Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by high expression of extracellular matrix in tumor tissue, which contributes to chemoresistance and poor prognosis. Here, we developed 3D pancreatic cancer spheroids, based on pancreatic cancer cells and fibroblast co-culture, which demonstrate innate desmoplastic properties and stay poorly permeable for model nanoparticles. Our study revealed that establishment of tumors by transplantation of spheroids significantly improved subcutaneous xenograft model of PDAC, which stays the most widely used animal model for testing of new drugs and drug delivery approaches. Spheroid based tumors abundantly produced different extracellular matrix (ECM) components including collagen I, fibronectin, laminin and hyaluronic acid. These tumors were highly reproducible with excellent uniformity in terms of ECM architecture recapitulating clinical PDAC tumors, whereas in more common cell based xenografts a significant intertumor heterogeneity in extracellular matrix production was found. Moreover, spheroid based xenografts demonstrated higher expression of pro-fibrotic and pro-survival PDAC hallmarks in opposite to cell based counterparts. We believe that future development of this model will provide an effective instrument for testing of anti-cancer drugs with improved predictive value.