In vitro response of a lung cancer cell model to differentiation-promoting conditions

Cancer cells are characterised by dysregulation of homeostatic and developmental pathways, leading to uncontrolled proliferation and impaired differentiation. Experimental approaches seeking to promote cell differentiation could enhance the effects of currently used cytotoxic, anti-proliferative therapies and reduce cancer pathogenicity by depleting tumours from undifferentiated, treatment-resistant cancer stem cells (CSC), which are responsible for cancer relapse after therapeutic cycles. In recent years, differentiation-focused approaches promoting differentiation into post-mitotic cell types have shown promising results in the hematologic malignancy acute promyelocytic leukemia (APL) and in some solid cancers. In particular, differentiation of triple negative breast cancer cells into post-mitotic adipocytes in vitro and in mouse models was observed to reduce the tumour metastatic outgrowth. Building on these observations, the present study aimed to apply a similar approach to a model of non-small cell lung cancer (NSCLC), which, despite the availability of different therapeutic options, remains a global health concern due to its aggressiveness, therapy resistance and metastatic spreading. Here, the NSCLC cell line A549 was exposed to differentiation media containing pro-adipogenic (AM) or pro-osteogenic (OM) factors. The effects of pro-differentiation treatments were analysed in terms of proliferative potential, cell cycle profile, clonogenic ability and anchorage-independent growth, a feature of malignant cells. Lung cancer migration and adhesion properties were also assessed, alongside stemness markers, after exposure to pro-differentiation media. Treatment of A549 cells with AM triggered substantial morphological changes with the appearance of numerous vesicles, and significant inhibition of cell proliferation and migration, while increasing adhesion properties. In addition, exposure to the pro-differentiation treatment significantly decreased the A549 clonogenic potential and the number of colonies formed in soft agar. It also reduced the expression of stemness markers including Sox2, Nanog, and CD44, and increased the expression of surfactant protein C (Sftpc) and alkaline phosphatase (ALP), markers of differentiated alveolar type II cells. In parallel, OM treatment was observed to induce morphological changes, albeit to a lesser extent, and significant reduction in proliferation and expression of stemness markers. As for AM, OM had a pronounced effect on A549 cell migration ability and induced mineral deposition in treated samples. These in vitro results on the A459 cell model indicate a rapid decrease in pathogenic features of NSCLC cells upon exposure to differentiation medium, which suggests that pro-differentiation treatments may represent a valuable option to pursue for further in vitro and preclinical testing.