Comparative Molecular Analysis of Cancer Behavior Cultured In Vitro, In Vivo, and Ex Vivo

Area of Science:

• Oncology
• Cancer Biology
• Biotechnology

Background:

• Pre-clinical cancer models often fail to accurately represent primary tumor cell behavior.
• The tumor microenvironment significantly influences cancer cell phenotype, but its effects are not fully understood.
• Existing in vitro models do not fully capture the complexity of in vivo tumor dynamics.

Purpose of the Study:

• To compare the transcriptional profiles and cellular heterogeneity of cancer cells across various pre-clinical models.
• To evaluate the suitability of 2D cultures, 3D cultures, allografts, and ex vivo tumoroids as cancer models.
• To identify key differences in gene expression and cell state transitions, particularly epithelial-to-mesenchymal transition (EMT), in different models.

Main Methods:

• Transcriptomic profiling (RNA-seq) of 4T1 murine mammary carcinoma cells.
• Comparison of cells from 2D cultures, 3D cultures, subcutaneous and orthotopic allografts (in immunocompetent and immunodeficient mice), and ex vivo tumoroids.
• Single-cell RNA sequencing (scRNA-seq) to assess cellular heterogeneity.

Main Results:

• 3D culture platforms exhibited transcriptional profiles more similar to in vivo tumors than 2D cultures.
• In vivo tumors showed more cells undergoing epithelial-to-mesenchymal transition (EMT), while in vitro cultures had cells in distinct epithelial or mesenchymal states.
• Ex vivo tumoroids retained EMT heterogeneity but shifted cancer cells towards a mesenchymal state, while losing immune cell components.

Conclusions:

• Improving in vitro cancer models is crucial for better recapitulating in vivo tumor characteristics.
• 3D cultures and ex vivo tumoroids offer more physiologically relevant models than traditional 2D cultures.
• Future models should aim to preserve EMT heterogeneity and include stromal subpopulations for greater accuracy.