Context-Specific Metabolic Alterations in HPRT1 Knockout Cells Within a 3D Culture System

Cancer cells reprogram their metabolism to sustain energy production and biosynthesis for malignant proliferation; however, their metabolic phenotypes vary significantly across different growth environments, creating discrepancies between in vitro and in vivo findings. These inconsistencies pose challenges for translating metabolic research into clinical applications. The emergence of 3D culture models as in vitro systems that accurately mimic the in vivo environment can mitigate these challenges by providing conditions that reflect physiological architecture and metabolic interactions. Therefore, we investigated the impact of the purine metabolism enzyme hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) on the proliferation and metabolism of SCLC cells using 2D and 3D culture models, with the goal of identifying context-specific metabolic regulation not captured in conventional 2D cultures.

We evaluated cell proliferation and performed metabolomic profiling of HPRT1-knockout (KO) SCLC cells grown in 2D cultures, two 3D culture systems, and mouse xenograft models. Metabolomic profiling was performed using CE-TOFMS, followed by PCA and pathway analysis. The expression of β-alanine (β-Ala) metabolism-related genes, including carnosine synthase 1 (CARNS1), was assessed by RNA-seq and RT-PCR. CARNS1 expression was further evaluated in publicly available lung cancer datasets, including both cell line and clinical tumor cohorts, to determine its association with patient prognosis and its correlation with HPRT1 expression.

The knockout of HPRT1 significantly reduced cell proliferation in 3D cultures and in vivo but had a minimal impact in 2D cultures. Comprehensive metabolomic analyses of HPRT1-KO cells revealed extensive alterations in amino acid and purine metabolism both in vitro and in vivo. Notably, the effect of HPRT1 KO on β-Ala metabolism differed between 2D and 3D cultures. In 3D cultures, HPRT1 KO led to increased expression of the endogenous antitumor metabolite carnosine and its biosynthetic enzyme CARNS1 within the β-Ala metabolic pathway. Furthermore, analysis of clinical databases showed that high CARNS1 expression correlated with improved prognosis in patients with lung cancer and negatively correlated with HPRT1 expression in tumor tissues.

This study highlights the potential of 3D culture systems to elucidate context-specific mechanisms of metabolic regulation, such as the suppressive effect of HPRT1 on carnosine production. Our findings demonstrate that metabolic phenotypes observed in 2D cultures may not fully capture the complexity of in vivo metabolism, whereas 3D models can reveal regulatory pathways that are otherwise overlooked, including context-dependent regulation of carnosine metabolism by HPRT1.