NCI-H460 gene expression analysis after in vivo treatment with anti-PD1 antibody

Immune checkpoint inhibitors (ICIs) targeting PD-1/PD-L1 axis have made a breakthrough in the treatment of non-small cell lung cancer (NSCLC). However, a paradoxical boost in tumor growth was reported in a fraction of NSCLC patients after ICIs administration: a novel pattern of cancer progression defined “hyperprogressive disease” (HPD). Because the mechanism of HPD onset has not completely elucidated yet, this study aimed at investigating the cellular and molecular bases of this clinical phenomenon. Pre-treatment histological samples obtained from NSCLC patients were evaluated by immunohistochemistry (IHC) for immune cell infiltrate. Tissue samples from all patients with HPD showed tumor-infiltration by M2-like macrophages. To validate these findings in preclinical models, we utilized athymic nude mice that, lacking T-cells that may cloud the results, represent a suitable model to evaluate the role of macrophages in determining HPD. Immunodeficient mice were injected with human NSCLC NCI-H460 cell line, treated with anti-PD-1 antibody and tumor growth was evaluated over time. Anti-PD-1 treated H460 tumor-bearing mice showed HPD-like tumor growth as well as an increase in macrophage infiltration compared to control group. Interestingly, in these in vivo models, HPD-like growth, triggered by anti-PD-1 treatment, was dependent of anti-PD-1 antibody Fc domain. These results suggest that macrophages are the major players in HPD development and that FcR engagement on macrophages may determine a functional reprogramming of these immune cells toward a more aggressive and pro-tumorigenic phenotype, eventually inducing HPD. NCI-H460 cells (2.5x106 cells/mouse) were injected subcutaneously (s.c.) on the mice right flank of 8- to 9-week-old female athymic nude mice. Mice were peritumorally (p.t.) treated with 200 ug of monoclonal antibody anti mouse PD-1 (clone RMP1-14) starting 3 days after tumor injection. Anti-PD-1 antibody was administered twice a week until the end of the experiment. Control group received saline. Tumor growth was monitored over time. At the end of the experiment, tumor-bearing mice were sacrificed and tumors were collected and immediately frozen in liquid nitrogen until RNA extraction.