Integrating Transcriptomics and 3D Spheroid Models Reveals Microenvironment-Dependent Purinergic Modulation in Hepatocellular Carcinoma

Background/Objectives: Dysregulation of purinergic signaling influences tumor progression and immune evasion in hepatocellular carcinoma (HCC). We aimed to characterize the transcriptional landscape of this system, identify prognostic markers, and investigate how the tumor microenvironment modulates pharmacological response to combined sorafenib and doxazosin in 3D spheroid models. Methods: We integrated RNA-seq data from the TCGA-LIHC to identify differentially expressed genes, pathway enrichment, gene co-expression networks, prognostic associations, and machine learning-based biomarker selection. Modulation of key targets was assessed in HepG2 and HepG2/LX-2 spheroids treated with sorafenib and doxazosin using qPCR and flow cytometry. Results: Transcriptomics revealed dysregulation and network fragmentation, where high expression of ADA, NT5E, and ADORA1 correlated with poor overall survival. In 3D models, co-treatment significantly downregulated NT5E and ADORA1 mRNA expression, while ADORA2A was specifically reduced in the co-culture setting. For ADA, effect size analysis revealed a large magnitude of inhibition in HepG2 spheroids. Although flow cytometry showed high CD73 protein expression remained stable across treatments in co-culture, the combination therapy successfully overcame stromal protection, significantly increasing apoptosis (active caspase-3) in both mono- and co-culture spheroids compared to vehicle and monotherapy. Conclusions: We identified a purinergic prognostic signature in HCC and demonstrated that the combination therapy of sorafenib and doxazosin targets the adenosine-generating axis and specific receptors. We show that the stromal microenvironment sustains CD73 protein expression even under transcriptional inhibition, highlighting the critical role of 3D co-culture models in deciphering therapeutic resistance mechanisms.