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

Abstract

Cutaneous squamous cell carcinoma (cSCC) accounts for the majority of non-melanoma skin cancer (NMSC) deaths, and its incidence continues to rise. The predominant risk factor for cSCC is solar radiation. Furthermore, evidence has shown that DNA damage-inducing pharmacological compounds, such as azathioprine (AZA), act synergistically with UVA radiation to photosensitise the skin of immunosuppressed individuals, including organ transplant recipients (OTRs). Although the effects of AZA and UVA on keratinocytes are known, their impact on the tumour microenvironment (TME) remains to be addressed. Fibroblasts are a major component of the TME that can switch from anti- to pro-tumourigenic phenotypes. Previous studies have shown that fibroblasts become senescent in response to UVA irradiation. Senescent cells resist apoptosis and secrete a plethora of pro-inflammatory factors that promote tumourigenesis. Thus, in this study, I hypothesised that AZA, or its analogue 6-thioguanine (6-TG), induces an enhanced UVA-induced senescent fibroblast phenotype, creating a pro-tumourigenic niche for cSCC. The study had three core objectives: 1) determine whether AZA/6-TG and UVA radiation induce fibroblast senescence; 2) investigate whether senescent fibroblasts promote cSCC growth; 3) assess how fibroblasts impact cSCC proliferation in the presence of inhibitors.

In this study, the phenotypic changes in fibroblasts following treatment with AZA/6-TG and UVA radiation were examined using senescence-associated-β-galactosidase (SA-β-gal) staining. The combination treatment resulted in a higher percentage of senescent fibroblasts compared with either treatment alone. To further validate fibroblast senescence, the secretome of these cells was analysed using a cytokine array, which showed that 6-TG/UVA-treated fibroblasts secrete higher levels of senescence-associated cytokines than controls. Additionally, bulk RNA-Sequencing (RNA-Seq) was performed to assess transcriptional changes in 6-TG/UVA-treated fibroblasts. The analysis demonstrated that treated fibroblasts overexpress genes implicated in senescence, fibroblast activation, and wound healing. The impact of senescent fibroblasts on cSCC cell growth was examined in 2D and 3D models, using both direct and indirect co-cultures. In 2D direct co-cultures, fibroblasts promoted cSCC cell growth and protected them from the anti-proliferative effects of the transcriptional enhanced associate domain (TEAD) inhibitor GNE-7883.

Together, these findings highlight that treatment with AZA/6-TG and UVA radiation not only impacts cancer cells but also affects the tumour stroma by inducing fibroblast senescence and providing a pro-tumourigenic niche for cSCC. Additionally, fibroblasts protect cSCC cell lines against the anti-proliferative effects of the TEAD inhibitor GNE-7883. Future work needs to further elucidate the signalling crosstalk between cancer and stromal cells and develop strategies to target senescent cells, as their persistence may contribute to disease recurrence, even after tumour excision.

Date of Award	25 Mar 2026
Original language	English
Awarding Institution	University of Bath
Supervisor	Ute Jungwirth (Supervisor), Gernot Walko (Supervisor), Robert Williams (Supervisor) & Charareh Pourzand (Supervisor)