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A New In Vitro Model of Breast Cancer Metastasis to Bone
Tumor Cell Biology I
Bone metastasis is a common sequela of breast cancer. The ensuing bone degradation leads to a poor quality of life. Administration of bisphosphonates to inhibit osteoclasts that resorb bone, reduces degradation but does not heal lesions. Using conventional cell culture we have shown that the osteoblasts, normal effectors of repair, do not function properly. In the presence of breast cancer cells, osteoblasts no longer make proteins required for repair, undergo increased apoptosis, and produce inflammatory cytokines. We hypothesize that metastatic breast cancer cells bring about a loss in osteoblast function. We propose to study how this occurs under physiologically relevant conditions in co-culture in a novel bioreactor where three-dimensional bone is formed and maintained. This system provides a non-invasive model for identifying reagents that support bone function while simultaneously destroying cancer cells. The aims are to determine the effects of metastatic breast cancer cells on 1 . the physiology of osteoblasts grown in three-dimension; 2 , the morphology of osteoblasts and organization of bone; and 3 . to develop means of selectively destroying cancer cells while maintaining osteoblasts. The bioreactor will be used as an in vitro tool to test the hypothesis by challenging ‘biosynthetic bone’ with breast cancer cells and following the metastatic process with a battery of biochemical and microscopy techniques. MC3T3-E1, an osteoblast line that differentiates and secretes an extracellular matrix that mineralizes, will be cultured in the presence of human metastatic breast cancer cells, MDA-MB-231 GFP . The medium will be tested for expression of osteoblast secreted proteins and cytokines. Morphology, apoptosis, and cell-cell interactions will be identified with microscopy: light, fluorescent, confocal, TEM and STEM. Finally, agents known either to positively affect osteoblasts or adversely affect breast cancer cells will be tested. We propose a new paradigm for understanding the growth of breast cancer metastasis in bone. This ground-breaking work applies bioengineering principles to the development of three-dimensional, tissue-like ‘biosynthetic bone’ that can be challenged with metastatic breast cancer cells. It provides a model to bridge the research between cell culture and animals. It has the potential to become a valuable tool with which to test new therapeutics early in the process from bench to patient.
Breast cancer frequently spreads to other locations in the body. One of the favored sites is the skeleton. Once the cancer has begun to grow in the bone, it is very difficult to treat. Five-year cure rates drop from 90% to 16%. Breast cancer in bone causes the bone to break down, which leads to fractures, pain, spinal cord compression, and high blood calcium levels that affect other organs. The current evidence is that the cancer cells themselves do not destroy the bone, but instead activate osteoclasts, the normal bone resorbing cells. Drugs such as bisphosphonates (Alendronate, Pamidronate), aimed at blocking osteoclasts, slow down lesion formation and progression. However, blocking the osteoclasts does not heal the existing lesions. Bone building cells, the osteoblasts, do not repair the bone. We asked why the osteoblasts do not carry out their repair function. We have evidence from experiments carried out in cell culture that osteoblasts in the presence of breast cancer cells do not differentiate to make the proteins needed to repair bone. Instead they make factors that attract and activate osteoclasts. These experiments are limited by conventional cell culture technology. There are no existing in vitro systems to study the long-term interactions of breast cancer under physiologically relevant conditions. We propose to use a bioreactor that we have developed as a tool to study the relationship of metastatic breast cancer cells with osteoblasts in long-term culture. We have already shown that we can grow osteoblast lines in this system for at least 120 days under conditions in which they form mineralized, collagenous connective tissue (synthetic bone). Now we plan to add metastatic breast cancer cells to the chamber to determine the fate of the osteoblasts. For aim 1, we will test osteoblast physiology in the presence of breast cancer cells. Culture medium sampled from the chamber over time will be analyzed for specialized osteoblast proteins and factors. In aim 2, we will use several microscopic techniques to detect the morphology and cell interactions of the bone cells co-cultured with breast cancer cells. In aim 3 we propose to use the bioreactor to study how potential therapeutic agents may affect both the cancer cells and the bone. This system offers a much needed bridge between conventional cell culture and in vivo animal studies. Furthermore, it has the potential to be used to study bone/tumor cell interactions for other types of cancers.