Mechanobiological origins of osteolysis during bone metastasis
Date
2023-03-28Author
Verbruggen, Anneke
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Abstract
Metastasis is the final, lethal stage of cancer where cells migrate from a primary
tumour site to colonise a secondary organ and is the primary cause of mortality in
cancer patients. Breast cancer is the leading cause of cancer death in women in the
world, with a recorded 627,000 deaths in 2018, and projected to reach 800,000 by
2030. In advanced breast cancer patients, cancer cells favour metastasis to bone tissue
70-80% of the time, primarily leading to osteolysis (bone destruction) and sometimes
unwanted tissue formation. The evolving mechanical environment during tumour
invasion might play an important role in these processes, as the activity of both bone
and cancer cells is regulated by mechanical cues. However, it is not yet known how
specific bone tissue composition is associated with tumour invasion. In particular, how
compositional and nano-mechanical properties of bone tissue evolve during
metastasis, where in the bone they arise, and how they influence the overall
aggressiveness of tumour invasion, are not well understood.
The first study of this thesis sought to develop an advanced understanding of
temporal and spatial changes in nano-mechanical properties and composition of bone
tissue during metastasis. Primary mammary tumours were induced by inoculation of
immune-competent BALB/c mice with 4T1 breast cancer cells, and microcomputed
tomography and nanoindentation were conducted to quantify cortical and trabecular
bone matrix mineralisation and nano-mechanical properties, respectively. Spatial
analysis was performed in proximal and distal femur regions of tumour-adjacent
(ipsilateral) and contralateral femurs after 3 weeks and 6 weeks of tumour and
metastasis development. By 3 weeks post-inoculation there was no significant
difference in bone volume fraction or nano-mechanical properties of bone tissue
between the metastatic femora and healthy controls. However, early osteolysis was
indicated by trabecular thinning in the distal and proximal trabecular compartment of
tumour-bearing femora. Moreover, cortical thickness was significantly increased in
the distal region, and the mean mineral density was significantly higher in cortical and
trabecular bone tissue in both proximal and distal regions, of ipsilateral (tumour bearing) femurs compared to healthy controls. By 6 weeks post-inoculation, overt
osteolysis, decreased bone volume fraction, cortical area, cortical and trabecular
thickness were reported in metastatic femora. Trabecular bone tissue stiffness in the proximal femur decreased in the ipsilateral metastatic femurs compared to
contralateral and control sites. This study uncovered changes in bone tissue
composition prior to and following overt metastatic osteolysis, local and distant from
the primary tumour site. On the basis of these findings, it was proposed that changes
in tissue composition may alter the mechanical environment of both the bone and
tumour cells, and thereby perpetuate the cancer vicious cycle during breast cancer
metastasis to bone tissue.
The objective of the second study was to quantify changes in the mechanical
environment within bone tissue, during bone metastasis and osteolytic resorption. This
study used finite element (FE) models reconstructed from micro-CT images obtained
during the first study of this thesis. In particular, the time-dependent changes in the
mechanical environment, local to and distant from an invading tumour mass, were
quantified to investigate putative mechanobiological cues for osteolysis during bone
metastasis. This study reported a decrease in strain distribution within the proximal
femur trabecular and distal cortical bone tissue in early metastasis (3 weeks after
tumour inoculation). These changes in the mechanical environment preceded
extensive osteolytic destruction, but coincided with the onset of early trabecular
thinning, cortical thickening and mineralisation of proximal and distal femur bone,
which were reported in the first study of this thesis. From these findings, it was
proposed that early changes in the mechanical environment within bone tissue may
activate resorption by osteoclast cells and thereby contribute to the extensive
osteolytic bone loss at later stage (6 weeks) bone metastasis.
To investigate this proposed adaptation of bone tissue upon breast cancer metastatic
invasion, the third and final study of this thesis sought to apply the mechanoregulation
theory, which predicts tissue adaptation on the basis of changes within the mechanical
environment. This was performed using a bone remodelling algorithm driven by
changes in mechanical strain. A user-defined field (USDFLD) subroutine was applied
to murine proximal femur models, with material properties obtained from the first
study, such that each individual element within an FE model adapted material density
and stiffness according to pre-defined strain stimuli criteria. In this way, this model
generated an iterative mechanoregulatory response to changes in strain distribution
throughout the bone mechanical environment, over a period of 3 weeks. This study predicted that bone tissue would undergo resorption in regions which corresponded to
those in the first study of this thesis upon overt osteolysis by 6 weeks of bone
metastasis. These findings further support the proposal that mechanobiology may play
a role in breast cancer bone osteolysis.
Together, the studies in this thesis report, for the first time, changes in bone mineral
content and mechanical properties prior to overt osteolytic destruction in an in vivo
animal model of breast cancer metastasis. Computational analysis revealed decreased
strain distribution at this early time point, prior to osteolysis, and on this basis it was
proposed that a mechanoregulatory response may contribute to subsequent osteolytic
destruction.