Osteosarcoma-on-a-chip model mimicking intra-tumoral heterogeneity to interrogate tumor-associated macrophage reprogramming for immunotherapeutics.

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.