Glucose and Oxygen Availability Shape Growth and Architecture of Tumor Spheroids in a Microfluidic 3D Model

Glucose and oxygen availability are fundamental drivers of tumor progression, deeply influencing cancer cell proliferation, metabolism, and spatial organization. In this study, we present a simplified microfluidic-based 3D model of tumorigenesis that allows for controlled and dynamic analysis of key tumor parameters under metabolic stress. Using this platform, lung adenocarcinoma (A549) spheroids embedded in a collagen-based extracellular matrix were monitored over time to assess growth kinetics, hypoxic response via HIF-1α expression, and structural organization. A multifactorial experimental design combining variations in glucose, glutamine, and oxygen availability revealed distinct and specific effects of each nutrient. Glutamine deprivation led to widespread cell death, while glucose limitation strongly arrests cell proliferation. Finally, oxygen restriction disrupted spheroid architecture, resulting in loss of the proliferative outer capsule and the formation of internal cavities. Our approach provides a flexible system to study how microenvironmental cues shape tumor behavior, offering insights into metabolic vulnerabilities and the morphofunctional consequences of nutrient stress in cancer.