Trajectories of partial and full EMT of mammary tumor cells in the MMTV-PyMT mouse model. Trajectories of partial and full EMT of mammary tumor cells in the MMTV-PyMT mouse model

Epithelial-mesenchymal transition (EMT) is a multistep process of cell de-differentiation which confers carcinoma cells with a variety of malignant characteristics. Due to the transient and reversible nature of the process, cancer cells may transit between various stages of an EMT continuum, including epithelial, partial EMT or full EMT, and the actual status of cancer cells, the kinetics of cellular state transition in vivo and the consequences thereof have remained elusive. We have established tamoxifen inducible dual recombinase lineage tracing systems combined with live cell imaging and 5-cell RNA-sequencing analysis to track cancer cells undergoing a partial or a full EMT in the MMTV-PyMT mouse model of metastatic breast cancer. In primary tumours, cancer cells infrequently undergo an EMT, and most of these cells transition between epithelial and partial EMT states, but rarely undergo a full EMT. Consequently, cells that have ever undergone a partial EMT dynamically transit between various EMT states, and they contribute to lung metastasis and chemoresistance. In contrast, full EMT cells mostly retain a mesenchymal phenotype and stably reside in perivascular regions, but fail to colonize the lungs. However, full EMT cancer cells are enriched in recurrent tumors upon chemotherapy. Our findings suggest that cancer cells in various stages of the EMT continuum contribute differentially to hallmarks of breast cancer malignancy, such as cell migration, invasion, metastasis and chemoresistance. Overall design: Partial and full EMT mammary tumor cells were sorted by flow cytometry from tumors of Tnc-CreERT2;MMTV-Flpo;RC::FrePe;MMTV-PyMT (Tnc-CreERT2) and Cdh2-CreERT2;MMTV-Flpo;RC::FrePe;/MMTV-PyMT (Cdh2-CreERT2) mice. The dual recombinase lineage tracing system allows to label all breast cancer cells as mCherry expressing cells (red). Upon tamoxifen administration cancer cells that have undergone either partial or full EMT switch to the expression of GFP (green). This red to green color switching is irreversible, which allows to trace all cancer cells that have ever undergone an EMT by the expression of GFP, even if they have reverted back to an epithelial state by undergoing a MET. In consequence, mCherry+ cells are breast cancer cells, GFP+ cells are breast cancer cells that have ever undergone either partial or full EMT and double positive cells are cells undergoing an EMT (transtioning cells). Based on the expression of GFP or mCherry in combination with the epithelial marker EpCAM expression, 3 populations of mCherry+ cells (P5, P17 and P18), 4 populations of GFP+ cells (P15, P10, P16 and P9) and 1 population of doble positive cells (P22) were collected from Tnc-CreERT2 mice, and 3 populations of mCherry+ cells (P5, P17 and P18) and 1 population of GFP+ cells (P15) were collected from Cdh2-CreERT2 mice. mCherry populations, P05: mCherry+ EpCAM-, P17: mCherry+ EpCAMhigh, P18: mCherryhigh EpCAMhigh. GFP populations, P15:GFP+ EpCAM-, P10: GFP+ EpCAMlow, P16: GFP+ EpCAMmed and P18: GFPhigh EpCAMmed. Double positive cells: P22: mCherry+ GFP+ EpCAMhigh

上皮间质转化（Epithelial-mesenchymal transition, EMT）是一类多步骤的细胞去分化过程，可赋予癌细胞多种恶性特征。由于该过程具有瞬时性与可逆性，癌细胞可在上皮间质转化连续体的不同阶段间动态转变，涵盖上皮型、部分上皮间质转化（partial EMT）与完全上皮间质转化（full EMT）。然而，癌细胞的实际状态、体内细胞状态转变的动力学过程及其后续效应，长期以来尚不明确。

本研究构建了他莫昔芬诱导型双重组酶谱系示踪系统，结合活细胞成像与5细胞RNA测序分析，在转移性乳腺癌的MMTV-PyMT小鼠模型中追踪发生部分或完全上皮间质转化的癌细胞。

在原发性肿瘤中，癌细胞极少发生上皮间质转化，其中多数细胞仅在上皮型与部分上皮间质转化状态间转换，极少完成完全上皮间质转化。因此，曾经历部分上皮间质转化的细胞可在多种上皮间质转化状态间动态转变，并参与肺转移与化疗耐药的发生。与之相反，完全上皮间质转化细胞大多维持间质表型，稳定定植于血管周围区域，但无法在肺部形成定植灶。然而，经化疗后的复发肿瘤中，完全上皮间质转化癌细胞的丰度显著升高。

本研究结果表明，上皮间质转化连续体不同阶段的癌细胞，对乳腺癌恶性表型的各项特征——如细胞迁移、侵袭、转移及化疗耐药——的贡献存在差异。

## 实验整体设计
通过流式细胞术，从Tnc-CreERT2;MMTV-Flpo;RC::FrePe;MMTV-PyMT（简称Tnc-CreERT2）及Cdh2-CreERT2;MMTV-Flpo;RC::FrePe;/MMTV-PyMT（简称Cdh2-CreERT2）小鼠的肿瘤中分选得到部分上皮间质转化与完全上皮间质转化乳腺肿瘤细胞。

本双重组酶谱系示踪系统可将所有乳腺癌细胞标记为表达mCherry（红色荧光）的细胞。给予他莫昔芬后，曾经历部分或完全上皮间质转化的癌细胞会转为表达GFP（绿色荧光）。这种红到绿的荧光转换不可逆，因此即便癌细胞通过间质上皮转化（mesenchymal-epithelial transition, MET）转回上皮状态，仍可通过GFP的表达追踪所有曾发生上皮间质转化的癌细胞。

据此，mCherry阳性细胞为乳腺癌细胞，GFP阳性细胞为曾经历部分或完全上皮间质转化的乳腺癌细胞，而双阳性细胞则为正在发生上皮间质转化的过渡态细胞。

基于GFP或mCherry的表达情况，结合上皮标志物EpCAM（上皮细胞黏附分子）的表达水平，我们从Tnc-CreERT2小鼠中分选出3群mCherry阳性细胞（P5、P17与P18）、4群GFP阳性细胞（P15、P10、P16与P9）以及1群双阳性细胞（P22）；从Cdh2-CreERT2小鼠中则分选出3群mCherry阳性细胞（P5、P17与P18）与1群GFP阳性细胞（P15）。

各细胞群具体信息如下：
mCherry阳性细胞群：P05为mCherry+ EpCAM-，P17为mCherry+ EpCAM高表达，P18为mCherry高表达且EpCAM高表达；
GFP阳性细胞群：P15为GFP+ EpCAM-，P10为GFP+ EpCAM低表达，P16为GFP+ EpCAM中等表达，P18为GFP高表达且EpCAM中等表达；
双阳性细胞群：P22为mCherry+ GFP+ EpCAM高表达。