P04 How fibroblasts and azathioprine prepare the soil for cutaneous squamous cell carcinoma

Abstract

Abstract Introduction and aims DNA damage-inducing immunosuppressive drugs such as azathioprine (AZA) act synergistically with ultraviolet (UV)A radiation and photosensitize the skin of immunosuppressed individuals, such as organ transplant recipients. These individuals have an increased risk of developing cutaneous squamous cell carcinoma (cSCC). The specific combination of AZA and UVA causes oxidative DNA damage that leaves behind a characteristic mutational signature in tumour keratinocytes of patients treated with AZA. However, as UVA radiation can penetrate the dermis, the impact of AZA/UVA on cells of the tumour microenvironment, such as fibroblasts, also need to be addressed. Fibroblasts are crucial for cSCC development and progression as they can switch to a protumourigenic phenotype. Methods Cell viability and senescence assays were performed to investigate the impact of AZA in combination with or without UVA on neonatal (nonsun-exposed) and adult (sun-exposed) human dermal fibroblasts. In addition, the impact of AZA/UVA-treated fibroblasts on the clonal growth properties of different cSCC cell models was investigated using three-dimensional (co)culture models. Results We found that AZA and UVA synergistically reduce the viability of both UV-naïve neonatal and sun-exposed adult dermal fibroblasts. In addition, combined AZA and UVA treatments lead to an increased percentage of senescent fibroblasts. We hypothesize that the aberrant secretome of these senescent fibroblasts can impact the growth behaviour of tumour cells. In a soft agar assay, the more malignant cSCC cells (MET-1) formed more and larger clonal spheroids and were less dependent on growth factors compared with spheroids formed by premalignant (PM-1) keratinocytes. Conclusions AZA and UVA act synergistically and potentiate a UVA-induced senescent fibroblast phenotype. The changed characteristics of these fibroblasts can impact the stem cell-like properties from premalignant and malignant keratinocytes. Combining keratinocyte and fibroblast culture models enables us to study how AZA/UVA promote the initiation and progression of cSCC.