Characterization of different osteoclast phenotypes in the progression of bone invasion by oral squamous cell carcinoma

Abstract

The present study aimed to characterize different phenotypes of osteoclasts in the progression of bone invasion by oral squamous cell carcinoma (OSCC). A local bone invasion model of OSCC was established by injecting SCC25 human OSCC cells into the center of calvariae in nude mice, and all mice were found to have a typical bone resorption area. Staining for tartrate-resistant acid phosphatase (TRAP) revealed various types of giant osteoclasts in the tumour-bone interface. Bone marrow cells (BMCs) were isolated from the nude mice for primary osteoclast culture, but only a few giant osteoclasts were generated. Additionally, special blood centrifuge tubes were utilized to obtain large numbers of peripheral blood mononuclear cells (PBMCs). Using magnetic activated cell sorting (MACS) and the cytokines colony-stimulating factor (CSF) and receptor activator of nuclear factor-κb ligand (RANKL), we differentiated human osteoclasts from CD14+ monocytes of PBMCs. Bone resorption was further confirmed by a bone resorption assay. Finally, Transwell inserts were used for indirect cell co-culture of SCC25 cells and CD14+ monocytes. Expression of specific osteoclast markers was detected by real-time PCR and western blotting. After co-culture for 3 and 6 days, conditioned medium (CM) of SCC25 cells stimulated the expression of osteoclast markers, and additional osteoclasts were detected through staining of TRAP and F-actin. In the present study distinct osteoclast phenotypes were observed in the established bone invasion animal model, and were confirmed using various primary osteoclast cultures. CM of OSCC cells may promote the expression of osteoclast markers and induce the differentiation of monocytes to mature osteoclasts, which can resorb adjacent bone tissue.

Figure 1.

Typical bone resorption was observed in calvarial bone invasion model of OSCC. (A) SCC25 cells were injected through the center of the calvaria, and the tumour formed in 1 month. (B) All nude mice showed typical bone resorption according to uCT analysis. Representative photos of the tumour formed and the bone invasion area.

Figure 2.

Various types of giant osteoclasts were observed at the tumour-bone interface in the animal model. (A) H&E staining suggested the formation of squamous cell carcinoma in vivo, which invaded adjacent bone tissue; (B) TRAP staining revealed various types of giant osteoclasts and different phenotypes including round, triangle, or slabstone shapes (arrow, TRAP). Representative photos of TRAP staining.

Figure 3.

Few osteoclasts were generated from BMCs from nude mice. (A) After continuous culture for 10 days, few osteoclasts were differentiated. TRAP staining showed several round and giant osteoclast precursors (arrow, TRAP). Representative photos of TRAP staining for osteoclasts. (B) Quantification of osteoclast numbers between normal control and tumour-bearing nude mice. No significant differences were found between groups (P>0.05).

Figure 4.

Special centrifuge blood tubes were utilized for monocyte collection and primary human osteoclast culture by MACS. (A) Schematic diagram of the centrifuge blood tube; (B) MACS selector; (C) TRAP staining indicated that typical round and giant osteoclasts were differentiated (arrow, TRAP), and IF confirmed the formation of F-actin rings (arrow, F-actin). Representative photos of osteoclasts.

Figure 5.

Bone resorption assay of mature osteoclasts. (A) TRAP staining of both cells with or without dentin slices revealed a normal color for TRAP; negative control dentin slice with only CD14⁺ monocytes. (B) A single giant osteoclast was found attached to the dentin slice. Negative control dentin slices with or without CD14⁺ monocytes were observed by SEM.

Figure 6.

SEM confirmation of the bone resorption pits of dentin slices degraded by mature osteoclasts. (A) Different magnification was utilized to observe resorption pits, which were in the form of either single (arrow), small resorption tracks or discrete areas of lacunar excavations. (B) Quantification of bone resorption pits. Significant differences were found between these two groups of CD14⁺ monocytes and mature osteoclasts (*P<0.05).

Figure 7.

Expression changes in osteoclast markers detected by real-time PCR and western blotting. (A) Indirect cell co-culture model, SCC25 cells were added to the top of the Transwell insert, while CD14⁺ monocytes were plated to the bottom of 24-well plates. (B) For monocytes without stimulation from CM of SCC25 cells, NFATc1 was expressed on day 3 and until day 6; proteinases MMP-9 and CTSK were increased on days 3 and 6. Similar expression trends in NFATc1 were observed in monocytes after stimulation with SCC25 CM. For MMP-9 and CTSK, SCC25 CM stimulated their expression from the start of co-culture, which reached a maximum on day 6; (C and D) Protein level changes in NFATc1, MMP and CTSK were further confirmed by western blotting.

Figure 8.

CM of SCC25 cells induces more differentiation of human osteoclasts. (A) Compared with the CD14⁺ monocytes cultured with cytokines CSF and RANKL, those cultured with CM of SCC25 cells yielded more osteoclasts on days 3 and 6 (arrow, TRAP). IF showed similar results, and greater numbers of F-actin rings were found in both groups on days 3 and 6 (arrow, F-actin). No osteoclasts were observed in the negative control group comprising CD14⁺ monocytes only. Representative photos of TRAP and F-actin staining of osteoclasts. (B) Quantification of osteoclasts and F-actin. Significant differences were found between these 4 groups (*P<0.05).