Abstract 2528: Determining gene expression variability between in vitro and in vivo cancer models: Monolayer, spheroids, and mouse allografts
Authors: Hum Nicholas R. , He Wei , Sebastian Aimy , Wheeler Elizabeth K. , Coleman Matthew A. , Loots Gabriela G. 
Published: August 2018

Abstract
An ideal pre-clinical environment that recapitulates in vivo growth conditions ex vivo is an essential pre-requisite for effective drug screening. Conventional monolayer culturing of cancer cells as a pre-clinical model have repeatedly failed to recapitulate responses seen clinically. While 3D culturing methods are able to generate some level of tumor heterogeneity including hypoxic core, cell-cell interactions, gradients of drug penetration, as well as cancer stem cell differentiation, these culturing methods fail to incorporate the complex heterogenous cell composition and transient fluxes in nutrients or drugs.
To investigate the effects of culturing conditions on gene expression of cancer cells, RNA sequencing was performed on a mouse mammary carcinoma (4T1) cell line grown in a variety of culture conditions: 2D (monolayer) or 3D (spheroid). Additionally, gene expression analysis was performed on tumors derived from 4T1 cells injected subcutaneously (SQ) into the murine flank or orthotopically (OT) into the mammary fat pad of BALB/c mice.
Pairwise analysis of RNA sequencing data identified 235 down- and 1029 up- regulated genes differentially expressed between the 2D and 3D culture methods. Differential expression identified genes involved in cell migration, extracellular matrix organization, cell adhesion, angiogenesis, hypoxic response, cell differentiation, as well as key cancer related pathways including TNF, Jak-STAT, and PI3K-AKt primarily upregulated in 3D culture. Similar differential expression was found for genes encoding extracellular matrix proteins, which may be reflective of the 3D environment. Both in vivo allograft models produced highly similar gene expression profiles with only 31 up- and 20 down- regulated genes differentially expressed between OT and SQ tumors. Down-regulated genes were enriched in transcriptional regulatory gene networks and up-regulated genes were enriched for signaling/secreted proteins. The gene expression profiles of in vivo tumors were significantly different when compared to the 2D (973 down regulated, 1971 upregulated genes); and 99 of these transcripts were only expressed in in vivo tumors, highlighting the increased heterogeneity of cell composition found in allografts.
3D culturing of cancer cells upregulates pathways known to be critical to tumor progression including genes known to be essential for adhesion, differentiation, and ECM remodeling however the gene expression profile of spheroids is significantly different than that of cancer cells expanded in vivo. Future culturing methods incorporating immune cells, cancer supporting cells such as fibroblasts and other stromal components are more likely to improve the phenotype of 3D cultured tumor cells.
This study received funding from LLNL LDRD grant 17-ERD-121. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344).
Citation Format: Nicholas R. Hum, Wei He, Aimy Sebastian, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Determining gene expression variability between in vitro and in vivo cancer models: Monolayer, spheroids, and mouse allografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2528.