Lipid Metabolic Remodeling in Heterotypic Colorectal Cancer Spheroids
MOREIRA I. 1, SOARES V. 1,2, WERNECK M. 1, DIAS S. 2, MASCARENHAS G. 1, CAMPOS M. 1, OLIVEIRA D. 1, ALVES M. 2, SOUZA T. 2, BASTOS N. 3, VIOLA J. 3, BOZZA P. 1,2
1 Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil; 2 Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil; 3 Instituto Nacional do Câncer (INCA), Rio de Janeiro, Brazil
Background: Colorectal cancer (CRC) remains a major global health burden, ranking among the most frequently diagnosed malignancies and leading causes of cancer-related mortality worldwide. Its high incidence, substantial mortality, and persistent resistance to current therapies underscore the urgent need to identify novel biological mechanisms that can be therapeutically exploited. Metabolic reprogramming is a defining hallmark of CRC and a critical driver of tumor growth and progression. Among altered metabolic pathways, lipid metabolism has emerged as a central regulator of tumor cell proliferation by supplying energy, membrane components, and bioactive signaling molecules essential for malignant behavior. Within the tumor microenvironment (TME), metabolic stressors such as hypoxia and nutrient limitation profoundly reshape lipid uptake, synthesis, and oxidation. Despite its recognized importance, the regulation of lipid metabolism within the three-dimensional tumor architecture remains incompletely understood.
Objective: This study aimed to investigate the temporal relationship between spheroid maturation, hypoxia establishment, and lipid metabolic remodeling using a heterotypic CRC spheroid model.
Methods: Heterotypic CRC spheroids were generated using a total of 6,250 cells composed of human HCT116 colorectal carcinoma cells and BJ5Ta fibroblasts at a 4:1 ratio (HCT116:fibroblasts). Spheroid growth kinetics, hypoxia development, and lipid metabolic alterations were evaluated over time.
Results: Spheroid growth was monitored from day 2 to day 10, revealing a sustained and progressive increase in spheroid size. From day 5 onward, the establishment of a hypoxic center was observed, indicating the formation of physiologically relevant intratumoral gradients. Lipid metabolic profiling revealed a gradual and time-dependent accumulation of intracellular lipid droplets between days 4, 6, and 10, consistent with enhanced lipid storage during spheroid maturation. Notably, advanced spheroids exhibited increased expression of the proliferation marker Ki-67 in the peripheral region, closely associated with elevated levels of the lipid droplet-associated protein PLIN2.
Conclusion: These findings demonstrate that spheroid maturation is accompanied by coordinated changes in growth dynamics, hypoxia, and lipid metabolic remodeling in a heterotypic HCT116 colorectal cancer model. This study highlights the relevance of three-dimensional heterotypic spheroids as a robust and biologically meaningful platform for investigating lipid metabolism in CRC and supports lipid metabolic pathways as promising targets for biomarker discovery and therapeutic intervention in colorectal cancer.