Recombinant mouse IL-1α was from Peprotech, Inc. (La Jolla, CA, USA). Anti-mouse CD34, CD45, CD90, CD105 and CD29 antibodies were from eBioscience (San Diego, CA, USA).

MSCs were generated from bone marrow flushed out of the tibias and femurs of 4–6-week-old mice. The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA). Non-adherent cells were removed after 72 h and adherent cells were maintained with medium replenishment every 3 days.

The murine prostate cancer cell line RM-1 was cultured at 37°C with 5% of CO₂ in RPMI-1640 with 10% FBS, supplemented with 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were subcultured every 3 days when they reached 70–80% confluence.

Male Balb/c and C57BL/6 mice, 6–8 weeks old, were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences, Shanghai, China. Mice were housed in pathogen-free conditions and all procedures were performed according to the guidelines of the Committee on Animals of the Chinese Academy of Sciences.

MSCs were cultured with an osteoinductive medium consisting of DMEM supplemented with 10% FBS, β-mercaptoethanol, 100 μM L-ascorbic acid, 10 nM dexamethasone and 10 mM β-glycerophosphate for 14 days. The cells were then stained with alizarin red to reveal calcium deposition, characteristic of osteoblasts. MSCs were induced to differentiate into adipocytes by culturing with DMEM supplemented with 0.5 mM isobutylmethylxanthine, 60 μM indomethacin, 10 nM dexamethasone and 10 μg/ml insulin for 14 days. The formation of adipocytes was verified by staining for triglycerides with Oil red O to detect intracellular lipid accumulation.

RM-1 cells and MSCs were prepared either as single-cell type suspensions (1×10⁶ cells in 200 μl PBS) or as a mixture of cells (1×10⁶ RM-1 cells and 2×10⁵ MSCs in 200 μl of PBS). RM-1 cells (alone or mixed with MSCs) were subcutaneously administered in the armpit area of Balb/c or C57BL/6 mice. The mice were examined every day and tumor growth was evaluated by measuring the length and width of the tumor mass. At the end of the experiment, the animals were sacrificed and the tumors were removed. The tumor masses were weighed and analyzed histologically.

MSCs were stimulated by culturing with IL-1α (20 ng/ml) for 12 h. The culture medium was then abandoned and replaced with DMEM culture medium without FBS. After culturing for a further 24 h, the conditioned medium was collected, as well as a 0.22-μm filtrate of the supernatant medium from the MSCs.

RM-1 cells and MSCs were prepared either as single-cell type suspensions (1×10⁶ cells in 200 μl of PBS) or as a mixture of cells (1×10⁶ RM-1 cells and 2×10⁵ MSCs in 200 μl PBS). Subcutaneous administration of RM-1 cells (alone or mixed with MSCs) was performed in the armpit area of C57/BL6 mice. Tumor incidence was evaluated 3 times per week.

Mouse spleens were disaggregated into 10 ml RPMI-1640 medium to isolate splenocytes. Erythrocytes were lysed with 0.84% NH₄Cl and subsequently washed 3 times with RPMI-1640. Trypan blue dye exclusion was used to assess cell count and viability. Splenocytes were incubated with 5 μg/ml concanavalin A (ConA; Sigma-Aldrich, St. Louis, MO, USA) for 72 h and then cultured with IL-2 (200 U/ml) for proliferation. Splenocyte cultures were maintained in RPMI-1640 medium supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 50 mM of β-ME (complete medium). MSCs were added to the MLR to provide a 200-μl final volume. Following 3 days of incubation, 1 μCi/well (0.037 MBq/well) ³H-thymidine was added overnight and thymidine incorporation was measured using a β-scintillation counter. The data were presented as the percentage of the relative proliferative response, corresponding to the mean counts per min (cpm) of a responder stimulator pair in the absence of MSCs which was attributed a 100% value.

MSCs were incubated with IL-1α (20 ng/ml) for 12 h and the total cell mRNA was collected with TRIzol reagent (Invitrogen). cDNA was synthesized using M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA) and 2 μg total RNA and oligo dT18-primers. PCR amplification was carried out using 2-μl aliquots of cDNA. Real-time PCR was performed in triplicate using the SYBR PrimeScript RT-PCR kit (Takara Bio, Inc., Shiga, Japan). The primer sequences for TGF-β were: forward, 5′-TGTCACCGGAGTTGTGCGGC-3′; reverse, 5′-CTCGGCGGCCGGTAGTGAAC-3′. Total sample RNA was normalized to endogenous β-actin mRNA. Thermocycler conditions included an initial hold at 50°C for 2 min and then 95°C for 10 min, followed by a two-step PCR program of 95°C for 15 sec and 60°C for 60 sec repeated for 40 cycles using an Mx 4000 system (Stratagene, La Jolla, CA, USA) on which data were collected and quantitatively analyzed. Expression levels of mRNA were presented as fold changes relative to an untreated control.

