Introduction: immunotherapy is a type of treatment that harnesses the power of the immune system by modulating the immune response and improving its ability to recognize and destroy cancer cells. In clinical practice, there are different types of immunotherapies used to treat cancer, including immune checkpoint inhibitors (ICIs). ICIs work by blocking proteins in cancer cells that prevent the immune system from recognizing and attacking them. ICIs immunotherapy could be combined with oncolytic virotherapy to enhance the overall therapeutic effect. Methods: in our study, we generated multicellular spheroids models of murine lung adenocarcinoma using 70 000 cells/spheroids. We selected the multicellular spheroids model for its ability to mimic the in vivo tridimensional structure. We then infected the multicellular spheroids with 5 000 multiplicities of infection (MOI) of two oncolytic adenoviruses, Ad5D24 and Ad5D24-anti-PD-L1-scFv. To mimic the immune system in vitro, we isolated naïve splenocytes from C57BL/6J mice spleen and performed a co-culture assay. Results: Infection of the murine lung adenocarcinoma spheroids with either oncolytic adenovirus induced a reduction of the area; the highest area reduction was obtained with a combination between Ad5D24-Anti-PD-L1-scFv and splenocytes. Conclusions: In line with the results obtained, we claim that the lung adenocarcinoma cell line is a suitable model to generate multicellular spheroids, contributing to standardize 3D culture models, and although the in vivo environment is more complex than in vitro, the 3D model could represent a good compromise.