vitro results, we assessed the potential therapeutic utility of MSC.TRAIL in vivo. First, we performed single subcutaneous injection of MSCs (s.c.; 4 × 10⁶ cells) in nude mice in order to verify the presence of Db-scTRAIL in the serum fraction after 1, 3, 7, 14, and 21 days by ELISA (Figure S6 in Supplementary Material). No TRAIL signals could be detected up to 3 days after injection, whereas after 7 and 14 days specific TRAIL signals were identified in the range of 1.5 ng/ml for MSC.TRAIL cells. As expected, all control cell injections gave no positive signal. Next, we investigated the antitumor activity of MSC.TRAIL in a Colo205 mouse xenograft tumor model, in which MSCs were peritumorally (p.t.) injected at three time points. The treatments started when tumors were palpable and vascularized, reaching a volume of ~100 mm³. At this time point, the first MSC (4 × 10⁶ cells) injection was performed, with Mock cells and PBS used as controls. In the combination treatment groups (MSC.TRAIL + BZB and MSC.Mock + BZB), 5 μg of BZB was intraperitoneally (i.p.) injected every other day. Up to 10 days after the first MSC injection, we observed no differences in tumor growth for all groups. However, from day 17 on, coincident with the second MSC administration, a slight, but increasingly significant reduction of tumor size was observed for the combination treatment group MSC.TRAIL + BZB over the whole observation period (31 days) (Figure 5A). Importantly, at day 26, serum analysis of Db-scTRAIL in animals receiving MSC.TRAIL revealed a concentration of ~1.5 ng/ml (Figure 5C) supporting a direct correlation of TRAIL activity and tumor reduction. Tumor response was maintained with the third application of MSC.TRAIL, although complete remission was achieved only in one case (Figure 5C). The observed MSC.TRAIL-dependent tumor response required co-administration of a low dose of the sensitizer BZB, corroborating the in vitro data. Thus, under the applied protocol, the MSC.TRAIL cells alone showed only a small, non-significant reduction in tumor growth, similar to the Mock-transfected MSC in combination with BZB (Figure 5B).

In order to get insights into potential off-target, systemic side effects of continuous Db-scTRAIL expression in tumor-bearing animals, we analyzed serum levels of the liver enzyme alanine aminotransaminase (ALT) as an established marker of acute liver toxicity. ALT serum levels were determined in tumor-bearing mice at day 31, after three MSC p.t. injections. The analysis showed for all MSC groups only slightly elevated serum ALT levels compared to PBS group (<50 U/l), but low compared to the Fas ligand treatment known to cause acute liver toxicity (Figure 5D). This result revealed that the applied treatment protocol (three MSC administrations with and without BZB) did not induce discernable acute hepatotoxic effects in vivo, in accordance with a recent study from Yan and colleagues (21). Additionally, all the other standard parameters, such as body weight, remained in the normal range for the entire period of treatment (data not shown).