Tumor suppressors in Sox2-mediated lung cancers promote distinct cell intrinsic and immunologic remodeling [scRNA-seq]

Non-small cell lung cancer (NSCLC) largely consists of lung squamous (LUSC) and lung adenocarcinoma (LUAD). While driver mutations are common in LUAD, LUSC has few if any actionable targetable drivers. Despite these distinct differences, alterations in the p53 and Pten tumor suppressors are common in both subtypes. While Sox2 is widely considered an LUSC proto-oncogene, the relationship of p53 and Pten with Sox2 is poorly understood. To evaluate the role of these tumor suppressors in Sox2-mediated NSCLC models on disease progression and the tumor microenvironment (TME), we deleted Trp53 or Pten in a C57BL/6J-Sox2hi, Nkx2-1-/-, Lkb1-/- (SNL) genetic background. In parallel, we generated a highly metastatic LUSC cell line (LN2A; derived from a Sox2high mouse model, followed by p53, Pten, and Cdkn2a deletion) that also grows in C57BL/6 mice. Histologic and single cell RNAseq analyses corroborated that SNL mice developed mixed tumors with both LUAD and LUSC histopathology while SNL-Trp53 and SNL-Pten mice developed LUAD and implanted LN2A tumors retained LUSC morphology. Compared with SNL mice, additional loss of p53 or Pten resulted in significantly reduced survival, increased tumor burden and altered tumor mucin composition. As the metastatic potential in the tumor models progressed from the least (SNL) to most (LN2A), we observed a progressive increase in cellular infiltrate, most notably high levels of recruited tumor-associated inflammatory monocytes (TIMs). We identified a sub-cluster of CD38+ TIMs in the LN2A model that significantly enriched for activation of the classical and alternative complement pathways. Importantly, Complement Factor B (Cfb) is associated with poor survival in LUSC patients, and we observed the LN2A model had significantly improved survival on a Cfb-/- background. Taken together, our findings demonstrate a cooperative role of p53 and Pten tumor suppressors in Sox2-mediated NSCLC tumor progression, mucin production, and remodeling of the immune TME. Single-cell RNA-Sequencing of tumors induced by four genetically engineered murine cell lines, consisting of: C57BL/6J-Sox2hi; Nkx2-1-/-; Lkb1-/- (SNL), Trp53 CRISPR deletion line in the SNL background (p53), Pten CRISPR deletion line in the SNL background (PTEN), and a highly metastatic LUSC line, LN2A.