The Cell Transformation Assay: A Historical Assessment of Current Knowledge of Applications in an Integrated Approach to Testing and Assessment for Non-Genotoxic Carcinogens

Abstract

The history of the development of the cell transformation assays (CTAs) is described, providing an overview of in vitro cell transformation from its origin to the new transcriptomic-based CTAs. Application of this knowledge is utilized to address how the different types of CTAs, variously addressing initiation and promotion, can be included on a mechanistic basis within the integrated approach to testing and assessment (IATA) for non-genotoxic carcinogens. Building upon assay assessments targeting the key events in the IATA, we identify how the different CTA models can appropriately fit, following preceding steps in the IATA. The preceding steps are the prescreening transcriptomic approaches, and assessment within the earlier key events of inflammation, immune disruption, mitotic signaling and cell injury. The CTA models address the later key events of (sustained) proliferation and change in morphology leading to tumor formation. The complementary key biomarkers with respect to the precursor key events and respective CTAs are mapped, providing a structured mechanistic approach to represent the complexity of the (non-genotoxic) carcinogenesis process, and specifically their capacity to identify non-genotoxic carcinogenic chemicals in a human relevant IATA.

Keywords: carcinogenesis; chemical-induced transformation; enrichment analysis; in vitro oncotransformation; mechanistic understanding; rodent cancer bioassay; rodent cell transformation assay; transcriptomics; transformics chemical-induced transformation; tumor microenvironment.

Figure 1

Milestones in the development of rodent cell transformation assay.

Figure 2

Family tree to show the origin of established 3T3 mouse embryonic fibroblasts.

Figure 3

Schematic simplified representation of the key molecular changes required for acquiring a fully malignant phenotype in human cancer, and in vitro oncotransformation.

Figure 4

Schematic representation of steps leading to human serrated adenocarcinoma, and SHE cells oncotransformation.

Figure 5

Priority biomarkers identified in transcriptomic prescreening approaches in the NGTxC IATA, biomarkers in the cell transformation assays.

Figure 6

Conceptual overview of the adaptive versus maladaptive critical data gaps for adverse outcome recognition in NGTxC, and how they can now be overcome using the CTAs. From adaptive to maladaptive disease progression: key data gaps in the testing and assessment of non-genotoxic carcinogenicity (updated from [3,6], with authors/copyright holders permission). There are numerous in vitro assays to address the early key events from receptor binding and transactivation, gene transcription, metabolism and cell proliferation (indicated by the green circle on the left of the figure) [3]. The CTAs will be able to address the key data gap for cell transformation, both for early (initiation) and later (promotion) phases (broken red line ellipse). A change in morphology represents the point at which adaptive (sustained) proliferation and hyperplasia/dysplasia become maladaptive, the CTAs are the crucial tests to ensure an evidence based in vitro translation from the in vivo hyperplasia to tumor formation (solid red lined ellipse). This tipping point is histopathologically characterized with cellular and/or structural atypia, and this change is often observed as abnormal nuclear division and disorganized cell proliferation with loss of cell polarity, which the CTAs now show that they can address.