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• W3043485353 abstract "•Generation of a genetic system for recording transient cell fate in vivo•Genetic tracing of EMT gene activity during breast-to-lung tumor metastasis•Vimentin is not involved during mammary tumor metastasis in MMTV-PyMT model•N-cadherin is activated and functionally required during tumor metastasis Genetic lineage tracing unravels cell fate and plasticity in development, tissue homeostasis, and diseases. However, it remains technically challenging to trace temporary or transient cell fate, such as epithelial-to-mesenchymal transition (EMT) in tumor metastasis. Here, we generated a genetic fate-mapping system for temporally seamless tracing of transient cell fate. Highlighting its immediate application, we used it to study EMT gene activity from the local primary tumor to a distant metastatic site in vivo. In a spontaneous breast-to-lung metastasis model, we found that primary tumor cells activated vimentin and N-cadherin in situ, but only N-cadherin was activated and functionally required during metastasis. Tumor cells that have ever expressed N-cadherin constituted the majority of metastases in lungs, and functional deletion of N-cad significantly reduced metastasis. The seamless genetic recording system described here provides an alternative way for understanding transient cell fate and plasticity in biological processes. Genetic lineage tracing unravels cell fate and plasticity in development, tissue homeostasis, and diseases. However, it remains technically challenging to trace temporary or transient cell fate, such as epithelial-to-mesenchymal transition (EMT) in tumor metastasis. Here, we generated a genetic fate-mapping system for temporally seamless tracing of transient cell fate. Highlighting its immediate application, we used it to study EMT gene activity from the local primary tumor to a distant metastatic site in vivo. In a spontaneous breast-to-lung metastasis model, we found that primary tumor cells activated vimentin and N-cadherin in situ, but only N-cadherin was activated and functionally required during metastasis. Tumor cells that have ever expressed N-cadherin constituted the majority of metastases in lungs, and functional deletion of N-cad significantly reduced metastasis. The seamless genetic recording system described here provides an alternative way for understanding transient cell fate and plasticity in biological processes. Cre-loxP technology has been widely used for genetically tracing cell lineages in development, tissue homeostasis, and diseases (Kretzschmar and Watt, 2012Kretzschmar K. Watt F.M. Lineage tracing.Cell. 2012; 148: 33-45Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). The temporally controlled genetic tracing utilizes tamoxifen-induced CreER-loxP recombination, such that the Cre-expressing cells and their descendants are genetically labeled after tamoxifen treatment (Tian et al., 2015Tian X. Pu W.T. Zhou B. Cellular origin and developmental program of coronary angiogenesis.Circ. Res. 2015; 116: 515-530Crossref PubMed Scopus (109) Google Scholar). Importantly, the selected temporal window of tamoxifen treatment depends on the critical information of CreER expression, which is determined by the gene activity indicative of specific cell fate. However, cell fate transition in vivo could be dynamic and transient, which is manifested by transient gene expression (Süel et al., 2006Süel G.M. Garcia-Ojalvo J. Liberman L.M. Elowitz M.B. An excitable gene regulatory circuit induces transient cellular differentiation.Nature. 2006; 440: 545-550Crossref PubMed Scopus (530) Google Scholar; Maamar et al., 2007Maamar H. Raj A. Dubnau D. Noise in gene expression determines cell fate in Bacillus subtilis.Science. 