Abstract
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.
Highlights
Cancer has a major impact on society and poses a significant financial burden globally [1].The American Cancer Society predicts that ~1.7 million new cases of cancer will be diagnosed this year and ~20% of all deaths will be cancer associated
In vitro methodologies profiled included a conventional 2D 4T1 monolayer culture on polystyrene tissue culture-treated flasks (Figure 1C), spheroids cultured in non-adherent well plates in culture media for 7 days following a 4-day initial spheroid formation (3DM) (Figure 1D) or spheroids encapsulated in a gelatin-fibrin hydrogel (3DG) (Figure 1E) for 7 days post-spheroid formation
Conventional transcriptomic analysis of whole tumor bulk RNA analysis can lead to erroneous interpretations of tumor biology; we found that only 39% of transcripts significantly changed in cancer cells purified from orthotopic immune-competent tumors (SBM) overlapped with genes significantly changed when whole tumor bulk RNA was performed (Figure 2E)
Summary
Cancer has a major impact on society and poses a significant financial burden globally [1]. The American Cancer Society predicts that ~1.7 million new cases of cancer will be diagnosed this year and ~20% of all deaths will be cancer associated. While the overall cancer death rate has dropped steadily since 1991, this decline is mostly due to changes in lifestyle (smoking cessation) and early detection (breast and colorectal). Cancer remains a challenging health burden as the second leading cause of death, in both men and women, with death rates continuing to rise for liver, pancreatic, endometrial, and brain cancers [2]. Cancers 2020, 12, 690 development of more effective therapies and improving survival outcomes for numerous cancer types [3]; advances have remained slow for patients at later stages of the disease or those with highly aggressive cancer subtypes [4]. Most significantly, >95% of new therapies that exhibit superior performance in animal models fail in the clinic due to therapeutic inefficacy or unwarranted toxicity [5]
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