A Novel Tumor Microenvironment Model that Combines Bioprinting and Tissue Culture to Investigate Cancer Cell and Microvascular Interactions

Abstract

A challenge in cancer research is the lack of a physiologically responsive in vitro model that allows for the investigation of cancer cells in a tissue‐like environment. A model that enables real‐time investigation of cancer cell migration, fate, and function during microvascular network growth does not currently exist. While current models such as 2D in vitro models or microfluidic systems incorporate real‐time cell tracking and multiple cell types, they do not mimic the complexity of intact networks and tissue environments. The objective of this study was to establish a novel tumor‐microvasculature model by demonstrating the feasibility of bioprinting cancer cells onto excised mouse mesometrium tissues. Pre‐labeled DiI mouse breast cancer (4T1) cells were inkjet‐printed onto mouse mesometrium tissues. The cell ink for printing comprised of 2% Na‐alginate mixed in minimum essential media with 1% PenStrep (MEM) containing 15 million 4T1 cells. A single cancer cell spot per tissue was created by printing 10 drops of cell ink in the same location. MEM was added on top of tissue 30 seconds after printing and then incubated for 5 minutes before being plated into 6‐well plates containing MEM supplemented with 20% serum. Tissues were cultured for 5 days with media being changed every day. The spot of DiI+ cells was imaged every 24 hours to then quantify cell number and area for Day 0, 1, and 2. At Day 2 or 5, tissues were fixed in methanol and labeled with platelet endothelial cell adhesion molecule (PECAM), and E‐cadherin, to identify endothelial cells and cancer cells, respectively. Co‐localization of DAPI+ nuclei confirmed that DiI+ cells remained intact post‐printing. Printed DiI+ 4T1 cells also remained viable after printing on Day 0 and after culture on Day 5. Time‐lapse imaging over 5 days in culture enabled tracking of cell motility and proliferation. The number of cells (Day 0: 159 ± 40, Day 1: 370 ± 78, Day 2: 889 ± 184, Day 5: 18,031 ± 1,695) and cell area (Day 0: 0.72 ± 0.19, Day 1: 1.89 ± 0.33, Day 2: 2.92 ± 0.44, Day 5: 5.93 ± 0.75 mm2) were significantly increased over time. Moreover, a proliferation assay of anti‐BrdU on Day 2 also highlighted that a subset of E‐cadherin+ cells are in the S‐phase of the cell cycle contributing to the increase in cell number and cell area. Also, microvessels in the tissue were angiogenic evident by PECAM+ sprouts. These results corroborate that cancer cells are mobile and proliferative in this novel ex vivo model. Further, it demonstrates the potential for bioprinting cancer cells onto live, intact tissues to investigate cancer dynamics within a physiologically relevant microenvironment.Support or Funding InformationNIH R01AG049821