2007; 317: 526-529Crossref PubMed Scopus (469) Google Scholar; Weinberger et al., 2008Weinberger L.S. Dar R.D. Simpson M.L. Transient-mediated fate determination in a transcriptional circuit of HIV.Nat. Genet. 2008; 40: 466-470Crossref PubMed Scopus (144) Google Scholar) and remains technically hard to capture in real time. For example, epithelial-to-mesenchymal transition (EMT) has been proposed as an essential step for tumor metastasis, but the EMT program and genes are usually transiently expressed in tumor cells (Nieto et al., 2016Nieto M.A. Huang R.Y. Jackson R.A. Thiery J.P. EMT: 2016.Cell. 2016; 166: 21-45Abstract Full Text Full Text PDF PubMed Scopus (1961) Google Scholar; Cano et al., 2000Cano A. Pérez-Moreno M.A. Rodrigo I. Locascio A. Blanco M.J. del Barrio M.G. Portillo F. Nieto M.A. The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression.Nat. Cell Biol. 2000; 2: 76-83Crossref PubMed Scopus (2691) Google Scholar; Yang et al., 2004Yang J. Mani S.A. Donaher J.L. Ramaswamy S. Itzykson R.A. Come C. Savagner P. Gitelman I. Richardson A. Weinberg R.A. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis.Cell. 2004; 117: 927-939Abstract Full Text Full Text PDF PubMed Scopus (2849) Google Scholar; Mani et al., 2008Mani S.A. Guo W. Liao M.J. Eaton E.N. Ayyanan A. Zhou A.Y. Brooks M. Reinhard F. Zhang C.C. Shipitsin M. et al.The epithelial-mesenchymal transition generates cells with properties of stem cells.Cell. 2008; 133: 704-715Abstract Full Text Full Text PDF PubMed Scopus (6153) Google Scholar; Ye et al., 2015Ye X. Tam W.L. Shibue T. Kaygusuz Y. Reinhardt F. Ng Eaton E. Weinberg R.A. Distinct EMT programs control normal mammary stem cells and tumour-initiating cells.Nature. 2015; 525: 256-260Crossref PubMed Scopus (388) Google Scholar). Due to its transient nature, tracking EMT in tumor metastasis by conventional Cre-loxP remains technically challenging. Whether EMT is involved in the tumor progression to the metastatic state remains as a significant yet highly contentious subject in the field for the past decade (Krebs et al., 2017Krebs A.M. Mitschke J. Lasierra Losada M. Schmalhofer O. Boerries M. Busch H. Boettcher M. Mougiakakos D. Reichardt W. Bronsert P. et al.The EMT-activator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer.Nat. Cell Biol. 2017; 19: 518-529Crossref PubMed Scopus (404) Google Scholar; Xu et al., 2017Xu Y. Lee D.K. Feng Z. Xu Y. Bu W. Li Y. Liao L. Xu J. Breast tumor cell-specific knockout of Twist1 inhibits cancer cell plasticity, dissemination, and lung metastasis in mice.Proc. Natl. Acad. Sci. USA. 2017; 114: 11494-11499Crossref PubMed Scopus (40) Google Scholar; Brabletz et al., 2018Brabletz T. Kalluri R. Nieto M.A. Weinberg R.A. EMT in cancer.Nat. Rev. Cancer. 2018; 18: 128-134Crossref PubMed Scopus (740) Google Scholar; Ledford, 2011Ledford H. Cancer theory faces doubts.Nature. 2011; 472: 273Crossref PubMed Scopus (108) Google Scholar; Fischer et al., 2015Fischer K.R. Durrans A. Lee S. Sheng J. Li F. Wong S.T. Choi H. El Rayes T. Ryu S. Troeger J. et al.Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance.Nature. 2015; 527: 472-476Crossref PubMed Scopus (1036) Google Scholar; Zheng et al., 2015Zheng X. Carstens J.L. Kim J. Scheible M. Kaye J. Sugimoto H. Wu C.C. LeBleu V.S. Kalluri R. Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer.Nature. 2015; 527: 525-530Crossref PubMed Scopus (1151) Google Scholar; Maheswaran and Haber, 2015Maheswaran S. Haber D.A. Cell fate: transition loses its invasive edge.Nature. 2015; 527: 452-453Crossref PubMed Scopus (22) Google Scholar; Ye et al., 2017Ye X. Brabletz T. Kang Y. Longmore G.D. Nieto M.A. Stanger B.Z. Yang J. Weinberg R.A. Upholding a role for EMT in breast cancer metastasis.Nature. 