Breast cancer has a predilection to metastasize to the skeleton. While the mechanism for preferential metastasis is unknown, the bone microenvironment likely provides a favorable environment for metastatic breast cancer cells. Besides affecting the activity of osteoblasts and osteoclasts, metastatic breast cancer cells create a unique bone niche by co-opting osteoblasts to increase the production of inflammatory cytokines.
I hypothesized that osteoblast-derived cytokines would increase in the presence of metastatic breast cancer cells, act as chemoattractants for metastatic breast cancer cells, and enhance osteoclast formation. I additionally hypothesized that treatment of osteoblasts with a bone-seeking metastatic breast cancer cell variant would elicit a larger increase in osteoblast-derived inflammatory cytokines than the parental line. I further hypothesized that the osteoblast-derived cytokines would be increased in the metaphyses of the femurs of cancer-bearing mice.
MC3T3-E1 cells were incubated with MDA-MB-231 metastatic or non-metastatic breast cancer cell variants, human epithelial cells, or their conditioned media, and assayed for osteoblast-derived cytokine expression. Femurs from mice inoculated via intracardiac injection with MDA-MB-231-GFP breast cancer cells were assayed ex vivo for bone-derived cytokine production. Tumor localization within femurs was examined using ìCT, densitometry, and MRI. The expression of cytokines IL-6, MCP-1, and VEGF were assayed via immunohistochemistry. The chemoattractant capability of osteoblast-derived IL-6, MCP-1, and VEGF on MDA-MB-231 metastatic breast cancer cell migration was assessed. The effect of culture supernatants from osteoblasts treated with breast cancer cells or their conditioned media on osteoclast formation was examined by staining for Tartrate Resistant Acid Phosphatase (TRAP).
In vitro, osteoblasts produced large quantities (~3 ng/ml) of MCP-1, while metastatic breast cancer cells did not. Both osteoblasts and metastatic breast cancer cells expressed IL-6, KC/GRO-α, MIP-2/IL-8, and VEGF in various concentrations. Osteoblast-derived cytokine production of IL-6, KC, MIP-2, MCP-1, and VEGF increased with direct co-culture of metastatic breast cancer cells or with treatment of their conditioned medium in vitro.
When metastatic breast cancer cells were inoculated into the left cardiac ventricle of athymic nude mice, MDA-MB-231 cells colonized the trabecular bone, as detected by ìCT and MRI ex vivo. The volume of the trabecular bone matrix of a tibia was 75% less in cancer-bearing mice than non-cancer-bearing mice. In addition, in the metaphyses of femurs cultured ex vivo, bone-derived cytokine production of IL-6, KC, MIP-2, MCP-1, and VEGF was increased in cancer-bearing mice compared to non-cancer-bearing mice as detected by a Bio-rad Bio-plex™ Murine Cytokine Assay. Immunohistochemistry revealed that MCP-1 and VEGF were localized in the matrix of trabecular bone. In contrast, IL-6 was expressed by cells throughout bone marrow. Murine MIP-2 and KC were expressed in negligible quantities in cultures of cancer-bearing and non-cancer-bearing femurs ex vivo, as determined by a Bio-rad Bio-plex™ Murine Cytokine Assay, and thus these cytokines were not assayed for by immunohistochemistry. Expression of murine MCP-1 and VEGF were not detected adjacent to metastatic breast cancer cells, but were present in increasing amounts extending away from the cancer cells. Human VEGF expression increased with increasing tumor size. In addition, it was noted that the number of megakaryocytes in the bone marrow cavity increased in cancer-bearing mice compared to non-cancer-bearing mice.
Osteoblast conditioned medium was a potent chemoattractant for metastatic breast cancer cells in a transwell assay, but neither IL-6, VEGF, nor MCP-1 were chemoattractants alone or in combination. The culture supernatant of osteoblasts treated with metastatic breast cancer cells or their conditioned media facilitated TRAP positive multi-nucleated osteoclast formation.
Data obtained during this study suggested that osteoblast-derived cytokines, IL-6, KC, MIP-2, MCP-1, and VEGF are important in the growth of metastatic breast cancer cells in the bone. Both cancer cells and osteoblasts produced VEGF, IL-6, IL-8/MIP-2, and GRO-α/KC, but only osteoblasts produced MCP-1. MCP-1 likely does not act directly on breast cancer cells, but rather on osteoclasts. As osteoclasts degrade bone, breast cancer maintenance factors such as TGF-β are released, which facilitate breast cancer cell colonization and survival. Thus, these findings indicated that metastatic breast cancer cells co-opt the cells of the bone into creating a unique niche supportive of breast cancer cell colonization. By identifying alterations in osteoblast-derived cytokines caused by metastatic breast cancer cells, it may be possible to block or disrupt these factors through the use of targeted drugs. Appropriate therapeutic treatment would allow for an improved quality of life and longer survival time for individuals with bone metastatic breast cancer.