H24 - Reusable Superhydrophobic Array Devices for Modeling Physiologically Relevant Microenvironments of Circulating Tumor Cells

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Ehsan Aalaei

PhD Student
Rice University
Houston, Texas, United States

Co-Author(s)

JL

Jonathan Largoza

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

Undergraduate Student
Rice University
Houston, Texas, United States

Primary Investigator(s)

Michael R, King

E.D. Butcher Chair of Bioengineering, Associate Vice President for Research
Rice University, United States

Introduction: : Metastatic progression begins when cancer cells detach from the primary tumor, often a hypoxic microenvironment, and enter the bloodstream as circulating tumor cells (CTCs), eventually forming secondary tumors. These CTCs may circulate as single cells or as clusters, with the latter exhibiting enhanced resistance to fluid shear stress, immune evasion, and metastatic potential. However, due to their relative rarity in patient blood, CTC clusters remain poorly understood, emphasizing the need for robust and scalable in vitro models. To address this, the King Lab developed the Superhydrophobic Array Device (SHArD), which enhances cell–cell adhesion and enables high-throughput formation of CTC-like clusters and tumor spheroids. While effective, SHArD was originally designed for single use. In this study, we present a reproducible cleaning protocol that allows the device to be reused multiple times under both normoxic and hypoxic conditions, preserving its performance and cluster-forming capabilities.

Materials and

Methods: : SHArD devices were fabricated using a multistep additive microfabrication process as reported in [1]. Briefly, SU-8 photolithography was used to define square microwells with a wall-to-wall distance of 100 μm and a wall depth of 75–85 μm. A ZnO nanoparticle suspension was spin-coated onto silicon wafers and annealed to form a crystalline thin film, followed by growth of ZnO nanorods to increase nanoscale roughness. A non-wetting Teflon-like polymer was then deposited via C₄F₈ plasma polymerization to complete the superhydrophobic coating. Devices were diced into 11 × 11 mm squares to fit into 24-well plates.
LNCaP prostate cancer cells were seeded at 120,000 cells per well onto SHArD devices placed in individual wells of a 24-well plate. Cells were suspended in 2 mL of culture media and centrifuged into the microwells at 100 × g for 3 minutes. Plates were incubated under normoxic (21% O₂) and hypoxic (1% O₂) conditions. After 3 days, cells were collected from the devices and fixed in 4% paraformaldehyde (PFA). Between each use, SHArD devices were cleaned by washing with HBSS, incubating with trypsin for 15 minutes, followed by a second HBSS wash. Devices were flipped upside down in fresh wells containing 2 mL HBSS and centrifuged at 1000 × g for 5 minutes to extract remaining debris. After rinsing with 70% ethanol, devices were sterilized under UV light for 30 minutes before reuse. This process was repeated four times. Fixed clusters were stained with DAPI and E-cadherin and imaged using confocal fluorescence microscopy.

Results, Conclusions, and Discussions:: LNCaP prostate cancer cells reliably formed 3D clusters within the SHArD device under both normoxic and hypoxic conditions, closely mimicking the morphology of CTC clusters found in patient blood. These clusters maintained a compact, spheroidal structure across four sequential uses of the same device, demonstrating the robustness and reusability of the SHArD platform. No visible deterioration of the nanostructured surface or decline in cluster-forming efficiency was observed over repeated cycles of seeding, incubation, and extraction. DAPI staining confirmed consistent cluster formation across all conditions (Fig. 1B–G), while E-cadherin staining revealed intact epithelial junctions, validating the preservation of cell-to-cell adhesion typical of CTC clusters. These findings indicate that the cleaning protocol, comprising sequential HBSS washes, trypsinization, high-speed centrifugation, ethanol rinse, and UV sterilization, effectively removes residual material and preserves device function for multiple rounds of use.
This study establishes SHArD as a reliable, reusable, and compatible with different oxygen conditions platform for modeling tumor cell aggregation in vitro. Future work will assess post-culture cell viability and metastatic potential of SHArD-derived clusters and expand the system to support co-culture with cancer-associated fibroblasts and immune cells for more physiologically relevant models.

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

References: [1] Lopez-Cavestany M, Wright OA, Reckhorn NT, Carter AT, Jayawardana K, Nguyen T, Briggs DP, Koktysh DS, Linares AE, Li D, King MR. Superhydrophobic array devices for the enhanced formation of 3D cancer models. ACS Nano. 2024;18(34):23637–54.