Novel mechanobiological modelling of bone metastasis reveals that substrate stiffness, biochemical bone cell signaling and mechanical stimulation alter metastatic activity
Kumar, Vatsal
Kumar, Vatsal
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Identifiers
http://hdl.handle.net/10379/17676
https://doi.org/10.13025/17414
https://doi.org/10.13025/17414
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Publication Date
2023-03-01
Type
Thesis
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Abstract
For more than 70% of breast cancer patients, cancer cells spread from their primary site and metastasize to bone. Although much research has been conducted to understand the pathogenesis of the metastatic bone disease, therapies are deficient and once cancer invades bone tissue the condition is widely untreatable. Hence, there is a distinct need to significantly advance our scientific understanding of bone metastasis. In bone metastasis, breast cancer and bone cells interact biochemically, which influences tumor growth and leads to significant bone destruction, releasing growth factors from the bone matrix that perpetuate tumor growth in a ‘vicious cycle’. As they evolve, metastatic bone tumors likely alter the biophysical cues acting on bone cells, through the exertion of tumor pressure and by altering matrix stiffness. Given the well established relationship between matrix properties, mechanical stimuli, and bone biology, such changes may activate mechanobiological responses in both native bone cells, and tumor cells, perpetuating the “vicious cycle” driving bone lysis and tumor growth. However, how the coupled mechanobiological responses of tumor and bone cells contribute to osteolysis and tumor growth is not fully understood. Although advanced 3D in vitro models have been developed that have significantly contributed towards our understanding of bone metastasis, these models either do not fully recapitulate the multicellular niche within a mineralized matrix or do not incorporate extrinsic mechanical loading to represent the role of daily physical activity in bone health. For this reason, the role of mechanical stimulation in the bone environment on the development of osteolytic bone metastases is not yet fully understood. In the first study of this thesis, a 3D in vitro model and a novel computational model were developed to investigate the effect of growth-induced stress on tumor spheroid growth. 4T1 breast cancer cells were cultured within hydrogels of three different stiffness (0.58 kPa, 0.85 kPa, and 1.1 kPa) and cultured for 7 days. Starting from single cell suspensions, these cells grew into spheroids whose size reduced with increasing stiffness of the hydrogels. The number of cells per spheroid was also found to be significantly higher in softer hydrogels. To investigate whether tumor spheroid growth was stress-dependent, an adaptive computational finite element model was developed where the proliferation of tumor cells in a spheroid was governed by spheroid stress induced by hydrogel deformation. The results of this model revealed that the circumferential and radial stresses generated by the growth of the spheroid in a stiff environment can reduce tumor cell proliferation. In a soft environment, these stresses were lower, and the spheroids grew larger, hence mechanical inhibition of proliferation was lower compared to the stiff environment. This chapter provided an advanced understanding of the mechanical cues governing tumor spheroid growth. However, in vivo bone cells play an important role in the ‘vicious cycle’ by interacting with the tumor cells and enabling them to invade the bone tissue, yet the coupled influence of tumor cell bone cell signalling and mechanical loading on tumor spheroid growth has not been widely studied. The second study investigated the coupled influence of tumor cell-bone cell signaling and hydrogel stiffness on tumor spheroid growth. Interestingly, a significant reduction in the tumor spheroid size occurred when tumor cells were co-cultured with osteoclast precursors. This effect was partially mitigated when osteoblasts were included in the culture with osteoclast precursors and tumor cells (i.e. tri-culture) in the soft and stiff hydrogels. The inhibitory influence of bone cells on tumor spheroid size was associated with a reduction in the overall cell population. A novel computational tumor growth model was developed here, which considered the level and dominance of certain biochemical signals (TNF-α, TGF-β, and IL-6) released in the culture of tumor cells and bone cells, and studied their effects on the tumor spheroid evolution across the different cell culture groups and hydrogels of different stiffness. The model predicted that the regulation of tumor spheroid size was dictated by a dominant inhibitory effect of TNF-α and an inhibitory effect of TGF-β, while IL-6 displayed a proliferative effect on tumor spheroid proliferation, in addition to the compressive stress resulting from growth. These results reveal for the first time a synergistic influence of osteoclast precursors, osteoblasts, and 3D matrix stiffness on tumor spheroid growth. However, these models did not fully recapitulate the multicellular niche within a mineralized matrix and did not incorporate extrinsic mechanical loading. Thus, the final study of this thesis sought to develop an advanced in vitro 3D bone like and metastatic models of the in vivo multicellular and mechanical environment. In the bone-like model, the presence of mineral, expression of osteogenic genes (OPN, BSP2, DMP1), and the presence of osteoclasts (TRAP, H&E, and CTSK) were confirmed. The metastatic model was able to capture the osteolytic metastatic activity as evident through an increase in the markers implicated in the tumor cell activity (PTHrP and IL-6), a decrease in osteoblast activity (BSP), and an increase in pro-osteoclastogenic genes (RANKL, OPG, and OSCAR). Hence the models developed here successfully recapitulated osteoblast, osteoclast, and metastatic activity within a mineralized matrix. These models were then applied to demonstrate the importance of mechanical loading in attenuating breast cancer cell activity, signaling for osteoclastogenesis, and rescuing osteoblast activity during early-stage bone metastasis. Altogether, the results from this thesis provide an advanced understanding of how tumor growth and osteolysis are regulated by the evolving mechanical environment during tumor invasion of the bone. These results also highlight a synergistic influence of bone cells and substrate stiffness on tumor spheroid growth. This work reported the development of an advanced biomimetic 3D in vitro mineralized multicellular model which successfully recapitulated the osteolytic-metastatic process. Using this model, the inhibitory effect of mechanical loading on osteolysis in early-stage bone metastasis was highlighted. Future studies could apply this model to investigate the inhibition of mechanobiological responses in attenuating tumor cell-bone cell signaling to mitigate the development of osteolytic bone metastases.
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NUI Galway