Human mesenchymal stem cells promote growth of osteosarcoma: involvement of interleukin-6 in the interaction between human mesenchymal stem cells and Saos-2

Abstract

Our previous study showed that exogenous human mesenchymal stem cells (hMSCs) targeted established osteosarcoma and promoted its growth and pulmonary metastasis in vivo. As a follow-up, the present study aimed to investigate how hMSCs would interact with Saos-2 through autocrine/paracrine communication. The results showed that co-injection of hMSCs with Saos-2 into the proximal tibia of nude mice could promote tumor growth and progression. In vitro, the proliferation of Saos-2 and hMSCs was promoted by each other's conditioned medium, in which interleukin-6 (IL-6) played an important role. Osteogenic differentiation of hMSCs could be inhibited by conditioned medium of Saos-2, in which IL-6 was also involved. Furthermore, decreased IL-6 secretion by hMSCs during its osteogenesis and increased IL-6 secretion in response to conditioned medium of Saos-2 were observed. Based on these data, we suggest that there was a positive feedback loop of IL-6 in the interaction between hMSCs and Saos-2.

Figure 1

Growth and progression of osteosarcoma (OS) in vivo in response to co‐injection of human mesenchymal stem cells (MSCs). (A) OS volume. *P < 0.05, hMSCs + Saos‐2 versus Saos‐2. (B) Alkaline phosphatase (ALP) in blood. *P < 0.05. (C,G,K) General observation of OS. (D,H,L) X‐ray examination. (E,I,M) Pathological analysis. (F,J,N) Fluorescent photographs. Green arrows, injected cells; red arrows, mean hMSCs labeled with GFP; yellow arrows, site of injection. Results are expressed as the mean ± SD.

Figure 2

Interaction between human mesenchymal stem cells (hMSCs) and Saos‐2 through paracrine in vitro. Conditioned medium (CM) of hMSCs (hMSCs‐CM), 293T (293T‐CM), and Saos‐2 (Saos‐2‐CM) was prepared and added to the culture medium of Saos‐2 and hMSCs. At the indicated time points, MTT assay was used to monitor hMSCs (A) and Saos‐2 (B) proliferation. Osteogenic induction medium was used to induce hMSCs to undergo osteogenic differentiation, in which Saos‐2‐CM or 293T‐CM was added. Alkaline phosphatase (ALP) quantification assay and real‐time PCR for osteopontin and osteocalcin were carried out (C). Results are expressed as the mean ± SD. *P < 0.05; **P > 0.05.

Figure 3

Screen for cytokines involved in the interaction between human mesenchymal stem cells (hMSCs) and Saos‐2. Conditioned medium of hMSCs and Saos‐2 were screened in the present study and plotted for their relative secretion levels (A). The results were expressed as standardized signal intensity. Interleukin‐6 (IL‐6) secretion volume by 10⁶ hMSCs and Saos‐2 for 2 h was assayed (B). Results are expressed as the mean ± SD. BDNF, brain‐derived neurotrophic factor; FGF‐6, fibroblast growth factor; IGFBP, insulin‐like growth factor binding protein; MCP‐1, monocyte chemotactic protein‐1; PDGF‐BB, platelet‐derived growth factor‐BB; RANTES, regulated upon activation, normally T‐expressed, and presumably secreted; SCF, stem cell factor; TIMP, tissue inhibitor of metalloproteinases; VEGF, vascular endothelial growth factor.

Figure 4

Involvement of interleukin‐6 (IL‐6) in the interaction between human mesenchymal stem cells (hMSCs) and Saos‐2. Human MSCs (A) and Saos‐2 (B) with and without IL‐6 knockdown were exposed to rhIL‐6 from 5 to 20 ng/mL, and MTT assay was used. These two cells were transfected with siRNA against IL‐6. At the indicated time points, real‐time PCR was carried out to assess knockdown of IL‐6 in hMSCs (C) and Saos‐2 (D). Human MSCs were exposed to osteogenic induction medium (OM) containing rhIL‐6 at 20 ng/mL. Alkaline phosphatase (ALP) quantification assay and real‐time PCR were carried out for osteopontin (OPN) and osteocalcin (OC) (E). Conditioned medium (CM) of hMSCs and Saos‐2 with and without IL‐6 knockdown was added to the culture medium of Saos‐2 and hMSCs. At indicated time points, MTT assay was used to monitor hMSC (F) and Saos‐2 (G) proliferation. Osteogenic induction medium was used to induce hMSCs to undergo osteogenic differentiation, in which Saos‐2‐CM in combination with anti‐IL‐6 antibody (whole IgG as control) was added. The ALP quantification assay and real‐time PCR were carried out for OPN and OC (H). Results are expressed as the mean ± SD. *P < 0.05; **P > 0.05.

Figure 5

Interleukin‐6 (IL‐6) secretion by human mesenchymal stem cells (hMSCs) during osteogenic differentiation and in response to Saos‐2 conditioned medium (CM). Osteogenic induction medium was used to induce hMSCs to undergo osteogenic differentiation. At the indicated time points, IL‐6 secretion was assayed (A). Human MSCs were exposed to culture medium containing Saos‐2‐CM at gradients from 100% to 0%. Interleukin‐6 secretion of hMSCs was assayed (B). Results are expressed as the mean ± SD. *P < 0.05; **P > 0.05.

Figure 6

Positive feedback loop of interleukin‐6 (IL‐6) in interaction between human mesenchymal stem cells (hMSCs) and Saos‐2 based on data in the current study.