Fibroblasts Attenuate Anti-Tumor Drug Efficacy in Tumor Cells via Paracrine Interactions with Tumor Cells in 3D Plexiform Neurofibroma Cultures

Plexiform neurofibromas (hereafter called pNF1) are often diagnosed in early childhood and occur in about 30% of neurofibromatosis type 1 (NF1) patients. pNF1 exhibits aggressive growth along a nerve in the body and has substantial potential for progression to malignant peripheral nerve sheath tumors that are rarely curable. There are two recently FDA-approved drugs, selumetinib and mirdametinib, for pNF1 patients who have symptomatic and inoperable plexiform neurofibromas; however, these treatments achieve only approximately 30% tumor shrinkage. Fibroblasts, the most abundant cell types within the pNF1 tumor microenvironment, are implicated in pNF1 growth and invasion; however, how fibroblasts affect a drug response of pNF1 remains poorly understood. In the present study, we focused on contributions of fibroblasts to the drug resistance in pNF1 via their secretome. We employed our established three-dimensional (3D) culture system incorporating human pNF1 tumor cells (<i>Nf1</i>^-/-) and primary fibroblasts (<i>Nf1</i>^+/-) grown in our patented microfluidic culture chips for monocultures and parallel cocultures in which 3D pNF1 structures and fibroblasts share their secretome without direct cell-to-cell contact. Three-dimensional pNF1 structures in 3D parallel cocultures with fibroblasts exhibited greater drug resistance than ones in monocultures. We found that pNF1 tumor cells showed increased P-glycoprotein expression when incubated with fibroblast-derived conditioned media or parallel cocultured with fibroblasts, compared to control conditions. Pharmacological inhibition of P-glycoprotein partially restored drug sensitivity. Additionally, fibroblasts showed higher resistance to selumetinib and mirdametinib than pNF1 tumor structures, likely due to elevated P-glycoprotein levels. This study is the first to define precise roles of fibroblasts in pNF1 drug resistance, emphasizing the potential of fibroblast-targeted therapies as a promising approach to improve pNF1 treatment outcomes.