Comparative molecular analysis of cancer behavior cultured in vitro, in vivo, and ex vivo

Current pre-clinical models of cancer fail to recapitulate the cancer cell
behavior in primary tumors primarily because of the lack of a deeper
understanding of the effects that the microenvironment has on cancer cell
phenotype. Transcriptomic profiling of 4T1 murine mammary carcinoma cells
from 2D and 3D cultures, subcutaneous or orthotopic allografts (from
immunocompetent or immunodeficient mice), as well as ex vivo tumoroids,
revealed differences in molecular signatures including altered expression
of genes involved in cell cycle progression, cell signaling and
extracellular matrix remodeling. The 3D culture platforms had more in
vivo-like transcriptional profiles than 2D cultures. In vivo tumors had
more cells undergoing epithelial-to-mesenchymal transition (EMT) while in
vitro cultures had cells residing primarily in an epithelial or
mesenchymal state. Ex vivo tumoroids incorporated aspects of in vivo and
in vitro culturing, retaining higher abundance of cells undergoing EMT
while shifting cancer cell fate towards a more mesenchymal state. Cellular
heterogeneity surveyed by scRNA-seq revealed that ex vivo tumoroids, while
rapidly expanding cancer and fibroblast populations, lose a significant
proportion of immune components. This study emphasizes the need to improve
in vitro culture systems and preserve syngeneic-like tumor composition by
maintaining similar EMT heterogeneity as well as inclusion of stromal
subpopulations.