Three-dimensional heterotypic colorectal cancer spheroid models for evaluation of drug response

Abstract

Colorectal cancer (CRC) is a leading cause of death worldwide. Improved preclinical tumor models are needed to make treatment screening clinically relevant and address disease mortality. Advancements in 3D cell culture have enabled a greater recapitulation of the architecture and heterogeneity of the tumor microenvironment (TME). This has enhanced their pathophysiological relevance and enabled more accurate predictions of tumor progression and drug response in patients. An increasing number of 3D CRC spheroid models include cell populations such as cancer-associated fibroblasts (CAFs), endothelial cells (ECs), immune cells, and gut bacteria to better mimic the in vivo regulation of signaling pathways. Furthermore, cell heterogeneity within the 3D spheroid models enables the identification of new therapeutic targets to develop alternative treatments and test TME-target therapies. In this mini review, we present the advances in mimicking tumor heterogeneity in 3D CRC spheroid models by incorporating CAFs, ECs, immune cells, and gut bacteria. We introduce how, in these models, the diverse cells influence chemoresistance and tumor progression of the CRC spheroids. We also highlight important parameters evaluated during drug screening in the CRC heterocellular spheroids.

Keywords: cancer associated fibroblast (CAF); colorectal cancer; drug screening; endothelial cell; gut microbiota; heterotypic 3D model; spheroid; tumor associated macrophages (TAMs).

Figure 1

Scheme of the main components of the tumor microenvironment that could be recapitulated in heterotypic 3D CRC spheroid models for drug screening. Created with Biorender.com.

Figure 2

Selected images of representative heterotypic 3D CRC models from original figures of published scientific articles. (A) Brightfield and confocal images of intra-spheroid localization of CRC cells (HCT116 and SW620), fibroblast, and endothelial cells over time. Spheroids were formed with a 1:1 ratio of cancer cells (HCT116, SSW620) and normal human colon fibroblasts (CCD18co) with 5% of human immortalized ECs (ECRF24) and used to study drug sensitivity (45). Scale bar = 200 µm. (B) Brightfield images of monoculture and heterotypic HT29 spheroids treated with different concentrations of 5-FU for 48 h. Heterotypic spheroids consisted of HT29 spheroids co-cultured with (2 x 10⁵) activated MRC-5 fibroblasts (70). Scale bar = 500 µm. (C) Brightfield images of 3D CRC models cultured for 48 h and consisting of cancer cells (HT29) and CD19^-CD14^- peripheral blood mononuclear cells (PBMC) from healthy donors to study immunomodulatory antibodies (43). Scale bar = 600 µm. (D) Confocal images of bacteria-spheroid co-culture consisting of HT29 cancer cells and Fusobacterium nucleatum (labeled in pink), and HT29 only spheroid at 12, 24, and 36 h. Scale bar = 200 µm. (E) High magnification (63 x) confocal images of 3D CRC spheroids at 12 and 36 h with and without F. nucleatum (in pink) (71). Scale bar = 20 µm.