Ubiquitin specific peptidase (USP37) mediated effects in microscaffold-encapsulated cells: a comprehensive study on growth, proliferation and EMT

Abstract

Though significant advances have been made in developing therapeutic strategies for cancer, suitable in vitro models for mechanistically identifying relevant drug targets and understanding disease progression are still lacking. Most studies are generally performed using two-dimensional (2D) models, since these models can be readily established and allow high throughput assays. However, these models have also been reported as the reason for unreliable pre-clinical information. To avoid this discrepancy, three-dimensional (3D) cell culture models have been established and have demonstrated the potential to provide alternative ways to study tissue behavior. However, most of these models first require optimization and cell cultures with a certain density, thus adding a prepping step in the platform before it can be used for any studies. This limits their use in studies where the fundamental understanding of biological processes must be carried out in a short time frame. In this study, we developed a 3D cell culture system that tests a less explored cancer therapeutic target-the deubiquitinating enzyme ubiquitin specific peptidase 37 (USP37)-in different cancer cell lines using sensitive carbon dot pH nanosensors, which provides a rapid model for studies compared to the parallel model available commercially. This enzyme is found to be elevated in different cancers and has been reported to play a role in cell cycle regulation, oncogenesis and metastasis. However, the confirmation of the role of USP37 downregulation in cellular proliferation via appropriate in vitro 3D models has not been demonstrated. To establish the applicability of the developed 3D platform in studying such oncogenes, classical 2D models have been used in this study for identifying the role of USP37 in tumor progression and metastasis. The data clearly suggests that this ingeniously developed 3D cell culture system is a better alternative to 2D models to study the growth and migration of different cancer cell lines on depletion of oncogenic proteins like USP37 and its effect on epithelial-mesenchymal transition (EMT) markers, and it can further be targeted as a viable therapeutic option.

Fig. 1. (A) Comparative expression analysis of USP37 in different cancers. The red boxplot indicates the tumor group. The grey boxplot indicates the normal group. Source: TCGA and GTEx data. (B) Survival analysis of patients with respect to USP37 expression in different gynecologic cancers (CESC and OV). (C) Comparative expression of USP37 in various cell lines. Relative mRNA expression of USP37 in ovarian cancer cell lines (IGROV-1 and PA-1) compared to the non-transformed cell line (MCF-10A). (D) Relative protein expression of USP37 in ovarian cancer cell lines (IGROV-1 and PA-1) compared to the non-transformed cell line (MCF-10A). (E) Graphical representation of western blot analysis between non-transformed (MCF10A) and ovarian cancer cell lines.

Fig. 2. (A) Representative image of colony formation capability after the knockdown of USP37 using siRNA in the PA-1 cell line. (B) Quantification of the number of colonies formed in the untreated control and cells with downregulated USP37. Brightfield microscopic image showing the scratch assay in untreated control cells and cells with the downregulated USP37 gene in (C) PA-1 and (D) IGROV-1 cell lines.

Fig. 3. (A and B) Fluorescence micrographs of the cells grown in tissue culture plates (2D) for USP37 (red), N-cadherin (green), E-cadherin (yellow) and DAPI (blue). The images were captured at 20× magnification and were pseudo coloured using ImageJ software (scale bar: 50 μm).

Fig. 4. (A) Cellular growth curves. The change in pH was plotted as a ratio of emission intensities at 450 nm (blue) and 550 nm (green) for various cell lines over five days. For comparison cell growth was also quantified using the Alamar Blue assay and plotted for various cell lines over 5 days. The growth curves for the ovarian cancer cell line PA I under two conditions, control and downregulated USP37, were compared using pH sensitive carbon dots and the Alamar Blue assay. (B) Fluorescent micrographs for the encapsulated PA I cells under two conditions, control and downregulated USP37, over a period of 5 days (scale bar for fluorescent micrographs: 100 μm).

Fig. 5. (A and B) Fluorescence micrographs of the cells encapsulated in the 3D microgels observed on day 1 and day 5 for USP37 (red), N-cadherin (green), E-cadherin (yellow) and DAPI (blue). The images were captured at 20× magnification and were pseudo coloured using the ImageJ software. (C) The fluorescence intensities were quantified for each image using the ImageJ software, normalized with respective DAPI and plotted using the GraphPad software. (D) Genetic expression of EMT markers (Snail, E-cadherin and N-cadherin) and USP37 in the ovarian cancer cell line (PA I) in both 2D and 3D platforms. The graphs plotted are an average of 2 biological replicates and 3 technical replicates. Scale bar for fluorescent micrographs: 100 μm.