Gas-Phase Fractionation Data-Independent Acquisition Analysis of 3D Cocultured Spheroid Tumor Model Reveals Altered Translational Processes and Signaling Using Proteomics.

Colorectal cancer (CRC) contains considerable heterogeneity; therefore, models of the disease must also reflect the multifarious components. Compared to traditional 2D models, 3D cellular models, such as tumor spheroids, have the utility to determine the drug efficacy of potential therapeutics. Monoculture spheroids are well-known to recapitulate gene expression, cell signaling, and pathophysiological gradients of avascularized tumors. However, they fail to mimic the stromal cell influence present in CRC, which is known to perturb drug efficacy and is associated with metastatic, late-stage colorectal cancer. This study seeks to develop a cocultured spheroid model using carcinoma and noncancerous fibroblast cells. We characterized the proteomic profile of cocultured spheroids in comparison to monocultured spheroids using data-independent acquisition with gas-phase fractionation. Specifically, we determined that proteomic differences related to translation and mTOR signaling are significantly increased in cocultured spheroids compared to monocultured spheroids. Proteins related to fibroblast function, such as exocytosis of coated vesicles and secretion of growth factors, were significantly differentially expressed in the cocultured spheroids. Finally, we compared the proteomic profiles of both the monocultured and cocultured spheroids against a publicly available data set derived from solid CRC tumors. We found that the proteome of the cocultured spheroids more closely resembles that of the patient samples, indicating their potential as tumor mimics.

Co-culturing multicellular tumor models: Modeling the tumor microenvironment and analysis techniques.

Advances in two-dimensional (2D) and three-dimensional (3D) cell culture over the last 10 years have led to the development of a plethora of methods for cultivating tumor models. More recently, cellular co-cultures have become a suitable testbed. The first portion of this review focuses on co-culturing methods that have been developed in recent years utilizing the multicellular tumor spheroid model. The latter portion describes techniques that are used to analyze the proteomes of mono- or co-cultured tumor models, with a focus on mass spectrometry (MS)-based analyses. Protein profiles are important indicators of the tumor heterogeneity. Therefore, there is a specific focus within this review on analysis by MS and MS imaging methods evaluating the proteomic profiles of 2D and 3D co-cultures. While these models are incredibly important for biological research, so far, they have not been widely explored on the proteomic level. With this review, we aim to introduce these systems to an analytical audience, with the goal of highlighting MS as an underutilized tool for proteomic analysis of tumor models.