Partial EMT takes the lead in cancer metastasis
2021; Elsevier BV; Volume: 56; Issue: 23 Linguagem: Inglês
10.1016/j.devcel.2021.11.012
ISSN1878-1551
Autores Tópico(s)Digestive system and related health
ResumoIn this issue of Developmental Cell, Lüönd et al. developed a tracing system, using the uncharacterized early epithelial-mesenchymal transition (EMT)-marker Tenascin C, to monitor cells undergoing partial EMT during malignant mammary cancer progression. They find that partial, but not full, EMT contributes to metastasis and that full EMT contributes to chemoresistance. In this issue of Developmental Cell, Lüönd et al. developed a tracing system, using the uncharacterized early epithelial-mesenchymal transition (EMT)-marker Tenascin C, to monitor cells undergoing partial EMT during malignant mammary cancer progression. They find that partial, but not full, EMT contributes to metastasis and that full EMT contributes to chemoresistance. Main textEpithelial-mesenchymal transition (EMT) is a well-characterized, progressive transition from an epithelial to a mesenchymal phenotype. Mesenchymal-like cells generated by EMT may regain their epithelial phenotype by undergoing the reverse process: mesenchymal-epithelial transition (MET) (Figure 1A; 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 (2567) Google Scholar). While EMT and MET have long been considered binary switches, it is now clear that epithelial cells may linger in a series of metastable, intermediate states that express both epithelial and mesenchymal markers. A 2020 consensus statement by EMT researchers favored the term epithelial mesenchymal plasticity to describe these dynamic cell states (Yang et al., 2020Yang J. Antin P. Berx G. Blanpain C. Brabletz T. Bronner M. Campbell K. Cano A. Casanova J. Christofori G. et al.EMT International Association (TEMTIA)Guidelines and definitions for research on epithelial-mesenchymal transition.Nat. Rev. Mol. Cell Biol. 2020; 21: 341-352Crossref PubMed Scopus (586) Google Scholar). Recently, single cell analysis confirmed this spectrum of epithelial mesenchymal states in circulating tumor cells and carcinomas. In absence of reliable post-EMT markers, classical epithelial and mesenchymal markers are used to match epithelial-mesenchymal traits: E-cadherin, EpCAM, Snail, ZEB1, N-cadherin, fibronectin, FSP-1, or Vimentin (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 (2567) Google Scholar; Yang et al., 2020Yang J. Antin P. Berx G. Blanpain C. Brabletz T. Bronner M. Campbell K. Cano A. Casanova J. Christofori G. et al.EMT International Association (TEMTIA)Guidelines and definitions for research on epithelial-mesenchymal transition.Nat. Rev. Mol. Cell Biol. 2020; 21: 341-352Crossref PubMed Scopus (586) Google Scholar). These markers label either the initial epithelial state or the terminal mesenchymal state. Interestingly, Tenascin C (TNC), a highly conserved extracellular matrix protein, was recently identified as a potential early and partial EMT marker (Saxena et al., 2018Saxena M. Kalathur R.K.R. Neutzner M. Christofori G. PyMT-1099, a versatile murine cell model for EMT in breast cancer.Sci. Rep. 2018; 8: 12123Crossref PubMed Scopus (11) Google Scholar).EMT is essential during embryogenesis and has been implicated in tissue regeneration and degeneration, cancer progression, and therapy resistance. Based on EMT-driven attenuation of cell adhesion and enhanced cellular motility in embryonic tissues and in vitro cancer cell lines, EMT has been proposed as a driver of metastasis formation. MET, on the other hand, is believed to contribute to the metastatic outgrowth of disseminated cancer cells (Goossens et al., 2017Goossens S. Vandamme N. Van Vlierberghe P. Berx G. EMT transcription factors in cancer development re-evaluated: Beyond EMT and MET.Biochimica et biophysica acta. Reviews on cancer. 2017; 1868: 584-591Crossref PubMed Scopus (163) Google Scholar; 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 (2567) Google Scholar). Lately, however, the effective in vivo contribution of binary EMT/MET switches in metastasis has been questioned (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 (1235) 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 (205) Google Scholar). Although intermediate EMT states have been proven highly metastatic in experimental metastasis models (Pastushenko et al., 2018Pastushenko I. Brisebarre A. Sifrim A. Fioramonti M. Revenco T. Boumahdi S. Van Keymeulen A. Brown D. Moers V. Lemaire S. et al.Identification of the tumour transition states occurring during EMT.Nature. 2018; 556: 463-468Crossref PubMed Scopus (694) Google Scholar), solid evidence on the role of partial EMT in spontaneous metastasis is limited.In this issue of Developmental Cell, Lüönd et al., 2021Lüönd F. Sugiyama N. Bill R. Bornes L. Hager C. Tang F. Santacroce N. Beisel C. Ivanek R. Bürglin T. et al.Distinct Contributions of Partial and Full EMT to Breast Cancer Malignancy.Dev. Cell. 2021; 56: 3203-3221Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar described an in vivo fate mapping of partial and late EMT states during cancer metastasis and chemotherapy in a mammary cancer mouse model. To mark with high precision mammary gland cancer cells that have undergone EMT, the MMTV-PyMT model was equipped with an innovative orthogonal dual recombinase lineage tracing system (Figure 1B). In this sensor system, expression of a red