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A challenge in cancer research is the lack of physiologically responsive in vitro models that enable tracking of cancer cells in tissue-like environments. A model that enables real-time investigation of cancer cell migration, fate, and function during angiogenesis does not exist. Current models, such as 2D or 3D in vitro culturing, can contain multiple cell types, but they do not incorporate the complexity of intact microvascular networks. The objective of this study was to establish a tumor microvasculature model by demonstrating the feasibility of bioprinting cancer cells onto excised mouse tissue. Inkjet-printed DiI + breast cancer cells on mesometrium tissues from C57Bl/6 mice demonstrated cancer cells' motility and proliferation through time-lapse imaging. Colocalization of DAPI + nuclei confirmed that DiI + cancer cells remained intact postprinting. Printed DiI + 4T1 cells also remained viable after printing on Day 0 and after culture on Day 5. Time-lapse imaging over 5 days enabled tracking of cell migration and proliferation. The number of cells and cell area were significantly increased over time. After culture, cancer cell clusters were colocalized with angiogenic microvessels. The number of vascular islands, defined as disconnected endothelial cell segments, was increased for tissues with bioprinted cancer cells, which suggests that the early stages of angiogenesis were influenced by the presence of cancer cells. Bioprinting cathepsin L knockdown 4T1 cancer cells on wild-type tissues or nontarget 4T1 cells on NG2 knockout tissues served to validate the use of the model for probing tumor cell versus microenvironment changes. These results establish the potential for bioprinting cancer cells onto live mouse tissues to investigate cancer microvascular dynamics within a physiologically relevant microenvironment.

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Laser-induced forward transfer (LIFT) is a well-established, versatile additive manufacturing technology for orifice-free printing of highly viscous solutions and suspensions. In order to improve the efficiency of point-wise LIFT printing, an optical scanner is integrated into the laser printing system to enable the formation of overlapping adjacent jets used for deposition. The objective of this study is to evaluate the ejection behavior and deposition performance under such conditions during LIFT printing for further improvement. The effects of the overlap of adjacent jets are investigated in terms of jet formation and material deposition processes, capturing the jet tilting phenomenon caused by the perturbance induced by previously formed jet(s). The feasibility of optical scanner-assisted LIFT printing of viscous metal-based ink suspension has been successfully demonstrated during conductive line printing with induced overlapping jets. Investigation of various overlap ratios of adjacent jets found that a 30% jet overlap and a time interval between laser pulses of 133 µs are optimal, in terms of deposition quality and ejection stability, even when a tilted jet ejection is present for the laser and material system in this study. Furthermore, multilayer polygonal and interdigitated structures are successfully deposited under these identified printing conditions. With the inclusion of an optical scanner, LIFT printing efficiency for viscous inks can be improved as the usage of higher laser frequencies is enabled, to provide a faster orifice-free laser printing methodology.

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. Prelabeled DiI mouse breast cancer (4T1) cells were inkjet-printed onto mouse mesometrium tissues. The cell ink for printing comprised 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, they demonstrate the potential for bioprinting cancer cells onto live, intact tissues to investigate cancer dynamics within a physiologically relevant microenvironment. Citation Format: Ariana D. Suarez-Martinez, Marc Sole-Gras, Samantha S. Dykes, Zachary R. Wakefield, Kevin Bauer, Arinola Lampejo, Dietmar W. Siemann, Yong Huang, Walter L. Murfee. A novel tumor microenvironment model that combines bioprinting and tissue culture to investigate cancer cell and microvascular interactions [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A23.