Abstract
Simple SummaryThree-dimensional (3D) culture of tumor spheroids (TSs) within the extracellular matrix (ECM) recapitulates solid tumors in vivo. This microtumor model is particularly useful for multiplex phenotypic analysis, but requires tissue optical clearing (TOC) for 3D visualization. We developed a transfer-free 3D microtumor culture-to-3D visualization system using a minipillar array chip combined with the TOC method. Our method succeeded in improving immunostaining and optical transmission in each TS as well as the entire microtumor specimen. The utility of this method was demonstrated by showing phenotypic changes, such as increased levels of membrane protrusion, single-cell dissemination, and ECM remodeling, and changes in the expression of epithelial–mesenchymal transition–related proteins and drug-induced apoptosis in TSs of human pancreatic cancer cells co-cultured with cancer-associated fibroblasts and M2-type tumor-associated macrophages.Three-dimensional (3D) culture of tumor spheroids (TSs) within the extracellular matrix (ECM) represents a microtumor model that recapitulates human solid tumors in vivo, and is useful for 3D multiplex phenotypic analysis. However, the low efficiency of 3D culture and limited 3D visualization of microtumor specimens impose technical hurdles for the evaluation of TS-based phenotypic analysis. Here, we report a 3D microtumor culture-to-3D visualization system using a minipillar array chip combined with a tissue optical clearing (TOC) method for high-content phenotypic analysis of microtumors. To prove the utility of this method, phenotypic changes in TSs of human pancreatic cancer cells were determined by co-culture with cancer-associated fibroblasts and M2-type tumor-associated macrophages. Significant improvement was achieved in immunostaining and optical transmission in each TS as well as the entire microtumor specimen, enabling optimization in image-based analysis of the morphology, structural organization, and protein expression in cancer cells and the ECM. Changes in the invasive phenotype, including cellular morphology and expression of epithelial–mesenchymal transition-related proteins and drug-induced apoptosis under stromal cell co-culture were also successfully analyzed. Overall, our study demonstrates that a minipillar array chip combined with TOC offers a novel system for 3D culture-to-3D visualization of microtumors to facilitate high-content phenotypic analysis.
Highlights
The three-dimensional (3D) culture of cancer cells provides an in vitro tumor model that best recapitulates the in vivo tumor pathophysiology [1,2]
We developed a protocol for the on-chip imaging of intact 3D microtumor specimens following in situ immunocytochemical staining using a pillar array platform, which allowed for 3D culture to 3D visualization of microtumors in a transfer-free manner
A 23% decrease was observed in the fluorescence intensity at a depth of 150 μm in the cleared samples, suggesting that partial light scattering remained in the deeper region of the microtumor. These results suggested that tissue optical clearing (TOC) was successfully adapted for 3D imaging of the extracellular matrix (ECM) of microtumors (z = 450 μm) grown on minipillar array chips (Figure 4C)
Summary
The three-dimensional (3D) culture of cancer cells provides an in vitro tumor model that best recapitulates the in vivo tumor pathophysiology [1,2]. The troublesome and time-consuming steps of these preparation procedures often cause the potential loss of sections, which may result in imprecise spatial reconstruction, leading to partial or incomplete sample analysis and incompatibility with high-throughput screening (HTS). To overcome these issues, immunocytochemical staining of the whole 3D sample with subsequent whole-mount 3D imaging can be considered as a better alternative. The limited diffusion of staining macromolecules in TSs and the light scattering of microtumors up to millimeter-scale specimens impose challenges in imaging and visualization of 3D cultures due to preventing the effective labeling of the inner layer of cells and intracellular proteins of interest [10,11]
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