Brain-metastatic Breast Cancer Exhibits Greater Resistance to Oxidative Damage and Enhanced Invasiveness than the Primary Breast Cancer

Introduction: Oxidative stress has been shown to modulate various processes in cancer biology. We hypothesize that increased reactive oxygen species (ROS) in the brain microenvironment may promote the spread of metastatic breast cancer into the brain.

Methods: We created a brain-metastatic cell line (“brain-seeking”) via serial intracardiac injection of mCherry+ 4T1 breast cancer cells in Balb/c mice and flow cytometry isolation of micrometastases. We used this cell line to define the metabolism of ROS in primary and brain-metastatic breast cancer.

Results: We exposed parental and “brain-seeking” cells to high levels of H2O2 (200 uM and 500 uM) to test their resistance to oxidative damage. Brain metastases displayed better adaptation to higher levels of ROS than the original breast cancer (p < 0.0001). We also used Matrigel assays to measure the invasiveness of both cell lines after the exposure. Our results showed that brain-seeking cells displayed significantly higher invasiveness when exposed to ROS than the 4T1 parental group (p=0.0028). In a parallel invasion experiment, we used low concentrations of H2O2 (100 uM) as a chemoattractant. Brain-seeking cells not previously exposed to ROS displayed decreased invasiveness, while those exposed for a long period of time (48h) became attracted to even low levels of peroxide and showed increased invasiveness (p < 0.0001). 4T1 parental cells invaded poorly when exposed to even low levels of H2O2.

Conclusion : This suggests that small levels of ROS may have antitumoral properties in brain metastases but once adapted to higher levels, this effect is reversed, and invasion is stimulated. We correlated transcriptomic data from both cell lines to our experimental findings to identify genes that may stimulate adaptability to highly oxidative environments. Our brain-seeking cells showed upregulation of Cdkn1a, a cyclin-dependent kinase which enhances metabolism of oxidative species. This offers a potential target for the reduction of ROS-mediated invasiveness of breast to brain metastases.