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. Overall design: 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.

非小细胞肺癌（Non-small cell Lung Cancer, NSCLC）主要分为肺鳞状细胞癌（Lung Squamous Cell Carcinoma, LUSC）与肺腺癌（Lung Adenocarcinoma, LUAD）两大亚型。肺腺癌中驱动突变较为常见，而肺鳞状细胞癌几乎不存在可靶向治疗的驱动突变。尽管二者存在上述显著差异，p53与Pten这两种肿瘤抑制基因的突变在两种亚型中均较为普遍。虽然Sox2被广泛认为是肺鳞状细胞癌的原癌基因，但目前学界对p53、Pten与Sox2之间的调控关系仍知之甚少。
为探究上述肿瘤抑制基因在Sox2介导的非小细胞肺癌模型中，对疾病进展及肿瘤微环境（Tumor Microenvironment, TME）的调控作用，我们在C57BL/6J-Sox2hi、Nkx2-1-/-、Lkb1-/-（以下简称SNL）遗传背景小鼠中敲除了Trp53或Pten基因。同时，我们构建了一株可在C57BL/6小鼠体内增殖的高转移性肺鳞状细胞癌细胞系（LN2A）：该细胞系源自高表达Sox2的小鼠模型，后续进一步通过CRISPR技术敲除了p53、Pten及Cdkn2a基因。
组织学与单细胞RNA测序分析证实，SNL小鼠会同时形成兼具肺腺癌与肺鳞状细胞癌组织病理学特征的混合肿瘤；而SNL-Trp53与SNL-Pten小鼠则仅形成肺腺癌，接种的LN2A肿瘤则保留了肺鳞状细胞癌的形态学特征。
与SNL小鼠相比，额外敲除p53或Pten可显著缩短小鼠生存周期、增加肿瘤负荷，并改变肿瘤黏蛋白的组成成分。随着肿瘤模型的转移潜能从最低（SNL模型）逐步升高至最高（LN2A模型），我们观察到肿瘤内细胞浸润程度逐渐加重，其中以招募而来的肿瘤相关炎性单核细胞（Tumor-associated Inflammatory Monocytes, TIMs）的富集最为显著。
我们在LN2A模型中鉴定出一个CD38+ TIMs亚簇，该亚簇显著富集经典与替代补体通路的活化特征。值得注意的是，补体因子B（Complement Factor B, Cfb）与肺鳞状细胞癌患者的不良预后密切相关，我们同时观察到，在Cfb-/-遗传背景下，LN2A模型小鼠的生存周期得到显著改善。
综上，本研究证实p53与Pten肿瘤抑制基因在Sox2介导的非小细胞肺癌进展、黏蛋白产生及免疫肿瘤微环境重塑过程中发挥协同调控作用。
整体实验设计：对4种基因工程改造的小鼠肿瘤细胞系开展单细胞RNA测序，分别为：C57BL/6J-Sox2hi、Nkx2-1-/-、Lkb1-/-（SNL）、SNL背景下经CRISPR敲除Trp53的细胞系（p53敲除组）、SNL背景下经CRISPR敲除Pten的细胞系（PTEN敲除组），以及高转移性肺鳞状细胞癌细胞系LN2A。