2017; 547: E1-E3Crossref PubMed Scopus (158) Google Scholar; Aiello et al., 2017Aiello N.M. Brabletz T. Kang Y. Nieto M.A. Weinberg R.A. Stanger B.Z. Upholding a role for EMT in pancreatic cancer metastasis.Nature. 2017; 547: E7-E8Crossref PubMed Scopus (129) Google Scholar), and there is a lack of clear in vivo genetic fate-mapping evidence for EMT. In this study, we generated a genetic approach to seamlessly record transient cell fate. Using dual recombinases-mediated genetic lineage tracing, we examined whether EMT genes, such as Vimentin and N-cadherin, were activated and functionally required from breast-to-lung tumor metastasis. The fate-mapping results revealed that Vimentin was not activated during tumor metastasis, nor was it functionally required for tumor EMT. Instead, tumor cells that have ever expressed N-cadherin constitute the majority of tumor metastases in lung, with increased expression of other EMT genes and cell invasion capability. Specific knockout of N-cadherin in mammary epithelial cells resulted in a significant reduction of tumor metastasis. Our study provided genetic evidence of the activity and function of EMT gene N-cadherin in tumor metastasis. A recent study demonstrated that the c-KIT tyrosine kinase receptor (encoded by the Kit gene) was expressed in mammary luminal epithelial cells (Lim et al., 2009Lim E. Vaillant F. Wu D. Forrest N.C. Pal B. Hart A.H. Asselin-Labat M.L. Gyorki D.E. Ward T. Partanen A. et al.Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers.Nat. Med. 2009; 15: 907-913Crossref PubMed Scopus (935) Google Scholar). To genetically trace these Kit+ mammary luminal epithelial cells in vivo, we generated a Kit-CreER mouse strain and crossed this strain with loxP reporter R26-tdTomato mice to track Kit expression in normal mammary glands (Figure 1A). Tamoxifen was induced when these mice were 6–7 weeks old, and tissues were collected 2 days after tamoxifen treatment. Flow cytometric analysis showed that Kit was mainly expressed in mammary luminal epithelial cells and not in basal epithelial cells (Figure 1B). Whole-mount fluorescence images of mammary gland showed the classic branching patterns of epithelial cells labeled by Kit-CreER (Figure 1C). Immunostaining for tdTomato, luminal epithelial cell markers K8 and K19, or basal epithelial cell markers K5 and K14, on tissue sections showed that tdTomato+ cells were luminal but not basal epithelial cells (Figures 1D and 1E). Of note, these tdTomato+ cells were mainly endogenous estrogen receptor (ER)-negative luminal epithelial cells (Figure 1F). Taken together, the above results demonstrated the specific expression of Kit in luminal epithelial cells, and Kit-CreER could be used for genetic tracing of Kit+ mammary luminal epithelial cells (Figure 1G). To trace Kit+ luminal epithelial cells during mammary tumor development and metastasis, we crossed Kit-CreER;R26-tdTomato mice with MMTV-PyMT (Mouse Mammary Tumor Virus-Polyoma virus Middle T antigen) mice, which form spontaneous breast adenocarcinomas (Lin et al., 2003Lin E.Y. Jones J.G. Li P. Zhu L. Whitney K.D. Muller W.J. Pollard J.W. Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases.Am. J. Pathol. 2003; 163: 2113-2126Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar; Guy et al., 1992Guy C.T. Cardiff R.D. Muller W.J. