High-throughput non-homogenous 3D polycaprolactone scaffold for cancer cell and cancer-associated fibroblast mini-tumors to evaluate drug treatment response

High-throughput screening (HTS) three-dimensional (3D) tumor models are a promising approach for cancer drug discovery, as they more accurately replicate <i>in vivo</i> cell behavior than two-dimensional (2D) models. However, assessing and comparing current 3D models for drug efficacy remains essential, given the significant influence of cellular conditions on treatment response. To develop <i>in vivo</i> mimicking 3D models, we evaluated two HTS 3D models established in 96-well plates with 3D polycaprolactone (PCL) scaffolds fabricated using two distinct methods, resulting in scaffolds with either homogenous or non-homogenous fiber networks. These models, based on human HeLa cervical cancer cells and cancer-associated fibroblasts (CAFs) cultured as mono- or co-cultures within the 3D scaffolds, revealed that anticancer drug paclitaxel (PTX) exhibited consistently higher inhibitory concentration 50 (IC₅₀) in 3D (≥ 1000 nM) compared to 2D (≥ 100 nM), indicating reduced toxicity on cells cultured in 3D. Interestingly, the toxicity of PTX was significantly lower on mini-tumors in non-homogenous 3D (IC₅₀: 600 or 1000 nM) than in homogenous 3D cultures (IC₅₀ exceeding 1000 nM). Microscopic studies revealed that the non-homogenous scaffolds closely resemble the tumor collagen network than their homogeneous counterpart. Both 3D scaffolds offer optimal pore size, facilitating efficient cell infiltration into the depth of 58.1 ± 1.2 µm (homogenous) and 86.4 ± 9.8 µm (non-homogenous) within 3D cultures. Cells cultured in the 3D non-homogenous systems exhibited drug treatment responses closer to <i>in vivo</i> conditions, highlighting the role of scaffold structure and design on cellular response to drug treatment. The PCL-based 3D models provide a robust, tunable, and efficient approach for the HTS of anti-cancer drugs compared to conventional 2D systems.