30 line summary: The objective of this proposal is to develop for the scientific community validated assays based on organ on chip systems that will enable interrogating the neuron dependent regulators of breast- to-brain metastasis and related complications such as vasogenic edema associated with blood brain barrier disruption. Brain metastases (with breast cancer being second largest cause) and their associated neurologic disabilities are particularly serious as they result in very short life expectancies and reduced quality of life. There is a significant lack of effective therapeutic regiments due to presence of blood brain barrier which precludes the delivery of multiple chemotherapeutic drugs into the central nervous system (CNS). We will use the NeuroVascular Unit (NVU) tissue chip, an organ-on-chip equivalent of the blood-brain-barrier comprised of neurons, astrocytes, brain pericytes and brain microvascular endothelial cells. We will evaluate its suitability to study the neuronal regulation of breast-to-brain metastasis with particular emphasis on measuring the specific role of neurons on metastatic disease expansion, colonization and the associated changes to the metastatic microenvironment. Furthermore, we will evaluate the NVU coupled to a Mammary Gland Thick Tissue bioreactor (MG-TTB) as a complete primary-to-secondary site oncology-on-chip system for advanced study of breast-to- brain metastasis. MG-TTB is a microfluidic bioreactor developed by our group for drug assessment of tumorigenic mammary epithelial cells that is currently in use for toxicant assessment, monitoring toxicant-dependent changes to cellular functions, and drug assessment of tumorigenic mammary epithelial cells cultured under defined physical and chemical tumor microenvironments. The MG-TTB is on the same physical scale and shares similar materials and hardware with NVU that will allow seamless integration of these units to probe primary-to- secondary tumor dissemination. Our aims to validate the suitability of NVU for study of breast-to- brain metastasis processes are as follows: 1) Determine the neuron dependent dynamic changes to metastatic tumor microenvironment driven by breast cancer brain metastases seeded within the NVU. 2) Determine the neuron dependent role into breast-to-brain metastasis by evaluating their contribution in tumor extravasation of the NVU blood brain barrier.

Narrative relevance/Lay Sentences: We propose to use an organ-on-chip system that recapitulates the blood brain barrier. We predict this will allow us to define the specific molecular and cellular events that neurons contribute to breast-to-brain colonization.