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease.Mol. Cell. Biol. 1992; 12: 954-961Crossref PubMed Scopus (1082) Google Scholar) that resemble the human luminal subtype and have high lung metastasis penetrance (Figure 1H). Tamoxifen was used to induce triple-positive mice at 6–7 weeks old, and tissue samples were collected at the early (8–12 weeks old) or late (18–24 weeks old) stages of tumor formation. In the hyperplastic mammary lesions at the early stage, we found that Kit+ luminal cells constituted the majority of PyMT+ tumor cells and these tdTomato+ tumor cells were E-Cad+ (Figures 1I and 1J). These Kit-derived cells expressed luminal cell marker K8, but not basal cell markers K5, indicating that these cancer cells kept their luminal epithelial phenotype at an early stage of tumor formation (Figure 1K). In the high-grade carcinoma at a later stage, the majority of PyMT+ tumor cells were tdTomato+ (Figures 1I and 1J). Of note, a subset of tdTomato+ cancer cells expressed basal cell markers K5 and K14, and quantification data showed that 2.65% ± 0.32% of these tdTomato+ cells expressed K14 (Figures 1L and 1M), indicating that luminal tumor cells could express basal epithelial genes in high-grade carcinoma. In the lung tissue at the late stage of tumor formation, we observed many tdTomato+ lung metastases (Figure 1N), and immunostaining data showed that these tdTomato+ cells in metastatic lung colonies were PyMT+ (Figure 1O). The original Kit+ cells residing in the lung are endothelial cells (Liu et al., 2015Liu Q. Huang X. Zhang H. Tian X. He L. Yang R. Yan Y. Wang Q.D. Gillich A. Zhou B. c-kit(+) cells adopt vascular endothelial but not epithelial cell fates during lung maintenance and repair.Nat. Med. 2015; 21: 866-868Crossref PubMed Scopus (40) Google Scholar). Notably, these tdTomato+ metastases expressed the epithelial cell markers E-Cad (Figure 1O), suggesting the epithelial phenotype of the cells in lung metastases. Thus, Kit+ mammary luminal epithelial cell contributes to tumor cells in primary tumor and lung metastasis (Figure 1P). In this study, we used these mouse strains to explore EMT activity during mammary tumor development and metastasis. Conventional lineage tracing is based on an inducible Cre-loxP system, in which tamoxifen treatment leads to CreER translocation into the nucleus and results in permanent genetic labeling (Figure 2AI ). Since the gene activity or expression could be transient and reversible, such as in EMT genes that are transiently expressed during metastasis, tamoxifen-induced Cre-loxP system may not capture these transient EMT gene activities (Figure 2AII). Therefore, it is technically challenging to capture transient and reversible EMT gene activity using this previously used lineage tracing strategy (Figure 2A). To seamlessly trace and permanently record gene activity in a defined time window (i.e., from primary tumor to remote metastasis), we took advantage of two orthogonal recombination systems, Cre-loxP and Dre-rox (He et al., 2017He L. Li Y. Li Y. Pu W. Huang X. Tian X. Wang Y. Zhang H. Liu Q. Zhang L. et al.Enhancing the precision of genetic lineage tracing using dual recombinases.Nat. Med. 2017; 23: 1488-1498Crossref PubMed Scopus (88) Google Scholar), to monitor EMT gene activities over time. We first generated Kit-CreER;EMTgene-LSL-Dre;NR1 triple knock-in mice, which were referred to as EMTracer mice throughout this study (Figure 2B). EMTgene-LSL-Dre was composed of a constitutive Dre recombinase driven by an EMT gene promoter, and there was a transcriptional Stop cassette (Stop) flanked by two loxP sites (LSLs) before Dre. For nested reporter 1 (NR1) (He et al., 2017He L. Li Y. Li Y. Pu W. Huang X. Tian X. Wang Y. Zhang H. Liu Q. Zhang L. et al.Enhancing the precision of genetic lineage tracing using dual recombinases.Nat. Med. 2017; 23: 1488-1498Crossref PubMed Scopus (88) Google Scholar), Cre-loxP recombination first resulted in ZsGreen labeling, and Dre-rox recombination switched the genetic labeling from