Novel rectal cancer spheroid models reveal metabolic and transcriptional adaptations driving radiotherapy response.

Abstract

Rectal cancer (RC) incidence is rapidly increasing, with over 50% of cases diagnosed at an advanced stage. Neoadjuvant radiotherapy (RT) is a key treatment, however, 20 to 40% of patients harboring rectal tumors have partial or no response to RT. Progress in understanding radioresistance has been hindered, especially by the limited availability of relevant in vitro models. We developed two high-throughput 3D spheroid models of RC using SW837 and SW1463 cell lines, optimized for reproducibility. The models were evaluated for 3D dynamics, tumor-like features, cell viability and proliferation, and response to a short course RT regimen of 5 Gray during 5 consecutive days, further dissected by assessing ATP levels, cell cycle, gene expression and metabolic activity. Both models exhibited distinct structural and functional features. Upon irradiation, SW837 spheroids displayed a necrotic core and suppressed ATP levels, maintaining peripheral Ki-67 expression, while SW1463 spheroids showed increased cell death levels. Importantly, the models showed distinct sensitivity to RT, as confirmed by the higher radioresistance in SW837 spheroids. Metabolomic profiling revealed that when irradiated, SW837 spheroids held increased levels of glutamate and citrate, while SW1463 spheroids presented significant consumption of metabolites linked to amino-acid pathways. Transcriptomic analysis revealed up-regulation of immunosuppressive pathways and cell cycle arrest genes in SW837 spheroids post-RT. This was supported by an accumulation of cells at G₂M state, while the SW1463 TS model presented more cells accumulated at S-phase. The RC spheroid models herein investigated represent valuable tools for dissecting differential metabolic and transcriptional adaptations to RT and reveal potential targets for battling RC radioresistance. Additionally, the two models display distinct phenotypes and responses support their use in pre-clinical studies, including screening and mechanistic studies in RC.