Abstract
Purpose :
Establishment of hepatic metastatic uveal melanoma (mUM) involves a complex series of reciprocal interactions between tumor cells and its microenvironment resulting in a metastatic niche supporting tumor cell colonisation, survival and growth. These interactions include exploitation of resident hepatic cells, immune cell dysregulation and extracellular matrix (ECM) remodelling. While focal adhesion kinase (FAK) is activated in UM as a consequence of perturbed Gαq signalling pathways, FAK activation also occurs as a consequence of ECM engagement, contributing to liver fibrosis via activation of hepatic stellate cells (HSC), suggesting a potential role in mUM. Here we investigate the role of FAK using 3D mono- and co-culture spheroids, and in combination with FAKi.
Methods :
UM cell lines (92.1, MP41, MP46, Mel270) and HSC cell line (LX2) were cultured alone/in combination in ultralow attachment plates to form 3D spheroids. At day 4, spheroids were dosed with increasing concentrations (1nM-100µM) of FAKi (AMP-945, Amplia Therapeutics Ltd). After 72 hours, spheroids were imaged, assessed for cell viability using 3D CellTiter GloTM and Western blotting detection of phosphoY397- and total- FAK, and α-smooth muscle actin (αSMA).
Results :
3D spheroids show increased Y397-FAK phosphorylation compared with 2D monolayer cells for 92.1, MP41 and MP46 which carry chr8q gain. TCGA PTK2 mRNA expression confirms PTK2 amplification above the median is associated with ≥4 copies chr8q. Co-culturing these cell lines with LX2 cells showed a 3D arrangement of an inner core of UM cells surrounded by LX2, which is absent in Mel270 (no chr8q gain), and representative of nodular mUM. αSMA expression increased in the presence of LX2 cells. FAKi caused morphological changes in spheroid appearance and reduced cell viability at concentrations ≥3µM, whilst Y397-FAK phosphorylation was decreased at sub-IC50 concentrations compared with untreated controls; total FAK remained constant. Co-culture spheroids showed decreased FAKi potency suggesting interaction of the two cell types affects drug diffusion and efficacy.
Conclusions :
FAK activation occurs in 3D spheroids of UM cell lines with chr8q gain and favours a cellular arrangement representative of nodular mUM. FAKi showed limited potency on cell viability, despite decreases in Y397-FAK phosphorylation. Further exploration of the effects of FAKi in these models is required.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.