Effect of Non-small cell lung cancer (NSCLC) cell line conditioned medium (CM) on bone marrow-derived macrophages (BMDMs). Effect of Non-small cell lung cancer (NSCLC) cell line conditioned medium (CM) on bone marrow-derived macrophages (BMDMs)

Hyperprogressive disease (HPD) is a newly described pattern of tumor progression characterizing a percentage of cancer patients receiving an immunotherapeutic regimen based on immune checkpoint inhibitors (ICIs), such as anti-PD1/PDL1 antibodies. Patients experiencing HPD are characterized by an unpredictable acceleration of tumor growth and a deterioration of the clinical conditions, rapidly leading to death. We have previously reported that macrophages play a pivotal role in contributing to HPD development. By analyzing non-small cell lung cancer (NSCLC) patient tumor samples, we observed that the tumor microenvironment (TME) of HPD-patients was infiltrated by particular clusters of macrophages. These macrophages are present before the initiation of ICI therapy and, therefore, we hypothesized that certain tumor cells may induce the acquisition of an “HPD-related” phenotype in macrophages that upon interaction with anti-PD1 antibody can unleash HPD. Since one of the strategies exploited by cancer cells to educate macrophage is to secrete molecules with immunomodulatory activity in the TME, bone marrow-derived macrophages (BMDMs) were exposed for 24 hours to the conditioned medium (CMs) obtained from five different NSCLC cell lines. We found that macrophages exposed to the CMs obtained from NCI-H460 and PC9 cell lines, reported to undergo a HPD-like growth in immunocompromised mice after anti-PD1 antibody treatment, have a gene expression profile completely different compared to the other experimental groups. Our findings suggest that soluble factors released by tumor cells can reprogram macrophages towards a phenotype potentially able to trigger HPD after interaction with anti-PD1 antibody. Overall design: RNA was extracted from bone marrow-derived macrophages (BMDMs) after 24 hours exposure to NCI-H460 (n=4), PC-9 (n=3), A549 (n=4), NCI-H1299 (n=4), Calu-1 (n=4) conditioned medium (CMs). Moreover, RNA was also isolated from LPS+IFN- (M1) and IL-4 (M2) stimulated BMDMs (n=3 for each experimental condition) and untreated macrophages (M0, n=4).