Study of innate immune responses during small cell lung cancer (SCLC) development

Abstract

SCLC is a highly aggressive neuroendocrine tumor with dismal prognosis. Inability to detect SCLC early renders surgical resections a rare event, limiting investigation of the early stages of tumor development. We sought to address this challenge using a genetically engineered mouse model (GEMM) to investigate how the innate immune system responds to SCLC development. The RPM (Rb1fl/fl, p53fl/fl, MycLSL/LSL) SCLC GEMM was selected to characterize the local immune microenvironment throughout the process of tumor development. Following tumor induction using an intranasally installed Cre bearing adenovirus, lung tissue was collected and analyzed using multi-parametric flow cytometry to assess changes in innate immune cell populations. Induction of SCLC resulted in late stage disease being present in animals approximately 7 weeks after virus exposure. Flow cytometric analysis of immune cell populations of the whole lung showed no significant differences in the overall levels of immune cell types, although an upwards trend in the levels of neutrophils in tumor-bearing mice was observed. The most significant finding was a decrease in the expression levels of major histocompatibility complex class II (MHCII) in both alveolar and interstitial macrophage populations. Further characterization, including separate analysis of the two interstitial macrophage subpopulations described by Chakarov et al. (Science 2019), and Schyns et al. (Nature Communications 2019), showed significant drops in levels of MHCII in all populations as measured by median fluorescent intensity (MFI) (p=0.004 in alveolar macrophages, p=0.0148 in vascular supporting CD206+ve interstitial macrophages, and p=0.0027 in CD206-ve interstitial macrophages). The striking difference between the changes seen in these populations was that alveolar and CD206+ve interstitial macrophages both showed a downward shift in MHCII expression as a whole, while CD206-ve macrophages acquired a novel population completely negative for MHCII accounting for approx. half of the CD206-ve macrophage subpopulation. The development of SCLC has been found to result in all macrophage subtypes expressing lower levels of MHCII, pointing towards a more immunosuppressive immune environment. The population of CD206-ve MHCII-ve interstitial macrophages is of particular interest, as it is contrary to the currently described interstitial macrophage phenotypes. A more in-depth characterization of all macrophage populations in the mouse lung, including their precise phenotype and localization is currently underway using CyTOF and IHC approaches. The precise timings of the observed changes are being elucidated through analysis of a disease time-course ranging from very early stage to late stage disease, in order to better understand the process of SCLC development.