Cells were washed with PBS solution, and protein was then extracted according to an established protocol. Furthermore, proteins were mixed with Laemmli sample buffer, heated at 65°C for 10 min, loaded (20 μg for each sample), separated by sodium dodecyl sulfate-polyacrylamide gel (7.5%) electrophoresis under denaturing conditions and electroblotted onto nitrocellulose membranes. The nitrocellulose membranes were blocked by incubation in blocking buffer (1% BSA in Tris-buffered saline-0.1% Tween 20), incubated with anti-TGF-β-antibody (Abcam, Cambridge, UK), and washed and incubated with anti-rabbit peroxidase-conjugated secondary antibody (Invitrogen). Signals were visualized by chemiluminescent detection. Blots were quantified using Quantity One software from Bio-Rad (Hercules, CA, USA), and TGF-β expression was normalized to values in the control group. Equal loading of samples was verified by Coomassie blue staining of simultaneously run gels. Gels were run 4 times and the images shown are representative.

Statistical analysis of the data was performed using GraphPad Prism 4 software. The Student's t-test was used to compare the mean values of the two groups. Data among 3 or more groups were compared using the one-way analysis of variance, followed by the Dunnett's post hoc test. Final values were expressed as the mean ± SEM. P<0.05 was considered to indicate a statistically significant result.

We identified long spindle-shaped fibroblastic cells isolated from bone marrow by examining their surface markers and ability to differentiate. The results demonstrated that these cells were positive for CD90, CD105 and CD29 and negative for CD34 and CD45. Furthermore, these cells differentiated into adipocytes and osteoblast-like cells. The results indicated that the cells isolated from the bone marrow had properties that were consistent with those of MSCs (Fig. 1A and B).

MSCs, which were either pretreated with inflammatory cytokines IL-1α or not, were co-injected with RM-1 cells into Balb/c mice. We found that a more rapid growth of the RM-1 cells that were mixed with non-pretreated MSCs in vivo than that of the RM-1 cells alone. Compared with the control group, MSCs pretreated with IL-1α demonstrated a tumor-promoting effect (Fig. 2). Conditioned media were collected from the MSCs and IL-1α-pretreated MSCs. The conditioned media were infused into subcutaneous RM-1 tumor-bearing mice via tail vein injection. Compared with the control groups, the conditioned medium from IL-1α-pretreated MSCs significantly enhanced the tumor growth in vivo (Fig. 2). These results indicate that IL-1α stimulates MSCs to promote the growth of RM-1 tumors in vivo.

To determine the immunosuppressive effect of MSCs in the promotion of tumor growth in vivo, we combined RM-1 cells with MSCs, which were either pre-incubated with IL-1α or not, and implanted subcutaneously in C57/BL6 mice. The RM-1 cells developed into tumors when implanted in Balb/c mice; however, these cells were rejected by the C57/BL6 mice. When RM-1 cells were co-injected with MSCs, the tumor incidence markedly increased. Furthermore, compared with the control group, IL-1α-pretreated MSCs further enhanced the tumor incidence (Fig. 3). In addition, we found that the only conditioned medium able to increase the RM-1 tumor incidence in the C57/BL6 mice was that obtained from IL-1α-pretreated MSCs (Fig. 3). Taken together, the results suggest that IL-1α induces the immunosuppressive action of the MSCs, which may help the RM-1 cells to escape from immune rejection by the C57/BL6 mice.

We employed the MLR to examine the immunosuppressive function of MSCs induced by IL-1α. We activated the splenocytes from Balb/c mice with Con A for 72 h and then expanded them with IL-2. The activated splenocytes were co-cultured with MSCs that were either pretreated with IL-1α or not. The results demonstrated that the MSCs did not inhibit the proliferation of splenocytes unless they were pretreated with IL-1α (Fig. 4). In addition, conditioned media collected from the MSCs were added to the activated splenocyte culture system and the proliferation of the splenocytes was examined. The conditioned medium obtained from IL-1α-pretreated MSCs effectively inhibited the proliferation of the splenocytes (Fig. 4).

To detect the mechanism by which IL-1α induced the immunosuppressive function of the MSCs, we examined the production of immunosuppression-related cytokines in the MSCs following exposure to IL-1α. Real-time PCR and western blotting were employed to detect the expression of immunosuppression-related cytokines in the MSCs. As shown in Fig. 5A and B, IL-1α effectively upregulated the expression of TGF-β in the MSCs. To confirm the role of TGF-β in the immunosuppressive function of the MSCs, TGF-β siRNA was used to inhibit the expression of TGF-β in the MSCs. In mixed co-cultures of splenocytes and MSCs pre-stimulated by IL-1α, splenocyte proliferation was restored to normal levels by TGF-β siRNA (Fig. 5C). These results suggest that TGF-β is the key factor that mediates the IL-1α-induced immunosuppressive effect of the MSCs on splenocyte proliferation.

We have demonstrated that IL-1α effectively induces the ability of MSCs to promote the growth of RM-1 cells in vivo and that this enhancement may be associated with the immunosuppressive function of MSCs. We have also shown that the IL-1α-induced immunosuppressive action of the MSCs was mediated by TGF-β. Therefore, we used TGF-β siRNA to confirm the role of TGF-β in the immunosuppressive effect of the MSCs. The results showed that the promotive effect of MSCs on tumor growth in vivo induced by IL-1α was inhibited by TGF-β siRNA (Fig. 6A). Furthermore, the enhancement of RM-1 tumor incidence in the C57/BL6 mice by IL-1α-pretreated MSCs was reduced following the use of TGF-β siRNA (Fig. 6B). These data suggest that TGF-β is a key factor in the immunosuppressive action of MSCs that enables RM-1 cells to escape from immune injury.