ZsGreen to tdTomato (Figure 2B). After tamoxifen treatment, Kit-CreER efficiently and specifically labeled luminal epithelial cells with ZsGreen by NR1 in mammary glands (Figures 2B and S1). Meanwhile, Kit-CreER also removed the Stop in the EMTgene-LSL-Dre allele, placing the constitutively active Dre directly under the control of the EMT gene promoter (i.e., EMTgene-Dre, Figure 2B). Thereafter, Dre would be expressed when the EMT gene was activated, subsequently switching the labeling from ZsGreen to tdTomato (Figure 2B). Thus, after removal of loxP-flanked Stop cassette, the activity of the constitutively active Dre would no longer depend on tamoxifen treatment, and EMTgene-Dre could seamlessly monitor and record EMT gene activity, even if the EMT gene activity was transient, with the permanent genetic marker tdTomato after Dre-rox recombination (Figure 2B). We first established a genetic lineage tracing system for EMT characterized by the activation of vimentin (hereafter abbreviated as Vim+ EMT). We generated a Vim-CreER knock-in mouse strain, and by labeling Vim+ cells, we found that Vim was actively expressed in both fibroblasts (mesenchymal cell lineage) and endothelial cells in multiple tissues, such as the heart valve, breast, and lung (Figures S2A–S2C), which was consistent with previous reports (Kalluri and Zeisberg, 2006Kalluri R. Zeisberg M. Fibroblasts in cancer.Nat. Rev. Cancer. 2006; 6: 392-401Crossref PubMed Scopus (3192) Google Scholar; Franke et al., 1979Franke W.W. Schmid E. Osborn M. Weber K. Intermediate-sized filaments of human endothelial cells.J. Cell Biol. 1979; 81: 570-580Crossref PubMed Scopus (161) Google Scholar). In the mammary and lung, Vim was active mainly in CDH5+ endothelial cells, and a subset of PDGFRa+ fibroblasts, but not in E-Cad+ epithelial cells (Figures S2D–S2F). We then generated a Vim-LSL-Dre knock-in mouse allele (Figure S3A) and then crossed this strain with Kit-CreER;NR1 mice to make Kit-CreER;Vim-LSL-Dre;NR1 triple knock-in mice to trace Vim+ EMT in Kit+ mammary epithelial cells (hereafter referred to as Vim-EMTracer, Figure S3B). As internal positive control, mesenchymal cells in the endocardial cushion were tdTomato+ cells, as they were mainly derived from endocardial cells after EMT in the early developing heart (Figure S3C). In the normal mammary tissues of the adult Vim-EMTracer mice, 0% and 75.42% ± 3.19% of mammary luminal epithelial cells were tdTomato+ and ZsGreen+, respectively (Figures 2C and S3D), demonstrating that there was no Vim+ EMT occurring in normal Kit+ mammary epithelial cells during homeostasis. As positive control, TGF-beta addition induced a substantial number of ZsGreen+ mammary tumor cells to switch into tdTomato+ (Figure S3E), indicating these ZsGreen+ epithelial cells were responsive to some EMT inducers. In the lungs of the same mouse, CDH5+ or PECAM+ endothelial cells, but not E-Cad+ epithelial cells or PDGFRa+ mesenchymal cells, were efficiently labeled with tdTomato by Vim-EMTracer (Figures 2D and S3F), indicating that there was vimentin expression in a subset of lung endothelial cells that also expressed Kit (Liu et al., 2015Liu Q. Huang X. Zhang H. Tian X. He L. Yang R. Yan Y. Wang Q.D. Gillich A. Zhou B. c-kit(+) cells adopt vascular endothelial but not epithelial cell fates during lung maintenance and repair.Nat. Med. 2015; 21: 866-868Crossref PubMed Scopus (40) Google Scholar). As technical controls, we performed the following three experiments. First, in Vim-EMTracer mice without tamoxifen induction (No Tam), no detectable fluorescence signal was found in the mammary or lung tissues (Figures 2E and 2F), indicating that there was no leakiness of Vim-EMTracer. Second, in Vim-LSL-Dre;NR1 