BACKGROUND: Drug resistance is a major challenge in the treatment of lung cancer. Increasing evidence indicates that the tumor microenvironment (TME), including cancer-associated fibroblasts (CAFs) and tumor endothelial cells (TECs), plays a significant role in determining the tumor sensitivity to therapeutic agents. There is, however, a dearth of models, two-dimensional (2D) or three-dimensional (3D), that represent the in vivo interaction of tumor and stromal components in the TME. The purpose of this study was to use conditionally reprogrammed lung cancer cells (CRLCs), and then use sodium alginate (Alg) and hyaluronic acid (HA) as a hydrogel matrix to establish an in vitro 3D model for co-culture of CRLCs, CAFs, and human umbilical vein endothelial cells (HUVECs). The application of this model in the sensitivity testing of anti-tumor drugs and the mechanism of drug resistance were studied, providing new means and ideas for individualized precise anti-tumor treatment and the development of new anti-tumor drugs. METHODS: We established direct 3D micro-beads of primary lung cancer using conditional reprogramming (CR) technology and co-cultured them with CAFs and HUVECs to evaluate the effect of the TME compartment on tumor sensitivity to chemotherapeutic agents and tyrosine kinase inhibitors (TKIs). RNA sequencing (RNA-seq) was performed on the 3D micro-beads in tissue, mono-culture, and co-culture conditions to uncover transcriptional changes induced by tumor-stroma interaction. RESULTS: The storage modulus of 3D hydrogel microbeads was shown to be 12 kPa, which is similar to that of lung tumor tissue and demonstrates good biocompatibility, making it suitable for constructing in vitro tumor models. RNA-seq data indicated that the co-culture of CAFs and HUVECs can upregulate the pathways related to extracellular matrix (ECM) remodeling, cell adhesion molecules, ECM-receptor interactions, cancer pathways, and the PI3K-Akt signaling pathway. Moreover, the results also showed that after co-culturing CRLCs with CAFs and HUVECs, the cytotoxicity induced by chemotherapeutic agents (cisplatin, paclitaxel, vinorelbine, and gemcitabine) as well as TKIs (gefitinib, afatinib) was reduced. Furthermore, protein expression analysis confirmed that cells seeded on 3D-3 co-culture models significantly overexpressed most of the stemness promoters tested compared to monoculture, including ALDH1A1, NANOG, and SOX9. CONCLUSIONS: These findings suggest that the patient-derived in vitro 3D-3 co-culture model, which highlighted the close association between tumor cell resistance and the TME, offers innovative ideas and methods for addressing treatment resistance in lung cancer patients. By closely mirroring human lung tumors, this model not only enhances our understanding of the disease but also paves the way for the development of more effective and personalized therapeutic strategies.