Gastric cancer represents a poor prognosis disease. Indeed, the 5-year survival rate in Europe is 32%. The heterogeneity of the disease represents a major issue. Molecular profiling studies have revealed the existence of four main different subtypes highlighting different targets for personalized medicine. Currently, chemotherapy is the standard-of-care for this disease, but its high heterogeneity and the discovery of new targets for personalized medicine calls for an urgent need for patient-derived preclinical models aimed at the fast identification of individualized combination therapies to improve clinical outcomes and avoid undesired side effects. The present project is aimed at generating a platform to test drug combinations active in gastric cancer. It is based on the setting-up of Gastric Cancer derived Organotypic Cultures (GC-OC) that retain the main characteristic of the primary tumour.

We took advantage of two syngeneic mice models of gastric cancer, YTN3 and YTN16 that differ in drug sensitivity. To generate GC-OC, tumors were subjected to mild dissociation followed by serial filtration. The obtained organotypic spheroids (40-100µm) were then seeded in a miniaturized microfluidic device to generate 3D-collagene embedded cultures in an appropriate medium. Flow cytometry and immunofluorescence analysis were performed to analyze GC-OC cell composition. Cell viability was evaluated by Acridine Orange (AO)/ Propidium Iodide (PI) staining and confocal imaging of 3D cultures. Cell number was monitored by staining of the nucleus via Hoechst.

We generated GC-OC as spheroids that keep neoplastic and stromal cells in their original matrisome, maintaining their spatial and functional organization. Immunofluorescence analysis of cell composition confirms the presence of the main components of the tumor microenvironment (e.g. cancer cells, immune cells, endothelial cells and fibroblasts) in the generated GC-OC. Immune cell composition was further investigated in the tumor and spleen of the mouse models leading to the identification of G-MDSC (Granulocyte-Myeloid Derived Suppressor Cell) as the main immune subpopulation that was retained by the GC-OC. Drug effects on cancer cell viability were also assessed, revealing differences in drug sensitivity with respect to the classical 2D culture.

Altogether, the data obtained in our lab has allowed the setting-up of a 3D cell culture system that is expected to result in the generation of tumor in-vitro-twins, a phenocopy of the patient tumor and its microenvironment. These in-vitro-twins could be exploited for therapy identification of effective personalized drug combinations in the clinical setting. Moreover, the eventual application of omics strategies will help to define the features of the tumors and its microenvironment representing novel drug vulnerabilities.