Metastatic dissemination of breast adenocarcinoma

Yun Zhang

doi:10.17918/00008123

Breast adenocarcinoma remains the second leading cause of cancer-related death in women. Although the majority of patients are presently diagnosed at an early stage, up to 30% will eventually develop metastatic tumors at distant sites such as bone, liver, lungs and brain. Curative therapeutic approaches for metastatic disease are currently lacking; therefore, the identification of molecular mediators of breast cancer dissemination and their validation as potential therapeutic targets are urgently needed. Early stage carcinoma is routinely treated with breast-conserving surgery; however, 20-50% of patients need re-excision to remove residual tumor before adjuvant therapies can be started. The surgical wound left by the excision of the primary tumor is associated with the production of a plethora of local tissue factors that regulate events such as angiogenesis, cell proliferation and survival. These alterations in the local tissue microenvironment may affect the micro-deposits of residual cancer cells, selecting for more aggressive phenotypes and promoting their access to the systemic blood circulation, therefore increasing the likelihood of distant spreading and colonization of secondary organs. A first aim of this proposal was to investigate the role of local recurrences in the metastatic dissemination of breast cancer. To this end, an appropriate animal model was developed in which fluorescent murine breast cancer cells were grafted in the mammary fat pad of immune-competent mice and the tumors that subsequently developed were surgically excised. By monitoring animals for local recurrences and distant metastases, it was found that only the animals that developed recurrent tumors presented also with secondary lesions in the lungs. Recurrent tumors showed a higher proliferation rate, increased angiogenesis and over-expression of the chemokine receptor CX3CR1, which has been previously implicated in the homing of breast cancer to the skeleton. Notably, all these results from our animal model were confirmed in human specimens from breast cancer patients. The dissemination of breast adenocarcinoma occurs predominantly via the hematogenous route. Circulating Tumor Cells (CTCs) shed by the primary tumor transit through the blood circulation to eventually home at distant sites, thereby converting into Disseminated Tumor Cells (DTCs). Interestingly, it has been proposed that CTCs can also derive from metastatic tumors already established at the skeletal level and generate secondary waves of dissemination and metastasis. The second aim of this proposal focused on the isolation, enumeration and molecular analysis of CTCs from animals harboring skeletal tumors, which has never been reported before. The third aim of this proposal aimed to both functionally validate the role of CX3CRI in the metastatic dissemination of breast cancer cells and test JMS 17-2, a novel small-molecule inhibitor of this receptor, as a potential drug candidate for counteracting the homing of CTCs to the bone. The experiments performed showed that blocking CX3CRI with a neutralizing antibody dramatically reduced the number of DTCs detected in the skeleton of animals inoculated with human breast cancer cells in the arterial circulation via the left cardiac ventricle. More importantly, it was shown that the novel compound JMS 17-2 was able to block the metastatic dissemination of breast cancer cells as effectively as the neutralizing antibody against CX3CRI. In conclusion, the work presented in this thesis indicates that the post-surgery stroma and local recurrences can support the spreading of breast cancer cells at distant sites. These observations provide a solid rationale for the development of preventive therapies to be used after initial interventions and prior to re-excision of the recurrent tumor. Furthermore, this thesis reports for the first time the successful isolation, enumeration and molecular analysis of CTCs isolated from animal models of metastasis. Finally, the study presented here provides compelling evidence for a pivotal role of CX3CRI in the homing of breast CTCs at the skeletal level and shows that its pro-metastatic activity can be counteracted with JMS 17-2, a novel, potent and specific inhibitor of this receptor.

Metastatic dissemination of breast adenocarcinoma

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Metastatic dissemination of breast adenocarcinoma

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