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. 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).

超进展疾病（Hyperprogressive disease, HPD）是一种新近被定义的肿瘤进展模式，特指接受以免疫检查点抑制剂（immune checkpoint inhibitors, ICIs，如抗PD1/PDL1抗体）为基础的免疫治疗方案的部分癌症患者所出现的疾病进程。出现超进展的患者表现为难以预测的肿瘤加速生长及临床状况恶化，可快速导致患者死亡。我们此前的研究表明，巨噬细胞在超进展疾病的发生发展中发挥关键作用。通过对非小细胞肺癌（non-small cell lung cancer, NSCLC）患者的肿瘤样本进行分析，我们观察到超进展患者的肿瘤微环境（tumor microenvironment, TME）中存在特定亚群的巨噬细胞浸润。这类巨噬细胞在接受免疫检查点抑制剂治疗前就已存在，因此我们推测，部分肿瘤细胞可诱导巨噬细胞获得“与超进展相关”的表型，这类巨噬细胞在与抗PD1抗体结合后可触发超进展疾病。由于癌细胞教育巨噬细胞的策略之一，是在肿瘤微环境中分泌具有免疫调节活性的分子，因此我们将骨髓来源巨噬细胞（bone marrow-derived macrophages, BMDMs）暴露于5种不同非小细胞肺癌细胞系所获得的条件培养基（conditioned medium, CMs）中培养24小时。我们发现，与其他实验组相比，暴露于NCI-H460和PC9细胞系条件培养基的巨噬细胞，其基因表达谱存在显著差异；已有研究显示，该两种细胞系在接受抗PD1抗体治疗后，可在免疫缺陷小鼠体内呈现类超进展生长表型。我们的研究结果提示，肿瘤细胞释放的可溶性因子可将巨噬细胞重编程为一种潜在可在与抗PD1抗体结合后触发超进展疾病的表型。本研究从暴露于NCI-H460（n=4）、PC-9（n=3）、A549（n=4）、NCI-H1299（n=4）、Calu-1（n=4）条件培养基的骨髓来源巨噬细胞中提取RNA。此外，我们还从经脂多糖+干扰素-γ（LPS+IFN-γ，诱导为M1型）、白细胞介素-4（IL-4，诱导为M2型）刺激的骨髓来源巨噬细胞（每组n=3）以及未处理的巨噬细胞（M0型，n=4）中分离得到RNA。