Cancer Stem cells (CSCs) are a subpopulation with the properties of extensive self-renewal, capability to generate differentiated cancer cells and resistance to therapies. We have previously shown that Malignant Pleural Effusions (MPEs) from patients with non -small cell lung cancer (NSCLC) represent a valuable source of cancer cells that can be grown as 3D spheroids enriched of stem-like features, which depend on the activation of the YAP-TAZ/Wnt- catenin/SCD1 axis. Here, we describe a novel support, called CytoMatrix, for the characterization of limited amounts of cancer cells isolated from MPEs of patients with NSCLC. Our results show that this synthetic matrix allowsan easy and fast characterization of several epithelial cellular markers. The use of Cyto Matrix to study CSCs sub-populations confirms thatSCD1 protein expression is enhanced in 3D spheroids as compared with 2D adherent cell cultures. YAP/TAZ nuclear-cytoplasmic distribution analyzed by Cyto Matrix in 3D spheroids is highly heterogeneous and faithfully reproduces what observed in tumor biopsies. Our results confirm and extend the robustness of our workflow for the isolation and phenotypic characterization of primary cancer cells derived from lung MPEs and underscore the role of SCD1.