The Interaction of Metastatic Breast Cancer Cells with Osteoblasts
Breast cancer cells frequently metastasize to the skeleton where they grow and cause bone loss. The cancer cells do not themselves destroy the bone but disturb the balance of the normal bone remodeling cells, osteoblasts and osteoclasts. We have focused on the intereactions of metastatic cancer cells with osteoblasts. We have used three different models, cell culture, a three-dimensional bioreactor, and animals to study this cell-cell interactions. We have found both in vitro and in vivo that the cancer cells cause increased osteoblast apoptosis, reduced production of matrix proteins, and increased production of a set of inflammatory cytokines, IL-6, IL-8, MCP-1, and VEGF. These same cytokines have been seen as part of an osteoblast stress response in other diseases. They are known to attract and activate osteoclasts, the cells that degrade the bone matrix. We currently are studying the control of transcription of these cytokines through NFκB and signal transducing molecules. We are examining their expression in bone early in the metastatic process. Finally, we are trying to mimic the bone environment in vitro by addition of osteoclasts to the three- dimensional osteoblast culture system. In this bioreactor model we have visualized cancer cells form single cell files and causing morphological changes in the osteoblastic tissue. These properties are not seen in standard tissue culture but are seen in pathological tissue. Pre-osteoclasts also differentiate into active osteoclasts in the bioreactor and bring about a loss of osteoblastic matrix. These three approaches will permit us to tease apart various aspects of the bone metastatic environment.
Exercise, diet and the immune system
For some time we have been interested in the cells of the immune system as influenced by exercise. In one study we followed the blood lymphocyte population of women following breast cancer chemotherapy. In another we examined the iron status of the elderly. In a current study we are examining the blood cells following high protein diets of several types and how exercise affects the lymphocyte subpopulations. In all of these, it is clear that diet and exercise are integral components of the immune system.
Figure 1. A, osteogenic tissue maturation in the bioreactor recapitulates development of native bone by systematic and reproducible phenotypic maturation of preosteoblasts through mineralizing osteoblasts to terminally differentiated osteocytes. B, MC3T3-E1 cells produce and mineralize a thick, engulfing extracellular matrix (ECM) that slowly decreases in thickness and number of cell layers through progressive apoptosis to a final stable state exhibiting no sign of tissue necrosis over 10 months of continuous culture (graph). C-F, interaction of MDA-MB-231GFP human cancer cells (green, GFP) with osteogenic tissue (red, osteoblasts; black, ECM) depends on tissue maturity (B, table) and exhibits stages of cancer cell adhesion (C), penetration (D), and alignment of cancer cell into files (E) that are reminiscent of events observed in pathologic tissue. F, filing is especially evident in corresponding 3D confocal reconstructions. G-I, for comparison, Indian Filing is shown in a section from bone (solid pink) with metastatic breast cancer (rows of cells with dark purple nuclei). The scale bars in A, C, and D represent 50 μm, the scale bar in F represents 100 μm, the scale bar in G represents 200 μm, and the scale bars in H and I represent 50 μm.
Figure 2: Murine MCP-1, IL-6, and VEGF are Localized in the Bone Microenvironment
Femurs were harvested from athymic nude mice inoculated with metastatic breast cancer cells were cryosectioned in 10 µm thick longitudinal sections, and stained for murine VEGF, murine MCP-1, or murine IL-6 via immunohistochemistry, and visualized using a brown DAB chromogen stain. Slides were counterstained using Gill’s Hematoxylin. a,c) Murine VEGF and b,d) murine MCP-1 were localized in trabecular bone of the proximal and distal femur. e,g) urine VEGF and f,h) murine MCP-1 were localized in cortical bone of the proximal and distal metaphyses. Neither i) murine MCP-1 nor j) murine VEGF were localized in cortical bone of the diaphysis. k) Murine IL-6 was localized throughout the bone marrow. Murine IL-6 was not present in the trabecular bone or the cortical bone matrix. At least three independent sections were stained per bone, and three bones examined per un-inoculated or inoculated MDA-MB-231-