Abstract
Purpose :
Uveal melanoma (UM) is a rare aggressive intraocular tumor that accounts for 13% of all melanoma deaths. Although the primary tumor is usually effectively treated, it metastasises in ~50% of patients, most commonly to the liver. Metastatic UM (mUM) has limited treatment options and overall survival is short. Treatment of hepatic mUM remains a significant challenge due to diffuse dissemination of UM cells in the liver, and a lack of effective systemic treatments, in most affected patients. Our understanding of the hepatic tumor microenvironment (TME) in mUM is limited. Morphological studies suggest that the mUM-TME, particularly the peritumoral fibrosis and extracellular matrix (ECM), promote mUM survival, progression and therapy resistance. The ECM is comprised of structural proteins, proteoglycans and glycoproteins that provide cellular scaffolds and molecular cues modulating cellular function. The aim of this study was to develop methods for tissue decellularisation allowing proteomic characterisation of UM-ECM.
Methods :
Eight samples of primary UM tissue were analysed: n=4 monosomy 3 samples, where the patients died of mUM (HR) and n=4 disomy 3 samples, all patients alive >7 years following diagnosis (LR). The samples underwent decellularisation over 72-hours using hypotonic (10nM Tris-HCl, pH 8, 5mM EDTA) and hyper-tonic (50mM Tris-HCl, 0.5M NaCl, pH 8, 10mM EDTA) (HH) solutions. Decellularised tissue was analysed histologically, and for residual DNA content using DAPI staining of tissue sections, compared with native non-decellularised tissue. Decellularised UM were subsequently analysed by mass spectrometry and immunohistochemistry.
Results :
HH solutions for 72-hours effectively removed cellular material with tissue architecture retention. Mass spectrometry of decellularised UM detected 488 proteins across all samples (1% FDR) identified by at least 3 unique peptides. 44 of these proteins are associated with the ECM according to the matrisome database (The Matrisome Project (mit.edu)) – 29 core matrisome proteins and 15 matrisome-associated. 33/44 (75%) are upregulated in HR-UM, including ECM glycoproteins associated with embryogenesis: nidogen, collagen VI, and agrin, as well as laminin and fibronectin.
Conclusions :
Future studies will expand on these preliminary results to better understand ECM remodelling in hepatic mUM and how this may impact tumour dormancy and metastatic outgrowth.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.