Deciphering the Mechanism of Lymphovascular Space Invasion Using a Lymphovascularized Bioengineering Breast Stromal Platform

Highly aggressive cancers are frequently characterized by tumor cell emboli within the lymphatic and blood vasculature. Termed lymphovascular space invasion (LVSI), this phenomenon has been of high interest to cancer researchers as it may represent one of the necessary events during progression from a localized to

metastatic cancer. Despite these biological implications, relatively little is known about the mechanisms that directly enable and promote LVSI formation. Existing in vitro systems lack the multi-tissue and vascular complexity to model these events. In vivo models, while available, are not amenable to mechanistic studies or

pharmacological screening because of the sheer number of animals required for such experiments. Therefore, a critical need in the field is to develop models that can faithfully recreate specific phenomena related to metastatic spread, such as LVSI. Using our novel, multi-cellular, vascularized 3D in vitro platform, we were able

to model intravasation of epithelial emboli and LVSI formation of inflammatory breast cancer (IBC) ex vivo for the first time. IBC is an aggressive breast cancer variant characterized by extensive LVSI. Gene expression data from IBC patients identified stromal infiltration and activation as a critical component of LVSI. Using our new in

vitro platform and animal models, we were able to confirm this finding when we discovered that macrophages in the microenvironment directly promote LVSI formation. Leveraging our new in vitro platform, we propose to answer three major questions relating to LVSI: What mechanisms promote 1) formation, 2) migration, and 3)

intra-vessel survival of tumor emboli? We hypothesize that LVSI formation is a two-step process where matrix properties and epithelial marker, E-cadherin which is strongly expressed in IBC, regulate tumor emboli formation and survival, while the cytokine axis, CCR7/CCL21, homes epithelial emboli to lymphatics. To test this

hypothesis, we propose to use our new in vitro platform in three aims: 1) Determine the role of matrix mechanics and lymphatic pumping in the temporal kinetics of LVSI, 2) Determine the role of E-cadherin in emboli formation and survival, and 3) Identify the mechanisms that promote emboli homing to vasculature. Despite the strong

clinical evidence that LVSI is a critical, pre-metastatic phenomenon, our inability to fully recreate LVSI in vitro has severely limited our mechanistic understanding of responsible pathways. For the first time, our team was able to recreate LVSI ex vivo using a novel microfluidic platform. Here, we propose to use our in vitro platform

to define the signaling steps that promote LVSI formation and survival in vasculature to better define critical targets related to cancer progression. To execute the proposal, we have assembled a team composed of experts in tissue bioengineering, clinical research, and preclinical models of breast cancer. If successful, our work will

offer novel and customizable platforms for studying LVSI and new biological discoveries that hold therapeutic potential for patients with advanced breast cancer.