mice treated with tamoxifen, no ZsGreen or tdTomato signal was detected in the mammary or lung tissues (Figures 2G and 2H), indicating that Dre was tightly controlled by CreER-mediated recombination. Third, in Kit-CreER;NR1 mice induced with tamoxifen, mammary luminal epithelial cells were ZsGreen+tdTomato– (Figure 2I), demonstrating that Cre targeted loxP sites but not rox sites in NR1 and confirming that Cre-loxP and Dre-rox were orthogonal systems for recombination (Anastassiadis et al., 2009Anastassiadis K. Fu J. Patsch C. Hu S. Weidlich S. Duerschke K. Buchholz F. Edenhofer F. Stewart A.F. Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice.Dis. Model Mech. 2009; 2: 508-515Crossref PubMed Scopus (171) Google Scholar; Zhang et al., 2016Zhang H. Pu W. Tian X. Huang X. He L. Liu Q. Li Y. Zhang L. He L. Liu K. et al.Genetic lineage tracing identifies endocardial origin of liver vasculature.Nat. Genet. 2016; 48: 537-543Crossref PubMed Scopus (49) Google Scholar). In the lungs of the same mouse, Kit+ endothelial cells but not E-Cad+ epithelial cells were ZsGreen+tdTomato– (Figure 2J). These data confirmed the establishment of Vim-EMTracer mice and demonstrated that there was no vimentin activity in Kit+ mammary epithelial cells in normal mammary tissues. To test if a Vim+ EMT program was activated during tumor growth and metastasis, we crossed Vim-EMTracer mice with MMTV-PyMT mice to generate Vim-EMTracer;MMTV-PyMT mice. Tamoxifen induction was performed when these mice were 6–7 weeks old, and tissue samples were collected at early (8–12 weeks old) or late (18–24 weeks old) stages of tumor formation (Figure 3A). We could readily detect ZsGreen+tdTomato– tumor nodules in the mammary tissues but not in the lung metastases in the early stage of tumor development (Figure 3B). Immunostaining for PyMT, ZsGreen, and tdTomato on mammary sections from early stage of tumor formation showed that 76.22% ± 3.54% of PyMT+ tumor cells were ZsGreen+, and only 0.11% ± 0.049% of PyMT+ cells were tdTomato+ (Figures 3C and 3D), indicating that very few, if any, tumor cells expressed vimentin in the early stage of tumor progression. In the late stage of tumor formation, mammary primary tumors exhibited robust ZsGreen and tdTomato fluorescence (Figure 3E). Immunostaining for PyMT, ZsGreen, and tdTomato in mammary sections showed that 74.42% ± 3.40% of PyMT+ cells were ZsGreen+, and 2.06% ± 0.48% of PyMT+ cells were tdTomato+ (Figures 3F and 3G), indicating that a subset of tumor cells exhibited Vim+ EMT during tumor growth. Immunostaining for tdTomato, ZsGreen, and Vimentin on mammary tumors showed that as subset of tdTomato+ exhibited spindle shape and expressed Vimentin in the tumor (Figure 3H). In the lung, we could readily detect ZsGreen+ nodules that ranged from a small (<100 μm) to a large (>0.5 mm) size (Figure 3I). During metastasis, we focused on the Kit-derived fluorescent tumor nodules in the lung for the subsequent analysis of potential Vim+ EMT. In small metastasis, all PyMT+ cells were ZsGreen+, and no tdTomato+ tumor cells were found (Figures 3J and 3K). In large metastasis, while the majority of PyMT+ cells were ZsGreen+, a very small number of PyMT+ cells in the periphery of nodules were tdTomato+Zsgreen– (Figure 3L). These tdTomato+ tumor cells constituted 0.42% ± 0.083% of PyMT+ cells in the lung metastases (Figure 3M), indicating that very few, if any, tumor cells exhibited vimentin activity after tumor colonization. To demonstrate that these tdTomato+ cells had undergone Vim+ EMT during tumor growth and expansion after but not during metastatic colonization, we also collected lungs before the mice were 18 weeks old, when there were only small metastatic