(D-122) Chimeric Invasion Assay (CIA) for Probing Collective Cancer Invasion in 3D Microenvironments

Introduction:: The development of 3D cancer invasion models that capture the cell-cell and cell-environment interactions will be critical for comprehending the intricate processes underlying collective cancer invasion and designing effective therapeutic interventions. To create multicellular tumor spheroids (MCTS), cancer cells can be assembled using the hanging drop method or round-bottom wells with a non-adherent coating. Following their formation, spheroids are embedded in extracellular matrices (ECM) that mimic the physiological microenvironment. Nonetheless, these techniques have a limited throughput for probing the heterogeneity of cancer cells and randomly position the spheroids in the z-plane, which limits high resolution imaging due to the working distance of optical microscopy. Here, we report a chimeric invasion assay (CIA) based on a novel liquid-infused surface coating technology that allows rapid self-assembly of cancer cells into 3D microtumors and selective adhesion of invasive cancer cells on the surface. We demonstrate that CIA (1) generates a large number of microtumors in a single step, (2) captures the collective invasion of cancer cells in the 3D microenvironment, (3) reflects the invasiveness of the tumor samples, and (4) allows controllable placement of the microtumors in the z-plane for high resolution imaging.


Materials and Methods:: The novel liquid-infused coating is constructed by assembling a thin, lubricated layer of polydimethylsiloxane (PDMS) on top of a flat glass bottom plate. The thickness of the substrate is adjustable, allowing for a small working distance in the z-direction and enabling high-resolution imaging. After PDMS solidification, a hydrophobic lubricant is infused onto the PDMS at 55ºC overnight. Excess lubricant is then removed, and the plate is sterilized. Subsequently, cells are seeded onto the CIA surface and incubated for 24 hours to self-assemble into microtumors. A biomimetic collagen/Matrigel matrix is then overlaid on top of the spheroids. The samples are imaged using a confocal microscope every 24 hours for three consecutive days. To understand the properties of CIA, multiple surface characterizations were performed. The wettability of the CIA surface was evaluated using contact angle and sliding angle measurements. The chemical structure of the CIA surface was analyzed using X-ray photoelectron spectroscopy (XPS) and FTIR. Primary disassociated cancer cells from muscle invasive bladder cancer patients (Stage II and III), as well as bladder cancer cell lines, were used to characterize the adhesive properties of CIA.


Results, Conclusions, and Discussions:: The CIA surface displayed both non-adherent and adherent properties for assembling 3D microtumors and probing cancer invasion. The non-adherent property of CIA allows self-assembly of a larger number of 3D microtumors on its surface 24 hours after seeding. The microtumors exhibited a high uniformity in sizes and circularity compared to untreated PDMS, which is also hydrophobic and non-adherent (Figure 1). Remarkably, the adherent property of CIA also captured the ability of the tumor samples to adhere and invade on the surface (Figure 1). The extent of cell adhesion reflects the invasiveness of the Stage II and IIIB samples, enabling rapid evaluation of the invasiveness of cancer samples.

We performed surface characterization to unravel the details of the microtumor formation and dissemination process. Firstly, contact angle data were analyzed after incubation with different components of the cell culture to identify the key factors causing the hybrid adherent/non-adherent properties. Figure 2 shows a significant reduction in contact angle when incubated with FBS. XPS analysis showed protein aggregation on PDMS, while no protein was detected on the CIA surface (Figure 3). Further testing of the surface was conducted using FTIR, which has a larger penetration depth (~500 nm). FTIR data revealed that protein retention was higher on PDMS compared to CIA. The data could potentially be explained by interactions of protein and lubricant on the CIA surface compared to direct deposition of protein on PDMS. Additionally, by examining F-actin structures of spheroids invading on CIA (Figure 4), we found that stage IIIB cells exhibited distinguished filaments, demonstrating their ability to adhere to the CIA surface. In contrast, stage II didn't show observable actin filaments. For PDMS, both stage IIIB and stage II cells didn't show adhesion and actin filaments. These results depict CIA's ability to capture the invasiveness of different tumor samples.

To conclude, we describe a novel invasion assay that addresses challenges of conventional 3D invasion assays. CIA allows high resolution imaging through control of the working distance and high throughput production of spheroids for dynamic monitoring of cancer invasion.


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References (Optional): : Zhu, N., Ahmed, M., Liao, J., & Wong, P. (2022). Long Noncoding RNA MALAT1 is Dynamically Regulated in Leader Cells during Collective Cancer Invasion. bioRxiv, 2022.2007. 2026.501593.