Gene expression profiling of murine SCLC tumor subpopulations with high and low autophagic flux

Small cell lung cancer (SCLC) is characterized by significant heterogeneity and plasticity, driving its aggressive progression and resistance to therapy. Understanding the underlying mechanisms of these features is crucial for improving treatment outcomes. Autophagy, a conserved cellular process, is involved in many cancers, but its role in SCLC remains unclear. Using a genetically engineered mouse model (Rb1fl/fl;Trp53fl/fl;GFP-LC3-RFP-LC3△G), we tracked autophagic flux in vivo to assess its effects on SCLC biology. Tumor subpopulations with high autophagic flux exhibited increased proliferation, enhanced metastatic potential, and neuroendocrine (NE) characteristics, whereas subpopulations with low autophagic flux exhibited more immune-related signals and non-NE traits. GFP-LC3-RFP-LC3ΔG-knockin mouse model was developed to facilitate in vivo monitoring of autophagic flux. This model was crossed with Rb1fl/fl; Trp53fl/fl (RP) mice to establish an animal model exhibiting molecular and histopathological features similar to human SCLC. Lung tumor tissues from the resulting RPΔG mice were harvested, digested into single cells, and red blood cells were removed. Cellular subpopulations were isolated using Fluorescence-Activated Cell Sorting (FACS). First, cellular debris was excluded, and DAPI-negative live cells were identified. From these live cells, EpCAM-positive and RFP-positive cells were sorted. Subsequently, GFP-positive and GFP-negative cells were separated from the RFP-positive population for downstream RNA sequencing analysis. Notably, the GFP-negative subpopulation represents cells with high autophagic flux, while the GFP-positive subpopulation represents cells with low autophagic flux.