colonies (Figure S4A). We found that all tumor metastatic cells in the lung were ZsGreen+tdTomato– (Figures S4B–S4E). Given that the tumor cells in small nodules were tdTomato− before they grew into larger nodules, and considering that very few tdTomato+ tumor cells were detected in only the large nodules, usually in the periphery of the nodule (Figures 3L and 3M), it was likely that ZsGreen+ metastatic cells activated in situ vimentin expression following their colonization, thus, switching from ZsGreen+ to tdTomato+ labeling in the tumor metastases. While Vim is not robustly activated during tumor metastasis, we asked if Vim could be functionally required for tumor metastasis. To address this possibility, we crossed Kit-CreER;Vim-LSL-Dre;NR1;MMTV-PyMT (heterozygous Vim control mice) with Vim-LSL-Dre to generate homozygous null allele for Vim. In Kit-CreER;Vim-LSL-Dre; Vim-LSL-Dre;NR1;MMTV-PyMT (homozygous Vim mutant mice), we could not detect any Vim expression in mammary tumors collected from Vim null mice, while Vim was readily detected in the mammary tumors collected from littermate control mice (Figure 4A). Similarly, we treated mutant mice with tamoxifen at 6 weeks, and collected mammary tissues at 18–24 weeks (Figure 4B), and could observe formation of large mammary tumors exhibiting ZsGreen and tdTomato (Figure 4C). Immunostaining for ZsGreen, tdTomato, and PyMT on mutant tumor sections revealed that a subset of PyMT+ tumor cells had switched from ZsGreen to tdTomato in the primary tumors (Figure 4D). In the lung, many small and large tumor metastases were detected, and virtually all of them were ZsGreen+ (Figure 4E), indicating that lack of Vim did not significantly inhibit tumor metastasis, nor was the Vim gene promoter activated during tumor metastasis. Similar to the control mice, a subset of tdTomato+PyMT+ cells was detected in the peripheral of some tumor colonies in the lung (Figure 4F), indicating Vim gene promoter was activated during the growth and expansion of metastases. The lack of detectable vimentin activity during the metastasis process was likely not due to inefficient Stop removal from the Vim-LSL-Dre allele by Kit-CreER, as vimentin activity could be readily detected in Kit+ endothelial cells (Liu et al., 2015Liu Q. Huang X. Zhang H. Tian X. He L. Yang R. Yan Y. Wang Q.D. Gillich A. Zhou B. c-kit(+) cells adopt vascular endothelial but not epithelial cell fates during lung maintenance and repair.Nat. Med. 2015; 21: 866-868Crossref PubMed Scopus (40) Google Scholar) throughout the lung (tdTomato+, Figures 3J and 3L), demonstrating that the Stop was efficiently removed from the Vim-LSL-Dre allele by Kit-CreER driver. In addition, we performed PCR analysis of the Stop DNA cassette in ZsGreen+ cells isolated from mammary tumors. ZsGreen+ cells had Stop-removed DNA signatures in the Vim-LSL-Dre allele (Figure 3N), indicating that the Kit-CreER-mediated Stop removal in the Vim-LSL-Dre allele was efficient in tumor cells. Furthermore, all the different types of internal technical controls confirmed the fidelity of the EMT genetic tracing in this tumor model (Figure S5). Taken together, these data demonstrated that there was a lack of Vim activation during the progression to tumor metastasis, indicating that breast tumors utilize Vim-independent EMT or an EMT-independent mechanism to metastasize. We next employed another commonly used EMT marker, N-cadherin, to generate a lineage tracing system for EMT characterized by the activation of N-cadherin (N-cad+ EMT) during tumor growth and metastasis. We generated the N-cad-LSL-Dre knock-in mouse strain (Figure S6A) and crossed it with Kit-CreER;NR1 mice to make the Kit-CreER;N-cad-LSL-Dre;NR1 triple