Tumor-Osteocyte Interactions under Fluid Flow Stimulation
Bone is one of the most common sites for breast cancer metastasis. Osteocytes compose approximately 90% of the cell population in bone matrix. Osteocytes are very sensitive to mechanical stimulation, and physical activities play an essential role in maintaining bone's health. Mechanical stimulation can alter the gene expression profile in osteocytes. However, little is known about the effects of mechanical stimulation on tumor-bone interactions. In this thesis, this question has been addressed: Does applying mechanical stimulation to osteocytes change tumor-osteocytes interactions? The hypothesis is that mechanical stimulation can change osteocytes secreting signals and contribute to higher proliferation and migration of tumor cells. In this thesis, fluid flow-driven shear stress has been used as the mechanical stimulator for osteocytes, and the interactions of tumor-osteocytes, with and without mechanical stimulation has been investigated. Monolayer cultures and 3D spheroids of breast cancer cells, including TMD and 4T1 cells were cultured in the conditioned medium (CM) isolated from MLO-A5 osteocytes, and fluid flow-treated conditioned medium (FFCM), and their migratory behavior, proliferation, and protein expression have been evaluated. The results showed that in response to MLO-A5 FFCM, tumor cells behave differently in Src expression, proliferation, and migration compared to MLO-A5 CM. As opposed to MLO-A5 CM, FFCM promoted migration, reduced proliferation, and upregulated Src expression in tumor cells. Moreover, by plasmid and siRNA transfection it has been shown that Src is upstream of Snail and their upregulation is causing epithelial-mesenchymal transition(EMT) responses in tumor cells. Furthermore, ELISA concentration assessment showed the involvement of TGF-β in Src upregulation. An in vivo study using seventeen mice was conducted to investigate the effect of mechanical stimulation on clinical conditions. Compressive loads were applied to tibia after intratibial injection of 4T1.2 cells. The results suggested that direct mechanical stimulation of metastasized bone, might not be advantageous, and cause more damage. Furthermore, the results indicated that direct mechanical loading can make the knee joint more fragile. This research showed mechanical stimulation can cause tumor cells to behave more migratory in bone microenvironment, and demonstrated its crucial role in tumor-osteocytes interactions.
Yokota, Purdue University.
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