Raman spectral analyses to investigate the physiological and metabolic development of a 3D hepatocellular carcinoma model

Three-dimensional (3D) cell culture models are increasingly attracting interest as powerful tools to recapitulate the in vivo tumor microenvironment, offering a more physiologically relevant alternative to traditional two-dimensional (2D) cultures. In this work, a 3D hepatocellular carcinoma (HCC) model using the HepG2 cell line was recreated and characterized over time. Gene expression analyses revealed changes in markers of proliferation (PCNA, Ki-67), differentiation (AFP), hypoxia (HIF-1α) and apoptotic regulators (BBC3), associated to the key adaptive phases of tumor development. Results exhibit a marked cells accumulation in the G0/G1 phase, indicative of a transition to quiescence. Raman analysis assessed biochemical composition and cellular response over time, allowing a non-invasive monitoring of metabolic states by detecting specific molecular vibrations. A clear correlation between the Raman spectral changes and the key genes involved in proliferation (PCNA, KI-67), differentiation (AFP), hypoxia (HIF-1α and apoptosis (BBC3) were found, so providing insights into the physiological evolution of 3D HepG2 spheroids. Thus, Raman approach could be a valuable tool to follow in real-time tumor adaptation and microenvironmental stress responses.