Scaffold-based　three-dimensional　(3D)　cell　culture　systems　have　gained　increased　interest　in　cell　biology,　tissue　engineering,　and　drug　screening　fields　as　a　replacement　of　two-dimensional　(2D)　monolayer　cell　culture　and　as　a　way　to　provide　biomimetic　extracellular　matrix　environments.　In　this　study,　microscale　fibrous　scaffolds　were　fabricated　via　electrohydrodynamic　printing,　and　nanoscale　features　were　created　on　the　fiber　surface　by　simply　leaching　gliadin　of　poly(-caprolactone)　(PCL)/gliadin　composites　in　ethanol　solution.　The　microstructure　of　the　printed　scaffolds　could　be　precisely　controlled　by　printing　parameters,　and　the　surface　nanotopography　of　the　printed　fiber　could　be　tuned　by　varying　the　PCL/gliadin　ratios.　By　seeding　mouse　embryonic　fibroblast　(NIH/3T3)　cells　and　human　nonsmall　cell　lung　cancer　(A549)　cells　on　the　printed　scaffolds,　the　cellular　responses　showed　that　the　fiber　nanotopography　on　printed　scaffolds　efficiently　favored　cell　adhesion,　migration,　proliferation,　and　tissue　formation.　Quantitative　analysis　of　the　transcript　expression　levels　of　A549　cells　seeded　on　nanoporous　scaffolds　further　revealed　the　upregulation　of　integrin-β1,　focal　adhesion　kinase,　Ki-67,　E-cadherin,　and　epithelial　growth　factor　receptors　over　what　was　observed　in　the　cells　grown　on　the　pure　PCL　scaffold.　Furthermore,　a　significant　difference　was　found　in　the　relevant　biomarker　expression　on　the　developed　scaffolds　compared　with　that　in　the　monolayer　culture,　demonstrating　the　potential　of　cancer　cell-seeded　scaffolds　as　3D　in　vitro　tumor　models　for　cancer　research　and　drug　screening.　©　2021　American　Chemical　Society.