Dev Cell: Use genealogical tracing techniques to capture the EMT process in tumor metastasis in the body.

Breast cancer is the highest cancer in the female population and poses a serious threat to the safety of female patients.
patients with primary breast cancer have good prognostic results after proper radiotherapy or chemotherapy after surgery to remove tumor situ.
However, because breast cancer cells lose their normal cellular characteristics, the connectionbetween cells becomes loose, easy to fall off, into the blood system or lymphatic system for metastasis, in the distant organs, such as lungs, bones, liver and other organs to form a metastasis, threatening the patient's life.
therefore, it is of great significance to study the way and mechanism of breast cancer metastasis. Whether or not the tumor cells that
metastasis have the transition of epithelial-mesenchymal cells (epithelial-mesenchymal transition, EMT) has been a hot topic in the field of tumor research.
and in recent years has been controversial about the role of EMT in tumor metastasis.
emtlicle seisphon lost its unique biological characteristics, lost cell polarity, lost the connection to the substrate membrane and was given the characteristics of some interstitial cells, the cell form is a spindle-shaped, with relatively strong migration and invasive ability, with the ability to resist apoptosis and degradation of the extracellular matrix.
early studies used in vitro cell culture and transplantation to think that emT process plays an important role in tumor cell metastasis, but lack of direct in vivo experimental evidence, in recent years, researchers have proposed that the metastasis of tumor cells in the body does not require emtly process.
, therefore, whether the metastasis of tumor cells has a shift to epithelial cells to interstitial cells needs further research to prove.
Zhou Bin Research Group of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, published an article online in the journal "Genetic Fate Mapping of Transient Fate Cell Cells N-Cadherin Activity and Function in Tumor Metastasis".
the results suggest that in the process of breast cancer metastasis, the epithelial cell conversion of tumor cells to interstitial cells has heterogeneous, in situ tumor cells will have N-Cadherin-dependent EMT, and the tumor cell contributing to the N-Cadherin-dependent EMT has formed most of the pulmonary metastasis, while the tumor cells in situ where Vimentin depends on EMT do not contribute to the formation of a pulmonary metastasis.
traditional single homologous recombination spectrum tracing technique spent on Cre-LoxP, although it can reveal the fate of cells in the body, but generally tracethed is the group of cells marked by tamoxifen induction, and the molecular markers of these cells are generally expressed steadily in the cells, while the induced single homologous recombinant is difficult to capture the genes of these transient expressions in the body.
therefore single homologous recombination systems are not suitable for transient, reversible EMT processes in tracer tumor models.
In this research project, Zhou Bin research team based on the double homologous recombination system to create induced seamless genealogy tracing technology, with spontaneous breast cancer tumor model mouse MMTV-PyMT as the research object, using Kit-CreER to mark the mouse breast cavity epithelial cells The gene used to track EMT was built to tap into mice N-Cad-LSL-Dre and Vim-LSL-Dre, using dual homologous reported gene mice NR1 to track both the occurring and the absence of EMT breast tumor cells.
first, the researchers established the induced, seamless genetic genealogy tracing technique, the EMTracer system, for body capture EMT, based on the pre-dual homologous recombination system.
to study EMT during the metastasis of breast tumor cells, the researchers referred to Kit-Creer; EMTgene-LSL-Dre; And NR1 (EMTracer mice, EMTgene can be a molecular marker of some recognized interstitial cells, such as Vimentin and N-cadherin) Spontaneous breast cancer tumor model mouse MMTV-PyMT together, after hemoxifen induction, Kit-Creer expressed the Cre identification report gene NR1 and EMTgene-LSL-Dre two genes on the LoxP site recombination.
The recombination of Kit-CreER and LoxP of the reporting gene NR1 caused Kit-Plus's breast tube cavity epithelial cells to be labeled with green fluorescent protein (ZsGreen), which could be traced to The cell fate of Kit-Plus epithelial cells in the stable state of the breast.
Kit-Creer and EMTgene-LSL-Dre's LoxP recombination, so that the stop sequence between the two LoxPs is cut off, when the transtheliosis cell to interstitial cell conversion, EMTgene will initiate expression, emtgene after the Dre expression, and the report gene NR1 Rox ahogenresave, the report The Rox-LoxP-stop-LoxP-ZsGreen-PolyA-Rox sequence, located between the two Rox sites, is cut off to initiate the expression of the red fluorescent protein (tdTomato), enabling the cells that have occurred EMTs to be converted from the previous green fluorescent protein (ZsGreen) to the red fluorescent protein (tdTomato) for the monitoring of EMT.
, after the STOP sequence of EMTgene-LSL-Dre is cut off, Dre will continue to express, although the EMT process can be transient and reversible, and the use of EMTracer mice to achieve continuous monitoring of the EMT process makes up for the shortcomings of the induced CreER in monitoring the EMT process, the researchers The collection of in situ tumors and lung tissue in the early and late stages of tumors for immunofluorescent staining analysis of EMT sesame status in tumor cells, and the identification of the molecular markers and transcription factors of these cells of highly expressed interstitial prosthesis cells by by streaming and sorting the mammograms of the mammograms of the mammogram cells that had occurred with N-Cadherin-dependent EMT.
and these in situ tumor cells of the breast that had been on the Ic-Cadherin-dependent EMT were more aggressive in in vitro culture.
, the researchers also used N-Cad flox mice to specifically knock out N-Cadherin in breast in situ tumor cells, reducing the formation of the pulmonary metastasis, while Vimentin's full-knocking breast tumor mice were still able to form a pulmonary metastasis.
the results suggest that EMT of tumor cells has heterogeneity in the process of tumor cell metastasis in breast cancer, and that the induced, seamless spectral tracing technique seissays created in this study can also be used in the study of EMT in development or other tumor models.
it is understood that the research work was completed by postdoctoral li Yan, Ph.D. student Lu Zan and other researchers under Zhou Bin's guidance.
the study was greatly assisted by Professor Shi Qihui of Fudan University, Professor Cai Dongqing of Jinan University, Professor Wu Mingfu of Albany University, Researcher Hu Guohong of Shanghai Institute of Nutritional Health, Professor Lu Ailan of the University of Chinese of Hong Kong, Professor Feng Jing of Southern Medical University and Professor Angela Nieto of the Spanish Institute of Neurology.
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