Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

Abstract

The prognosis of osteosarcoma (OS) has remained stagnant over the past two decades, requiring the exploration of new therapeutic targets. Cytokines, arising from tumor-associated macrophages (TAMs), a major component of the tumor microenvironment (TME), have garnered attention owing to their impact on tumor growth, invasion, metastasis, and resistance to chemotherapy. Nonetheless, the precise functional role of TAMs in OS progression requires further investigation. In this study, we investigated the interaction between OS and TAMs, as well as the contribution of TAM-produced cytokines to OS advancement. TAMs were observed to be more prevalent in lung metastases compared with that in primary tumors, suggesting their potential support for OS progression. To simulate the TME, OS and TAMs were co-cultured, and the cytokines resulting from this co-culture could stimulate OS proliferation, migration, and invasion. A detailed investigation of cytokines in the co-culture conditioned medium (CM) revealed a substantial increase in IL-8, establishing it as a pivotal cytokine in the process of enhancing OS proliferation, migration, and invasion through the focal adhesion kinase (FAK) pathway. In an in vivo model, co-culture CM promoted OS proliferation and lung metastasis, effects that were mitigated by anti-IL-8 antibodies. Collectively, IL-8, generated within the TME formed by OS and TAMs, accelerates OS proliferation and metastasis via the FAK pathway, thereby positioning IL-8 as a potential novel therapeutic target in OS.

Fig. 1. Expression and quantification of TAM in human osteosarcoma tissues from primary and lung metastasis sites.

A Immunohistochemical analysis of Iba1-positive cells in primary tumors and lung metastases of patients with OS and lung metastases. Scale bars represent 50 μm. B Quantification of Iba1-positive areas per high-power fields (400× magnification). C Violin plot depicting Iba1-positive cells in primary and lung metastases from each sample (N = 5). D Immunohistochemical analysis of CD163-positive cells in primary tumors and lung metastases of OS patients with lung metastases. Scale bars represent 50 μm. E Quantification of CD163-positive areas per high-power fields (×400 magnification). F Violin plot illustrating CD163-positive cells in primary and lung metastases from each sample (N = 5). For the analysis of infiltrating macrophages, ten non-overlapping high-power fields (400× magnification) were randomly selected in tumor areas and the proportion (%) of positive areas was evaluated using the ImageJ software. For cell size analysis, 100–150 cells per sample that had their nucleus in the pictures were encircled, and the size of encircled area was measured based on the scale bar. ns: not significant, OS: osteosarcoma. Data are presented as mean ± standard deviation. *p < 0.05 and **p < 0.01.

Fig. 2. Effect of co-culture CM on proliferation, migration, and invasion in osteosarcoma cells.

A Schematic diagram illustrating the method for differentiating monocytes into macrophages and co-culturing them with OS cells. B Cell viability assay for OS cells treated with or without co-cultured CM using the Cell Counting Kit-8 assay. C and D Scratch and invasion assays for OS cells treated with or without co-culture CM. The scratch assay was conducted for 12 h, and the invasion assay was performed for 24 h. Scale bars represent 500 μm. CM conditioned medium, HMDMs human monocyte-derived macrophages, OS osteosarcoma, PBMCs peripheral blood mononuclear cells, PMA phorbol 12-myristate 13-acetate, THP-1 Mφ THP-1-derived macrophage. Data are presented as mean ± standard deviation. **p < 0.01. All data were obtained from at least three independent experiments.

Fig. 3. Interaction between osteosarcoma and macrophages increased IL-8 production.

A Cytokine arrays of 143B-Luc cells, human macrophages, and co-culture CM. B IL-8 production during the co-culture of OS cells (143B-Luc, SJSA-1) and macrophages (THP-1 Mφ, HMDMs). C IL-8 expression in macrophages stimulated with OS-CM. D IL-8 expression in OS cells stimulated with macrophage CM. E UMAP plot of OS lung metastases. F UMAP plot of Iba1, CD163, and IL-8 expression in myeloid cell clusters. G Violin plot depicting IL-8 expression in Iba1^+/− or CD163^+/− myeloid cell clusters. CM conditioned medium, HMDMs human monocyte-derived macrophages, OS osteosarcoma, THP-1 Mφ THP-1-derived macrophage. Data are presented as mean ± standard deviation. **p < 0.01. All data were obtained from at least three independent experiments.

Fig. 4. Effect of IL-8 in co-culture CM on proliferation, migration, and invasion of osteosarcoma cells.

A Cell viability assay for OS cells treated with or without recombinant IL-8 (rIL-8, 10 ng/mL) using Cell Counting Kit-8. B, C Scratch and invasion assays for OS cells treated with or without co-culture CM. The scratch assay was performed for 12 h, and the invasion assay was conducted for 24 h. Scale bars represent 500 μm. D Cell viability assay for OS cells treated with or without co-culture CM and with or without anti-IL-8 antibodies (IL-8 Abs, 1 µg/mL) using Cell Counting Kit-8. E, F Scratch and invasion assays for OS cells treated with or without co-culture CM and with or without IL-8 Abs (100 ng/mL). The scratch assay was performed for 12 h, and the invasion assay was conducted for 24 h. Scale bars represent 500 μm. CM: conditioned medium, OS: osteosarcoma, ns: not significant. Data are presented as mean ± standard deviation. *p < 0.05 and **p < 0.01. All data were obtained from at least three independent experiments.

