Generation of a CRISPR/Cas9 EO771-tumor platform to study breast cancer cell – immune system interactions

Paula Aguirre*1, Marcos Palavecino*2, Lilian Castillo*3, Roberto Meiss4, Santiago Rodriguez-Seguí2, Omar Coso2, Eva Wertheimer5, Edith Kordon2, Marina Simian3, Andrea Errasti6, Albana Gattelli2, Manuel De la Mata2, Eugenio Carrera-Silva7, Juan Pablo Fededa8 *Equal contribution

The main immunosuppressive mechanism by which cancer avoids eradication by the immune system is the expression of PD-L1, the ligand for T-cell inhibitory receptor PD-1. Despite their success in the treatment of different types of solid tumors, PD-1/PD-L1 blockade therapies have been ineffective in triple negative breast cancer (TNBC). Thus, it remains unclear which is the role of tumor PD-L1 and other immune checkpoint ligands in TNBC immune evasion. To address this, we developed a CRISPR/Cas9 expressing EO771 cell line as a platform to genetically study tumor-immune system interactions. As a first step, we confirmed the TNBC behavior of the EO771 model and characterized the immune response associated with the progression of EO771 tumor growth using flow cytometry. We found that EO771 tumor progression is hormone-independent and correlates with an increase in M2 macrophage polarization, a decrease in MHCII+ Antigen Presenting Cells (APCs), a marked increase in the CD4+/CD8+ ratio and CD4+ T cell exhaustion, which are consistent with tumor-mediated immunosuppression associated with poor prognosis in TNBC patients. In preliminary experiments, PD-L1 KO EO771 tumors show a significant but partial reduction in tumor growth rates, consistent with tumor PD-L1 being not sufficient to suppress anti-tumor immunity. By screening this platform, we will be able to massively test tumoral PD-L1 synthetic interactions to identify candidate genetic targets to overcome PD-1/PD-L1 resistance in TNBC.