Bone invading NSCLC cells produce IL-7: mice model and human histologic data

Abstract

Background: Bone metastases are a common and dismal consequence of lung cancer that is a leading cause of death. The role of IL-7 in promoting bone metastases has been previously investigated in NSCLC, but many aspects remain to be disclosed. To further study IL-7 function in bone metastasis, we developed a human-in-mice model of bone aggression by NSCLC and analyzed human bone metastasis biopsies.

Methods: We used NOD/SCID mice implanted with human bone. After bone engraftment, two groups of mice were injected subcutaneously with A549, a human NSCLC cell line, either close or at the contralateral flank to the human bone implant, while a third control group did not receive cancer cells. Tumor and bone vitality and IL-7 expression were assessed in implanted bone, affected or not by A549. Serum IL-7 levels were evaluated by ELISA. IL-7 immunohistochemistry was performed on 10 human bone NSCLC metastasis biopsies for comparison.

Results: At 12 weeks after bone implant, we observed osteogenic activity and neovascularization, confirming bone vitality. Tumor aggressive cells implanted close to human bone invaded the bone tissue. The bone-aggressive cancer cells were positive for IL-7 staining both in the mice model and in human biopsies. Higher IL-7 serum levels were found in mice injected with A549 cells close to the bone implant compared to mice injected with A549 cells in the flank opposite to the bone implant.

Conclusions: We demonstrated that bone-invading cells express and produce IL-7, which is known to promote osteoclast activation and osteolytic lesions. Tumor-bone interaction increases IL-7 production, with an increase in IL-7 serum levels. The presented mice model of bone invasion by contiguous tumor is suitable to study bone-tumor cell interaction. IL-7 plays a role in the first steps of metastatic process.

Figure 1

Histologic analysis of human bone implants. Representative H&E-stained section of human bone fragment implanted subcutaneously in NOD/SCID mice 12 weeks post implantation (A). Magnifications show newly synthesized bone, osteocytes, fat cells, areas of solid mineralized bone and osteoblast lining cells, as labelled by the arrows (B and C). Human bone marrow remains localized in the marrow spaces, as indicated in D. Magnification: A 100×, B 250×, C 250×, D100×.

Figure 2

Immunohistochemical staining of human bone implants. Immunohistochemistry was performed on serial sections using human-specific CD34 antibodies to delineate human blood vessels, which are shown within and around bone xenografts (arrows). Magnification 250×.

Figure 3

Histologic analysis of A549 metastases in human implanted bone. H & E-stained sections show large areas of invading tumor cells surrounding live human bone, with presence of fat cells, blood vessels, and osteoblasts, as labeled by the arrows. Magnification 100×.

Figure 4

IL-7 expression by NSCLC bone invading cells in mouse and human biopsies. A strong IL-7 level expression is shown by tumor cells in invaded bone (A). In contralateral bone, IL-7 level expression is detectable on stromal cells and it is lower than in bone invaded by tumor cells (B). In human biopsies, IL-7 is highly expressed by bone invading cells (C). Magnification 100×.

Figure 5

Serum IL-7 levels in mice. In mice injected with A549 cells close to the bone implant IL-7 is significantly higher than in mice injected with A549 cells in the opposite flank, p < 0.001. There was not significant difference between opposite flank and the control group without tumor cells.