knock-in mouse for the lineage tracing of N-cad+ EMT during tumor growth and metastasis (N-cad-EMTracer, Figure S6B). As internal control, we could detect tdTomato+ mesenchymal cells in the endocardial cushion of developing heart (Figure S6C), demonstrating that endocardial EMT could be genetically traced by N-cad-EMTracer. Whole-mount fluorescence and immunostaining of mammary tissues derived from N-cad-EMTracer mice showed that the mammary epithelial cells were ZsGreen+tdTomato– (Figures 5A, 5B and S6D–S6G), indicating that there was no N-cadherin activity in Kit+ mammary epithelial cells in the normal mammary gland during homeostasis. Moreover, no tdTomato+ cells were detected in the lung (Figure 5A). Immunostaining for ZsGreen, tdTomato, and E-Cad on lung sections verified that there were no tdTomato+ epithelial cells in the lung (Figure 5B), demonstrating that the N-cad+ EMT program was not active in normal mammary and lung tissues. We then crossed N-cad-EMTracer mice with MMTV-PyMT mice to generate N-cad-EMTracer;MMTV-PyMT mice to study N-cad+ EMT in a tumor model. After tamoxifen induction when the mice were 6–7 weeks old, we collected mammary tissues at early (8–12 weeks old) or late (18–24 weeks old) stages of tumor formation for analysis. ZsGreen+tdTomato− tumor nodules were readily detected at the early stage of tumor development in the mammary tissues (Figure 5C). Immunostaining for PyMT, ZsGreen, and tdTomato on mammary sections showed that 77.67% ± 2.63% and 0.22% ± 0.035% of PyMT+ cells in hyperplastic lesions were ZsGreen+ and tdTomato+, respectively, while there were no tdTomato+ epithelial cells in the morphologically normal glands of the same mammary tissues (Figures 5D and 5E). These tdTomato+ tumor cells still expressed epithelial cell marker E-Cad (Figure 5F). Of note, N-cad could be detected in subset of tdTomato+ tumor cells (Figure 5G). At the late stage of tumor formation, when high-grade carcinomas had formed, the tumor nodules were ZsGreen+ and tdTomato+ (Figure 5H). Immunostaining for PyMT, ZsGreen, and tdTomato in mammary sections showed that 73.38% ± 3.76% and 9.46% ± 1.57% of PyMT+ cells were ZsGreen+ and tdTomato+, respectively (Figures 5I and 5J). These tdTomato+ tumor cells also expressed epithelial cell marker E-Cad (Figure 5K), and a subset of tdTomato+ tumor cells expressed N-cad (Figure 5L). The appearance of tdTomato+ tumor cells at the early stage of tumor formation and the increase in their number of these cells at the late stage demonstrated that the N-cad+ EMT program occurred and increased in frequency over a wide time span during primary tumor growth and expansion based on a genetic reporter (tdTomato). To study tumor metastasis, we collected the lungs of N-cad-EMTracer;MMTV-PyMT mice at the late stages of tumor development to examine N-cad+ EMT (Figure 6A). Whole-mount fluorescence imaging of the lung showed that most tumors were tdTomato+, while a subset of tumors were ZsGreen+ (Figure 6B). These data indicated that most lung metastases (tdTomato+) had activated EMT gene N-cad, while ZsGreen+ tumor cells either employed N-cad-independent EMT or an EMT-independent mechanism to metastasize. These tdTomato+ cells were derived from lung metastases labeled by the EMT tracing system, as the lungs collected from N-cad-EMTracer siblings remained tumor free and tdTomato− with the same tamox" @default.
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• W3043485353 date "2020-09-01" @default.
• W3043485353 modified "2023-10-17" @default.
• W3043485353 title "Genetic Fate Mapping of Transient Cell Fate Reveals N-Cadherin Activity and Function in Tumor Metastasis" @default.
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