Effect and mechanism of tumor-associated macrophages on facilitating development of osteosarcoma mediated by nuclear factor-κB

Objective To investigate the effect and mechanism of tumor-associated macrophages (TAMs) on facilitating development of osteosarcoma mediated by nuclear factor-κB(NF-κB). Methods Fifteen Balb/c male SPF nude mice (4-6 weeks old) were selected and divided into 3 groups of human osteosarcoma cells (MG63), human monocyte-macrophages (THP-1), and the mixture of MG63 and THP-1 in accordance with the random number table, there were 5 mice in each group. Different cell suspensions with the concentration of 1.0×108/mL were injected into the right side of armpit under the skin of nude mice to establish a xenotransplanted tumor model in nude mice. 0.2 mL MG63 cell suspension and 0.2 mL THP-1 cell suspension were injected into the nude mice in MG63 cell group and THP-1 cell group respectively, and 0.2 mL mixture of MG63 and THP-1 cells (9∶1) were injected into the nude mice in MG63+ THP-1 group. The development of xenografts in nude mice after injection of different cells was observed every day. Since xenografts showed up in both MG63 group and MG63+ THP-1 group, the length and width of the transplanted tumor were measured by vernier caliper every 5 days, then the volume of the transplanted tumor was calculated and compared. The nude mice were sacrificed on the 25th day after injection, and subsequently, the weight of the transplanted tumors was detected. The transplanted tumors were fixed in 10% formaldehyde and paraffin sections were prepared. The changes of tumor characteristics of xenografts were observed using HE staining. Immunohistochemical SP staining was used to observe the expression of nuclear transcription factor NF-κB protein in transplanted tumors in different groups. The protein expression levels of NF-κB in transplanted tumors in different groups were quantitatively determined using Image-Pro Plus 6.0 (IPP 6.0) image software. Results There was no transplanted tumor after injecting THP-1 cells alone. The transplanted tumors began to be observed on 10th day and 7th day after injection of MG63 cells and MG63+ THP-1 mixed cells, respectively; the transplanted tumors grew bigger as injection time went on. The volumes of transplanted tumors formed by MG63+ THP-1 mixed cells were (474.4±56.1), (945.0±79.7), (2 886.0±462.3), (3 319.6±388.4) mm3 on the 10th, 15th, 20th and 25th day, respectively, which were all increased significantly compared with those of MG63 cells [(233.3±28.2), (669.6±75.9), (1 464.0±135.2), (2 068.0±223.2) mm3, respectively] on the 10th, 15th, 20th and 25th day (t=3.536, 2.504, 2.952, 2.794; all P values<0.05). On the 25th day after subcutaneous injection, nude mice were sacrificed, the tumor weight of MG63 and THP-1 mixed cells group [(0.920 ±0.134) g] was heavier than that of MG63 cells group [(0.544 ±0.079) g] (t=2.404, P<0.05). HE staining demonstrated that there were obvious changes of tumor characteristics in transplanted tumors in MG63 group and MG63+ THP-1 group. Immunohistochemical SP staining showed that NF-κB protein was stained in the tissue and tissue space of transplanted tumors in MG63 group and MG63+ THP-1 group, and the absorbance value of NF-κB protein expression in the mixed cells of MG63 and THP-1 group was (0.362±0.006), which was significantly higher than that in MG63 cells group alone (0.326±0.006) using IPP 6.0 software (t=9.895, P<0.05). Conclusions Tumor-associated macrophages could facilitate the development of osteosarcoma, which is mediated by high expression of NF-κB protein. Key words: Osteosarcoma; Tumor-associated macrophages; Nude mice; Human MG63 cells; Human THP-1 cells