X14 - Modeling Physiologically Relevant Microenvironments for Circulating Tumor Cells via the Superhydrophobic Array Device

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

JL

Jonathan Largoza

Undergraduate Student
University of Pennsylvania
San Antonio, Texas, United States

Co-Author(s)

Ehsan Aalaei

PhD Student
Rice University
Houston, Texas, United States

Primary Investigator(s)

Michael King

Professor
Rice University, United States

Introduction: : Cancer tumors are hypoxic microenvironments due to the uncontrollable growth rate of tumors and abnormal tissue organization. This leads to the metastatic cascade where cancer cells undergo epithelial-mesenchymal transition (EMT); epithelial cells are normally held to each other via strong junctions through E-cadherin, but these connections are disrupted during EMT, allowing cancer cells to mobilize and become invasive. Cells called circulating tumor cells (CTCs) detach from the primary tumor and enter the bloodstream as singlets or clusters with the ability to migrate to distant sites and form secondary tumors. CTC clusters are better equipped to survive under the fluid shear stress of blood flow and evade immune response. However, CTC clusters are rare in patient blood and not well understood, which presents a need to culture artificial CTC clusters in a high-throughput manner. The King Lab has recently developed the superhydrophobic array device (SHArD) to form thousands of CTC clusters and tumor-mimicking spheroids (Fig 1. A). The superhydrophobic features of the SHArD promotes greater cell-to-cell adhesion than existing 3D culture methods. The device has been proven to artificially make clusters representative of real clusters found in patient blood.

Materials and

Methods: : For culture in the SHArD, devices were gently placed in wells of a 24-well plate with a pair of forceps and sterilized by UV irradiation for 30 min. 120,000 cells of human prostate cancer cell line LNCaP were seeded in each well. Additionally, a monolayer control well with no SHArD device was implemented, and 2 well plates were seeded for normoxic (21% oxygen) and hypoxic conditions (1% oxygen). Cells were suspended in 2 mL of media and centrifuged into the microwells at 300xg for 5 min. After 3 days of incubation time, cells were collected and fixed in 4% PFA. The SHArDs were thoroughly cleaned and underwent UV irradiation before the next set of cells were seeded during this reusability evaluation. This protocol was conducted 4 times to collect and fix 4 sets of LNCaP control and SHArD-made clusters. These fixed cells were fixed and stained for DAPI marking the nucleus and E-Cadherin marking cell-to-cell adhesions. Lastly, these cells were placed onto slides and observed under a fluorescent confocal microscope.

Results, Conclusions, and Discussions:: Prostate cancer cells cultured in the SHArD consistently formed compact 3D clusters in both hypoxic and normoxic conditions resembling circulating tumor cell (CTC) aggregates observed in patient blood. These clusters maintained a relatively spheroidal morphology across repeated uses of the device, demonstrating SHArD’s durability and reusability. There was no visible degradation of nanostructure performance or loss in cluster-forming efficiency was observed after 4 rounds of seeding, incubation, and extraction. DAPI staining confirmed cell aggregation across conditions (Fig 1. B-G), while E-cadherin staining highlighted epithelial junctions within clusters, supporting the vital cell-to-cell adhesion representative of CTC clusters found in cancer patients. These results indicate the cleaning protocol for SHArDs consisting of vigorous washing, spinning, and UV sterilization is effective in removing leftover debris before the next use. This study reinforces the reproducibility and adaptability of the SHArD across multiple uses and also different oxygen conditions as a versatile in vitro platform for modeling tumor cell aggregation. Future work will aim to confirm the metastatic potential of SHArD-made CTC clusters and utilize the SHArD culture heterotypic CTC clusters with cancer-associated fibroblasts and immune cells.

Acknowledgements and/or References (Optional): : This research was funded by the NSF-supported Rice ENGMED REU Program and the Cancer Prevention and Research Institute of Texas (CPRIT) to M.R.K., Grant No. RR230029.