Hydrogel Microwell Arrays Allow the Assessment of Protease-Associated Enhancement of Cancer Cell Aggregation and Survival

Abstract

Current routine cell culture techniques are only poorly suited to capture the physiological complexity of tumor microenvironments, wherein tumor cell function is affected by intricate three-dimensional (3D), integrin-dependent cell-cell and cell-extracellular matrix (ECM) interactions. 3D cell cultures allow the investigation of cancer-associated proteases like kallikreins as they degrade ECM proteins and alter integrin signaling, promoting malignant cell behaviors. Here, we employed a hydrogel microwell array platform to probe using a high-throughput mode how ovarian cancer cell aggregates of defined size form and survive in response to the expression of kallikreins and treatment with paclitaxel, by performing microscopic, quantitative image, gene and protein analyses dependent on the varying microwell and aggregate sizes. Paclitaxel treatment increased aggregate formation and survival of kallikrein-expressing cancer cells and levels of integrins and integrin-related factors. Cancer cell aggregate formation was improved with increasing aggregate size, thereby reducing cell death and enhancing integrin expression upon paclitaxel treatment. Therefore, hydrogel microwell arrays are a powerful tool to screen the viability of cancer cell aggregates upon modulation of protease expression, integrin engagement and anti-cancer treatment providing a micro-scaled yet high-throughput technique to assess malignant progression and drug-resistance.

3D Cell Culture—Can It Be As Popular as 2D Cell Culture?

“…[ 36 ] Therefore, they can be used as an efficient 3D cell culture substrate. Hydrogels can be used either alone or in combination with other materials or devices (such as solid scaffolds, [ 37 ] cell microarrays, [ 38 ] and microfluidic devices [ 39 ] ). There are many ways to use hydrogels in the 3D culture system; for example, they can be used to coat a variety of cell culture on surfaces of solid scaffolds, or to wrap the cells or sandwich cells in the matrix.…”

Reality Check for Nanomaterial‐Mediated Therapy with 3D Biomimetic Culture Systems

“…Most notably, there was a distinct down‐regulation of key cytoskeleton protein such as cytokeratin and vimentin when cells were cultured in 3D. Conversely, adhesion proteins such as E‐cadherin were mostly unregulated as the cells were coerced to aggregate and form multi‐cellular spheroids in the confined 3D space . The transition from a predominately cell‐matrix adhesion in 2D culture, to a cell‐cell type adhesion in 3D culture has also been highlighted as an important phenotypic alteration that is responsible for chemoresistance in several cancer cells lines …”

Living Cell Microarrays: An Overview of Concepts

“…Thus, at the beginning of a drug development process, a huge number of drug candidates and their in-situ enzyme-generated metabolites can be screened, resulting in a more efficient development of drugs [ 43 , 44 , 45 ]. Effects on gene or protein expression can be investigated after the extraction of DNA, RNA, or proteins from the cells [ 46 , 47 ]. However, such a miniaturized format can represent a challenge for the isolation of sufficient amounts of analytes to be studied.…”

“…In addition, the variation in microgel composition provided control of the stiffness for an investigation of the influence of the microenvironment on cell survival and function. Another working group used functionalized PEG hydrogel-based cell microarrays for the cultivation and study of epithelial ovarian carcinoma cell aggregates [ 47 ]. Here, the combination of two 3D culturing approaches (termed spheroids in hydrogels) was developed to study cell aggregation and to screen anti-cancer therapeutics.…”