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The Role of Circulating Monocytes in the Development of Brain Metastases

Periodic Reporting for period 1 - Brain Metastases (The Role of Circulating Monocytes in the Development of Brain Metastases)

Reporting period: 2015-05-01 to 2017-04-30

Brain metastasis of breast cancer is a devastating condition and carries a very poor prognosis. Patients that are diagnosed with spread to the brain survive between 3 and 23 months from diagnosis. Our understanding of how brain metastases develop is limited and closing this gap in knowledge will allow for improved therapies to be developed.

One of the main gaps in our knowledge is how cells in the microenvironment contribute to brain metastasis, particularly those of the innate immune system such as monocytes/macrophages.

The goal of this fellowship was to determine how monocytes and macrophages originating from the blood participate in the development of brain metastases from breast cancer and identify potential therapeutic routes. To study the blood-derived population specifically, we have used a mouse strain where the monocytes and macrophages have been genetically engineered to express the protein eGFP, which fluoresces green, allowing us to trace the origin of the monocytes and macrophages in these mice.

The objectives in this fellowship were as follows:

(1) Characterize the recruitment of blood-derived monocytes and macrophages into brain metastases, and determine how this correlates disease progression.

(2) Determine if inhibiting blood-derived monocyte and macropahge infiltration into the brain will reduce both the initiation and progression of metastases.

(3) Determine if the blood-derived monocytes and macrophages are pro-inflammatory (tumour killing) or anti-inflammatory (tumour promoting) and if changing their characteristics will slow brain metastasis progression.
We have completed a study where mouse breast tumour cells were injected intracerebrally into our eGFP monocyte/macrophage mice and the generated metastases were analysed at days 7, 14, and 21. These animals were also imaged using gadolinium enhanced MRI and these images were registered with histological images of the eGFP positive cells within the brain. The eGFP positive cells were detected at all timepoints and correlated with blood-brain barrier breakdown detected using MRI. We also found that the amount of eGFP positive cell infiltration increased significantly before significant changes could be seen in the vasculature within the metastases.

The phenotype of the eGFP positive cells in the brain metastases was analysed for iNOS (pro-inflammatory marker) and Arg1 (anti-inflammatory marker) expression to determine the initial phenotype any changes that occurred during growth of the lesion. The phenotype of the infiltrated GFP positive cells was initial a mixed phenotype of pro- and anti-inflammatory but leaning towards anti-inflammatory, which then shifted to a more predominantly anti-inflammatory phenotype at the later time points.

In vitro studies were performed where mouse macrophages were stimulated with either be pro-inflammatory, anti-inflammatory or left unstimulated and then co-cultured with mouse breast tumour cells in a transwell plate system that allows us to measure the invasiveness of the tumour cells. We found that tumour cells cultured with anti-inflammatory macrophages were more invasive than those that were cultured with pro-inflammatory macrophages.

We are currently conducting a study using miniature pumps loaded with PBS (a control), Macrophage Inhibitory Peptide (TKP, a general inhibitor of macrophage activation) or a CSF-1R neutralizing antibody (inhibits the anti-inflammatory expansion of macrophages) to modulate the function of blood-derived monocytes and macrophages and evaluate the how these different inhibitors affect the metastases’ growth. This study has been delayed due to poor breeding performance of our mouse colony.
This fellowship has advanced our knowledge on the generation and progression of brain metastases, particularly the role of the innate immune system. With this work, we have begun to uncover the role of circulating monocytes/macrophages in brain metastases. We have shown that these cells can make up a significant proportion of the brain metastases’ microenvironment and that this population of cells becomes more anti-inflammatory (or tumour-supporting) as the metastases progress. We expect to show with our current studies that inhibiting and modulating the function of this population specifically will lead to reduced growth.

This work has begun to address a fundamental gap in knowledge and research in cancer: the biology of brain metastasis development. Cancer patients that are currently diagnosed with spread to the brain have a poor prognosis primarily because these lesions are typically detected at later stages of development where current therapeutic strategies used clinically have limited effectiveness and may only have a palliative benefit.

This work begins to uncover the role of the innate immune system in brain metastases and will provide base knowledge for furthering the understanding of brain metastasis development and will provide new targets for novel and more effective therapeutic approaches which will benefit patients in the long term.
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