Evaluating Impact of Endothelial Cells on Drug Response of Lung Cancer Spheroids Using Microfluidic Chip

Drug resistance continues to be a major limitation in the clinical management of non-small cell lung cancer (NSCLC). While most of the existing research has focused on genetic mutations of tumor cells, increasing attention is being paid to the surrounding tumor microenvironment (TME) as a key factor in therapeutic response. In particular, the presence of stromal cells such as endothelial, fibroblast, and immune cells can alter cancer cell behavior by releasing soluble factors or through direct physical contact. However, the exact role in this context remains largely unclear. The objective of this work is to better understand and quantify how stromal cells, and in particular endothelial cells, impact drug resistance in NSCLC spheroids. To achieve this, we used both conventional in vitro systems and a microfluidic platform. The standard in vitro model developed in previous work was used for spheroid generation and as a baseline for comparative analysis. However, it is based on conventional culture format (such as polystirene 24-well plates) which limits control over cell positioning and interactions. Conversely, the microfluidic chip enables co-culture of multiple 2D and 3D cell types within confined microenvironments, which offers enhanced spatial control for drug response studies. In this work, NSCLC spheroids were either cultured either with endothelial cell supernatant or in direct contact (the so called tangled culture) with endothelial cells. This dual approach allowed us to investigate the impact of both paracrine and juxtacrine interactions. By comparing these two approaches, we aimed to determine whether endothelial-mediated drug resistance is primarily driven by secreted signaling molecules or by more complex cell-cell interactions. Fluorescence imaging and quantitative analysis were used to assess spheroid viability and size after treatment with targeted therapies. These data provide insights into how endothelial cells and their secreted factors may protect tumor cells from drug-induced cytotoxicity. Ultimately, this work aims to quantify the role of endothelial cells within the TME and compare their impact to other previously studied components.