Transcriptomic profiling of Sox11 wildtype and mutant intestinal organoids upon treatment with TGF-ß1

Colorectal cancer (CRC) cells infiltrating surrounding tissue commonly undergo partial epithelial-mesenchymal transitions (pEMT) and employ a collective mode of invasion. How these phenotypic traits are regulated and possibly interconnected remains underexplored. Here, we used intestinal organoids with CRC driver mutations as model system to investigate the mechanistic basis of TGF-ß1-induced pEMT and collective invasion. By scRNA-seq we identified multiple cell subpopulations representing a broad pEMT spectrum, where the most advanced pEMT state correlated with the transcriptional profiles of leader cells in collective invasion and a poor prognosis mesenchymal subtype in human CRC. Bioinformatic analyses pinpointed Sox11 as a transcription factor whose expression peaked in the potential leader/pEMThigh cells. Immunofluorescence staining confirmed Sox11 expression in cells at the invasive front of TGF-ß1-treated organoids. Loss-of-function and overexpression experiments showed that Sox11 is necessary, albeit not sufficient, for TGF-ß1-induced pEMT and collective invasion. In human CRC samples, elevated Sox11 expression was associated with advanced tumor stages and worse prognosis. Unexpectedly, aside from orchestrating the organoid response to TGF-ß1, Sox11 controlled expression of genes related to normal gut function and tumor suppression. Apparently, Sox11 is embedded in several, distinct gene regulatory circuits, contributing to intestinal tissue homeostasis, tumor suppression, and TGF-ß-mediated cancer cell invasion. Overall design: Sox11 wildtype and knockout small intestinal organoids (lines 931 and 1308; Apc-/-, KrasG12D/+, Trp53R172H/+ )were treated with solvent or TGF-ß1 for 72 h, followed by bulk RNA-seq analysis.

结直肠癌（Colorectal cancer, CRC）细胞浸润周围组织时，通常会发生部分上皮间质转化（partial epithelial-mesenchymal transitions, pEMT）并采取集体侵袭模式。目前，关于这些表型特征的调控机制及其潜在关联仍有待深入探索。本研究以携带CRC驱动突变的肠道类器官为模型体系，探究转化生长因子β1（transforming growth factor-β1, TGF-β1）诱导的pEMT与集体侵袭的分子机制。通过单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），我们鉴定出涵盖宽泛pEMT谱系的多个细胞亚群；其中，最成熟的pEMT状态与集体侵袭中领头细胞的转录谱以及人类CRC中预后不良的间质亚型显著相关。生物信息学分析将Sox11确定为一个关键转录因子，其表达在潜在领头细胞/pEMT高表达细胞中达到峰值。免疫荧光染色实验证实，在经TGF-β1处理的类器官侵袭前沿的细胞中，可检测到Sox11的表达。功能缺失与过表达实验结果表明，尽管Sox11不足以单独诱导该过程，但它是TGF-β1诱导的pEMT与集体侵袭所必需的。在人类CRC样本中，Sox11的高表达与肿瘤晚期分期及不良预后显著相关。出乎意料的是，除了介导类器官对TGF-β1的应答之外，Sox11还调控与正常肠道功能及肿瘤抑制相关的基因表达。由此可见，Sox11嵌入多个不同的基因调控环路，参与维持肠道组织稳态、肿瘤抑制以及TGF-β介导的癌细胞侵袭过程。总体实验设计：将Sox11野生型与敲除型小肠类器官（细胞系931与1308；基因型为Apc-/-、KrasG12D/+、Trp53R172H/+）分别用溶剂或TGF-β1处理72小时，随后进行批量RNA测序（bulk RNA-seq）分析。