Three Dimensional Mixed-Cell Spheroids Mimic Stroma-Mediated Chemoresistance and Invasive Migration in hepatocellular carcinoma

Abstract

Interactions between cancer cells and cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME) play an important role in promoting the profibrotic microenvironment and epithelial-mesenchymal transition (EMT), resulting in tumor progression and drug resistance in hepatocellular carcinoma (HCC). In the present study, we developed a mixed-cell spheroid model using Huh-7 HCC cells and LX-2 stellate cells to simulate the in vivo tumor environment with respect to tumor-CAF interactions. Spheroids were cultured from cancer cells alone (monospheroids) or as a mixture (mixed-cell spheroids) in ultra-low-attachment plates. Compact, well-mixed, and stroma-rich mixed-cell spheroids were successfully established with heterotypic cell-cell contacts shown by the presence of gap junctions and desmosomes. Mixed-cell spheroids showed enhanced expression of collagen type-I (Col‐I) and pro‐fibrotic factors such as, transforming growth factor beta1 (TGF-β1), and connective tissue growth factor (CTGF) compared to the levels expressed in mono-spheroids. The EMT phenotype was evident in mixed-cell spheroids as shown by the altered expression of E-cadherin and vimentin. Differential drug sensitivity was observed in mixed-cell spheroids, and only sorafenib and oxaliplatin showed dose-dependent antiproliferative effects. Simultaneous treatment with TGF-β inhibitors further improved sorafenib efficacy in the mixed-cell spheroids, indicating the involvement of TGF-β in the mechanism of sorafenib resistance. In 3D matrix invasion assay, mixed-cell spheroids exhibited fibroblast-led collective cell movement. Overall, our results provide evidence that mixed-cell spheroids formed with Huh-7 and LX-2 cells well represent HCC tumors and their TME in vivo and hence are useful in studying tumor-stroma interactions as mechanisms associated with drug resistance and increased cell motility.

A Spheroid Killing Assay by CAR T Cells

“…This method is so far only limited to a certain type of adherent cell line (in this publication HCT 116); further development is required to generalize this protocol to every cancerous cell line. Although a wide range of methods is already available for a greater number of target cells 12,13,14,15 , most of them rely on the use of expensive and/or complex procedures. Our method, although limited to a few types of target cells so far, is interesting as it requires very limited inputs from the experimenter and permits a fast and easy screen of various drugs and/or immunotherapy treatments (here CAR CD19 cells).…”

Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

“…Thus, 3D cell cultures, such as spheroids, have received great attention from the research community, as these in vitro models can represent more accurately the different properties of human tissues, such as liver (Yoon, Lee, Lee, & Lee, 2015), thyroid *Elisabete C. Costa and Daniel N. Silva contributed equally to this work. (Cirello et al, 2017), cartilage (Jukes et al, 2008), pancreatic tissue (Lumelsky et al, 2001), cardiac muscle (Kehat et al, 2001) or of solid tumors (e.g., breast, colon, pancreas, prostate, ovary, among others (Eiraku et al, 2008;Eiraku et al, 2011;Ham et al, 2018;Ham, Joshi, Luker, & Tavana, 2016;Hamilton, 1998;Khawar et al, 2018;Lazzari et al, 2018;Suga et al, 2011)). Spheroids are microtissues with a diameter within hundreds of micrometers to few millimeters that present a spatial architecture, cellular organization, cell-cell, and cell-extracellular matrix interactions quite similar to those found in the human tissues (as reviewed in detail elsewhere (Costa et al, 2016;Duval et al, 2017)).…”

Investigating TNS4 in the Colorectal Tumour Microenvironment Using 3D Spheroid Models of Invasion

“…The use of 3D in vitro models may fill in this gap by providing an enriched tumour microenvironment with improved cell interactions and ECM which better mimic the in vivo context and which may provide a better measure of drug response [14][15][16][17][18] . Addition of stromal cell types such as cancer-associated fibroblasts (CAFs) to 3D models substantially supports growth of cancer cell lines and may also promote invasion and metastasis by guiding cancer cells at the invasive edge [19] .…”