Fig. 5. Role of the FAK pathway in IL-8 production in co-culture CM.

A Western blot assays for FAK phosphorylation in OS cells treated with co-culture CM and quantification of western blot bands. B Western blot assays for the phosphorylation of FAK (p FAK) in OS cells treated with co-culture CM + isotype IgG (IgG) or + anti-IL-8 antibodies (IL-8 Abs, 1 µg/mL) and quantification of western blot bands. The FAK phosphorylation levels were also compared between the same times. C Cell viability assay for OS cells treated with or without co-culture CM and with or without FAK inhibitor (PND-1186, 1 µM) using Cell Counting Kit-8. D, E Scratch and invasion assay for OS cells treated with or without co-culture CM and with or without FAK inhibitor (PND-1186) (1 µM). The scratch assay was performed for 12 h, and the invasion assay was conducted for 24 h. Scale bars represent 500 μm. CM conditioned medium, FAK focal adhesion kinase, ns not significant, OS osteosarcoma. Data are presented as mean ± standard deviation. *p < 0.05 and **p < 0.01. All data were obtained from at least three independent experiments.

Fig. 6. Effect of IL-8 in co-culture CM on primary growth.

A Schematic showing the experimental design for subcutaneous transplantation of OS cells. 143B-Luc cells (2 × 10⁶/mouse) were subcutaneously transplanted in 6–7-week-old BALB/c nu/nu mice, and DMEM or co-culture CM was injected into the para-tumor thrice per week. B Tumor volume with or without co-culture CM measured thrice per week after tumor transplantation. C, D Tumor weight and image of the excised tumor with or without co-cultured CM 14 days after tumor transplantation. E, F Immunohistochemistry and quantification of Ki-67 and phospho-FAK in tumor sections with or without co-culture CM 14 days after tumor transplantation. Two fields of view per sample were randomly selected, and quantification was performed in 10 fields. E Ki-67-positive cells in the field were counted and indicated as Ki-67 labeling index. Scale bars represent 50 μm. G Schematic showing the experimental design for subcutaneous OS transplantation using anti-IL-8 antibodies (IL-8 Abs). H Tumor volume after pretreatment with co-culture CM with or without IL-8 Abs (10 µg/mouse) measured thrice per week after tumor transplantation. I and J Weight and image of the excised tumor pretreated with co-culture CM with or without IL-8 Abs 14 days after tumor transplantation. K, L Immunohistochemistry and quantification of Ki-67 and phospho-FAK in tumor sections with co-culture CM with or without IL-8 Abs 14 days after tumor transplantation. Scale bars represent 50 µm. CM conditioned medium, DMEM Dulbecco’s modified Eagle’s medium, FAK focal adhesion kinase, OS osteosarcoma. Data are presented as mean ± standard deviation; *p < 0.05, **p < 0.01. Each group contained five animals.

Fig. 7. Effect of IL-8 in co-culture CM on lung metastasis.

A Schematic showing the experimental design for OS tail vein injection. 143B-Luc cells were treated with DMEM or co-culture CM for 12 h prior to tail vein injection; 143B-Luc cells (1 × 10⁶/mouse) were injected into the tail vein of 6–7 week-old BALB/c nu/nu mice. B IVIS imaging and quantification of lung metastasis 14 days after the injection of tumors pretreated with or without co-culture CM. C Hematoxylin and eosin staining of lung sections with or without co-culture CM and quantification of lung colonies. Scale bars represent 1 mm and 500 µm. D Immunohistochemistry of Ki-67 positive cells in lung colonies with or without co-culture CM and quantification of the Ki-67 labeling index. Two colonies per sample were randomly selected, and quantification was performed for 10 colonies. Ki-67-positive cells in the colonies were counted and shown as the Ki-67 labeling index. Scale bars represent 100 µm. E Schematic showing the experimental design for OS tail vein injection using anti-IL-8 antibody. F IVIS imaging and quantification of lung metastasis 14 days after the injection of tumors pretreated with co-culture CM with or without IL-8 antibodies. G Hematoxylin and eosin staining of lung sections with co-culture CM with or without IL-8 Abs and quantification of lung colonies. Scale bars represent 1 mm and 500 µm. H Immunohistochemistry of Ki-67-positive cells in lung colonies with co-culture CM with or without IL-8 Abs and quantification of Ki-67 labeling index. Scale bars represent 100 µm. CM conditioned medium, DMEM Dulbecco’s modified Eagle’s medium, IVIS, in vivo imaging system, OS osteosarcoma. Data are presented as mean ± standard deviation; *p < 0.05, **p < 0.01. Each group contained five animals.