1:Oncoimmunology Research Unit, Navarrabiomed-Fundación Miguel Servet, Universidad Pública de Navarra (UPNA), Complejo Hospitalario de Navarra (CHN), Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, 31008, Spain.; 2:Division of Gene Therapy and Regulation of Gene Expression, Cima Universidad de Navarra and Instituto de Investigación Sanitaria de Navarra (IdISNA), Pamplona,31008, Spain; 3:Medical Oncology Unit, Complejo Hospitalario de Navarra (CHN), Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, 31008, Spain.; 4:Proteomic Research Unit, Navarrabiomed-Fundación Miguel Servet, Universidad Pública de Navarra (UPNA), Complejo Hospitalario de Navarra (CHN), Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona,31008, Spain.

Therapies based on PD1/PD-L1 blockade have shown remarkable clinical outcomes and durable responses, but are not efficient in a significant number of patients. One approach to increase the potency of immunotherapy is to modulate the tumor microenvironment (TME) by reprogramming myeloid cells to stimulate anti-cancer activity, since these cells constitute the major TME component. For this purpose, it is important to identify molecular signatures associated to cancer-promoting myeloid cells. In this work we defined the phenotype and proteome of tumor associated myeloid cells generated ex vivo using a lung cancer model (3LL). We identified differences in proteomic signatures between monocytic myeloid-derived suppressor cells (M-MDSC), granulocytic-MDSC, and tumor-associated macrophages (TAMs) related to lineage and cancer-driven polarization. The proteomic atlas of tumor-associated cells revealed important routes that could be altered to reprogram cancer-associated myeloid cells. Based on these data we evaluated the effects of several compounds at the differentiation, maturation, and immunosuppressive levels in MDSCs and TAMs. Furthermore, we confirmed the effects of selected compounds in myeloid cells from non-small cell lung cancer (NSCLC) patients in vitro. Finally, to assess this therapy in vivo, we evaluated the combination of two selected compounds, administered intraperitoneally, with immunotherapy based on a Semliki Forest virus (SFV) vector expressing an anti-PD-1 antibody locally in 3LL tumors in mice. This combination strategy showed potent antitumor effects, higher than the ones obtained with individual agents. Moreover, we observed similar effects when combining the most effective compound with systemic delivery of anti-PD-1 mAb.