Interaction between human osteosarcoma and mesenchymal stem cells via an interleukin-8 signaling loop in the tumor microenvironment

Abstract

Background: Osteosarcoma (OS) is the representative primary malignant bone tumor with the highest incidence. It is known that malignant phenotypes of OS, such as proliferation, invasion, and metastasis, are significantly influenced not only by characteristics of the tumor itself, but also by the surrounding microenvironment. In other words, OS is considered to utilize cells in the vicinity of the tumor by changing the characteristics of these cells. Direct intercellular contact is believed to be important for this phenomenon. In the present study, we hypothesized that an interaction mediated by a humoral factor, requiring no cellular contact, might play a significant role in the progression of OS.

Methods: We developed a new co-culture model, using OS cells and mesenchymal stem cells (MSCs) without cellular contact, and found that both cell types expressed IL-8 at a high level, and FAK in OS cells was phosphorylated leading to an increase in the metastatic potential of the tumor in the co-culture condition.

Results: It was revealed that OS cells formed a loop of signal cross-talk in which they released IL-8 as a paracrine factor, stimulating MSCs to express IL-8, and received IL-8 released by MSCs to accelerate IL-8 expression in OS cells. Administration of anti-IL-8 antibody resulted in the inhibition of FAK expression, its downstream signaling, and the invasive potential of the OS cells, resulting in decrease in metastatic lesions.

Conclusion: The present study might lead not only to the clarification of a new molecular mechanism of invasion and metastasis of OS, but also to the development of a new therapeutic strategy of blocking IL-8 in OS.

Keywords: Interleukin-8; Mesenchymal stem cells; Osteosarcoma; Tumor proliferation and metastasis.

Fig. 1

The expression of IL-8 in mono-cultured and co-cultured osteosarcoma and normal cells. a Relative expression of IL-8 mRNA in hMSC and MG63. b Relative expression of IL-8 mRNA in MRC 5 and MG63. c Changes in gene expression in hMSCs after the co-culture with MG63. d Changes in gene expression in MG63 after the co-culture with hMSCs

Fig. 2

Changes in IL-8 expression in MG63 and hMSCs induced by the co-culture condition and rIL-8 administration. a Changes in IL-8 expression in MG63 were assessed by qRT-PCR. The co-culture and rIL-8 addition significantly increased the expression of mRNA of IL-8. (**) p < 0.01. b Changes in IL-8 expression in hMSCs were assessed by qRT-PCR. The co-culture and rIL-8 addition significantly increased the expression of mRNA for IL-8. (**) p < 0.01. c Changes in IL-8 protein expression of intra-cellular hMSCs and MG63 were assessed by western blot analysis. The co-culture and addition of rIL-8 increased the expression of IL-8 protein in MG63 and hMSCs. d The quantification of western blot analysis. Data represents represent the mean ± SD of three independent experiments. p < 0.05 was considered to indicate significance: (*) p < 0.05, (**) p < 0.01

Fig. 3

Effects of neutralizing anti-IL-8 Ab on IL-8 expression in mono-cultured and co-cultured MG63 and hMSCs. a Changes in IL-8 expression in MG63 were assessed by qRT-PCR. The addition of nIL-8 Ab to MG63 decreased the expression of IL-8 mRNA. b Changes in IL-8 expression in hMSCs were assessed by qRT-PCR. The addition of nIL-8 Ab to hMSCs decreased the expression of IL-8 mRNA. c Changes in IL-8 protein expression in hMSCs and MG63 were assessed by western blot analysis. The addition of nIL-8 Ab to MG63 and hMSCs decreased the expression of IL-8 protein in these cells. d The quantification of western blot analysis. Data represents represent the mean ± SD of three independent experiments. p < 0.05 was considered to indicate significance: (*) p < 0.05, (**) p < 0.01

Fig. 4

Changes in cell growth of MG63 cells and hMSCs induced by co-culture conditions and rIL-8 addition. a Co-culture with hMSCs significantly increased the cell growth of MG63 (MG63(Co)). (*) p < 0.05, (**) p < 0.01. b Co-culture with MG63 significantly increased the cell growth of hMSCs (hMSCs(Co)). (*) p < 0.05, (**) p < 0.01. c rIL-8 was administered to mono-cultured MG63. There was a significant increase in cell growth with rIL-8 at 10 ng/ml. (*) p < 0.05, (**) p < 0.01

Fig. 5

Effects of co-culture condition and administration of rIL-8 or anti-IL-8 Ab on motility of MG63. a The cell motility of MG63 was assessed in each group at 48 h after the challenge with or without rIL-8 and neutralizing anti-IL-8 antibodies (nIL-8 Ab). b The amount of MG63 cells that crossed the membrane was measured. A significant decrease in motility was found in the group given nIL-8 Ab. (*) p < 0.05, (**) p < 0.01. c The cell invasion in MG63 was assessed in each group after 48 h. d The amount of the cells of which the membrane with Matrigel was crossed by MG63 was measured. Decreased migration ability was found in the group administered anti-IL-8 Ab. (*) p < 0.05, (**) p < 0.01

Fig. 6

Changes in expression of FAK and its downstream factors related to invasive potential. a Changes in phosphorylation and the expression of protein factors relating to invasive potential were analyzed. Decreased phosphorylation of FAK, paxillin, Src, and Akt in MG63 cells was noted in the group administered nIL-8 Ab. b The quantification of western blot analysis. Data represents represent the mean ± SD of three independent experiments. p < 0.05 was considered to indicate significance: (*) p < 0.05, (**) p < 0.01. c Immunofluorescence staining of cultured MG63 cells showed decreased phosphorylation of FAK and paxillin in the group administered nIL-8 Ab. d The number of IL-8 and p-FAK positive cells per unit area. Data represents represent the mean ± SD of three independent experiments. p < 0.05 was considered to indicate significance: (*) p < 0.05, (**) p < 0.01

Fig. 7

Changes in the lung nodules and the expression of IL-8 and phosphorylated FAK in pulmonary metastatic lesions. a The group given nIL-8 Ab showed a significant suppression of the size of the pulmonary metastatic lesion. (*) p < 0.05. b Immunostaining of the tissues collected from the pulmonary metastatic lesion. Decreased expression of IL-8 and phosphorylation of FAK was observed in the group administered nIL-8 Ab. Original magnification, × 400; Scale bars: 50 μm. c The number of IL-8 and p-FAK positive cells per unit area. Data represents represent the mean ± SD of three independent experiments. p < 0.05 was considered to indicate significance: (*) p < 0.05, (**) p < 0.01