Professor University of Minnesota Minneapolis, Minnesota, United States
Introduction: In breast cancer, patient survival rates drop from 90% to 27% once cancer cells metastasize to distant tissues. Previous work documents the phenomenon of tumor-favoring distant organs, termed pre-metastatic niches (PMN), that are formed prior to metastatic seeding in extracellular matrix (ECM) and are highly conducive to colonization by circulating tumor cells. Several factors dictate the formation of the pre-metastatic niche, such as primary tumor conditioning and immune cell infiltration. Identifying the factors and understanding the biological cues that regulate PMN formation and cancer metastasis is critical for therapeutic development, as 90% of cancer-related deaths are caused by secondary, not primary, tumors.
Numerous technical hurdles have impeded our ability to study the drivers of PMN initiation and modulation, particularly in vivo models that are time-intensive, costly, immunocompromised, limited in optical accessibility, and hindered by interspecies differences. Traditional 2D in vitro systems lack sophisticated 3D tissue architecture, and necessary cell-cell and cell-ECM interactions required to study real-time PMN formation and metastasis. In the absence of an ideal model system, we have lacked the ability to perform robust mechanistic studies that are required to dissect the fundamental factors during the PMN regulation and to develop novel therapeutics that specifically disrupt metastasis. To overcome the limitations of existing models, we engineered a comprehensive microfluidic device that allows upstream 3D tumor conditioning and visual monitoring of cancer cell-endothelial cell interactions in the downstream vascular system.
Materials and
Methods: The upstream tumor chamber was designed to incorporate large tumor samples, such as multicellular tumor spheroids (MCTS) or organoids, in collagen ECM, while the downstream PMN chamber was a self-assembled 3D vascularized tissue site that connected to the primary chamber via microchannels (Fig 1. A-C). To carry out the evaluation of the angiogenesis in the PMN chamber in response to the primary tumors, total vessel length, vessel diameter, number of vessel branches, number of junctions, and number of endpoints were quantified every 24 hours. To validate the functionality of the vascular system in the PMN, vessel integrity, and adhesion ability, as well as cancer extravasation, will be measured in our platform. The expression of endothelial junction markers, such as E-Cadherin, N-Cadherin, VE-Cadherin, and ZO-1, and adhesion markers, such as VCAM, ICAM, and E-selectin can be characterized by IF staining (Fig 1. D). The vessel permeability can be visualized and quantified by 70kDa FITC-dextran solution over time (Fig 1. E). Cancer cells were injected into the microchannels, and cancer extravasation events can be visualized and quantified by imaging for 24 hours (Fig 1. G-H).
Results, Conclusions, and Discussions: Analysis of multiple secretions from the media collected from devices showed an increase in pro-angiogenesis factors, such as VEGF, from the devices with MCTS/collagen ECM than plain collagen condition. In the downstream PMN site, the vascular system was formed under the impact of such soluble factors from the primary chamber (Fig 2. A-B). In our microfluidic model, the tumors in the primary chamber can lead to hyperpermeability by the downregulation of VE-cadherin on endothelial cells (Fig 1. C-F) and alter the morphology of the vascular system (Fig 1. F-G).
Our platform separates the primary tumor chamber and the downstream vascular system to perform robust mechanistic studies through endocrine signaling. The vessel quantification matrix provides us with information about vessel growth and structure under the impact of the primary chamber. Future studies using our device include evaluating anti-cancer therapeutics, in particular NK and T cell-based therapies, administered through the blood vessel-like channels to target both primary tumor and secondary metastatic growth. Additionally, this platform provides more opportunities for chemotherapeutic drug screening that targets either primary tumors or downstream PMN modulation.
Our platform incorporated the large-scale tumors into collagen ECM and connected the tumor site to the downstream vascular system. The tumor secretion can support angiogenesis and change the vessel morphology as well as permeability.
