骨肉腫の腫瘍内不均一性を模倣したオンチップモデルによる腫瘍関連マクロファージのリプログラミングと免疫療法の検討

The osteosarcoma tumor microenvironment (OS-TME) exhibits pronounced cellular and biophysical heterogeneity, arising from infiltrating immune cells, primarily tumor-associated macrophages (TAMs) and mechanical stress gradients, respectively. TAMs significantly contribute to OS progression through various mechanisms; hence, targeting TAMs could improve treatment outcome in OS patients. This study presents a novel immunocompetent tri-culture osteosarcoma (iTC-OS) model developed using a porous 3D silk fibroin blend-hydroxyapatite (SF-HA) scaffold seeded with human OS cells, human blood derived TAMs, and human umbilical vein endothelial cells (HUVECs). The physiological relevance of the iTC-OS model is further enhanced by integrating into a physiomimetic microfluidic bioreactor (iTC-OS-on-a-chip), featuring dynamic perfusion to simulate intra-tumoral mechanical stress gradient, validated through computational fluid dynamic (CFD). Additionally, we employed pexidartinib and tenalisib to evaluate TAMs reversal in the iTC-OS-on-a-chip model by selectively inhibiting CSF1R and PI3Kγ, respectively. TAMs reprogramming from tumor promoting M2 to tumor suppressing M1 phenotype is confirmed through gene expression analysis of M1 (CCR7, IL-1β, IL-6) and M2 (CD206, CD163, IL-10) macrophage markers, alongside quantification of secreted cytokines via ELISA assay. This advanced iTC-OS-on-a-chip model offers a robust platform for investigating OS-immune cell interactions, enabling pre-clinical evaluation of chemo/immunotherapeutics and improving the translational relevance in OS research.