fluorescent protein (mCherry) and green fluorescent protein (GFP) is prevented by a set of stop cassettes. The MMTV promotor is used to control expression of a murine-optimized Flippase (Flpo). Flpo causes recombination between FRT sites, resulting in expression of mCherry in mammary epithelial cells and cancer cells derived thereof. To irreversibly label cells which have undergone partial or full EMT, a Cre-recombinase fused to the estrogen receptor (CreERT2) was placed under control of the Tenascin C promotor (Tnc-CreERT2 mice), active during early EMT, or the N-cadherin promotor (Cdh2-CreERT2 mice), active only after full EMT. As such, cells reaching partial or full EMT will express the CreERT2 recombinase. In the presence of tamoxifen, functional CreERT2 causes recombination of the LoxP sites, switching mCherry for GFP. As a result, cells which have reached a partial or full EMT state during tamoxifen treatment will permanently express GFP, irrespective of their current differentiation state. Importantly, recombination by CreERT2 in mCherry-negative cells will not cause GFP expression. Therefore, cells that express N-cadherin or Tenascin C—but which are not derived from the mammary epithelium, like mesenchymal stromal cells—will remain unlabeled. Furthermore, because the system is inducible, timely tamoxifen treatment allows assessment of the kinetics and reversibility of early and late EMT-states. Using such a system, the authors found that in primary tumors, cells infrequently undergo partial EMT. However, the majority of partial EMT cells revert to an epithelial state by MET within 7 days. In contrast, mammary cancer cells only very rarely reach a full EMT state. Once reached, however, these cells retain their mesenchymal phenotype.To assess if different EMT states contribute to chemoresistance, mice were treated with paclitaxel, cyclophosphamide, or doxorubicin. While primary tumors seemed to be enriched for both partial and full EMT cells following treatment with chemotherapeutics, metastases were enriched in full EMT cells only. Based on these data, the authors claim that partial—but especially full—EMT contributes to chemoresistance which is consistent with earlier publications (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 (1235) Google Scholar).Compared to the primary tumors, lung metastases were significantly enriched in cells which had undergone partial EMT. Compellingly, elegant ex vivo imaging identified that Tenascin C-positive partial EMT cells act as highly migratory leader cells, promoting collective invasion (Figure 1B). Furthermore, in vivo transplantation of aggregates of epithelial and partial EMT cells resulted in significantly more metastases compared to aggregates consisting of any single population. Lastly, ablation of partial EMT cells reduced the metastatic burden. Together, the experimental data in this mammary cancer model strongly supports that cells undergoing partial EMT cooperate with fully differentiated epithelial cells to promote metastatic tumor progression. In contrast, cells undergoing full EMT did not noticeably contribute to the metastatic process.Currently, several attempts have been made to map EMT in vivo during cancer progression in the PyMT mammary cancer model (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 (1235) Google Scholar; Li et al., 2020Li Y. Lv Z. Zhang S. Wang Z. He L. Tang M. Pu W. Zhao H. Zhang Z. Shi Q. et al.Genetic Fate Mapping of Transient Cell Fate Reveals N-Cadherin Activity and Function in Tumor Metastasis.Dev. Cell. 2020; 54: 593-607.e5Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar; Lüönd et al., 2021Lüönd F. Sugiyama N. Bill R. Bornes L. Hager C. Tang F. Santacroce N. Beisel C. Ivanek R. Bürglin T. et al.Distinct Contributions of Partial and Full EMT to Breast Cancer Malignancy.Dev. Cell. 2021; 56: 3203-3221Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The use of these different complex genetic tracing tools has resulted in discrepancies regarding the role of EMT in metastasis formation. FSP-1-based lineage tracing showed that mesenchymal-like mammary cancer cells do not contribute to pulmonary metastases in the PyMT model (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 (1235) Google Scholar). However, this conclusion was recently disputed using an in vivo real-time EMT sensor, which showed that FSP1 is not expressed in the majority of the PyMT cancer cells (Bornes et al., 2019Bornes L. van Scheppingen R.H. Beerling E. Schelfhorst T. Ellenbroek S.I.J. Seinstra D. van Rheenen J. Fsp1-Mediated Lineage Tracing Fails to Detect the Majority of Disseminating Cells Undergoing EMT.Cell Rep. 2019; 29: 2565-2569.e3Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). In 2020, Li et al. reported that continuous N-cadherin mediated tracing of c-KIT-positive luminal cells fate mapped the majority of pulmonary PyMT metastasis (Li et al., 2020Li Y. Lv Z. Zhang S. Wang Z. He L. Tang M. Pu W. Zhao H. Zhang Z. Shi Q. et al.Genetic Fate Mapping of Transient Cell Fate Reveals N-Cadherin Activity and Function in Tumor Metastasis.Dev. Cell. 2020; 54: 593-607.e5Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). This in sharp contrast to the results of Lüönd et al. that show complete absence of metastases labeled by N-cadherin-mediated tracing (Lüönd et al., 2021Lüönd F. Sugiyama N. Bill R. Bornes L. Hager C. Tang F. Santacroce N. Beisel C. Ivanek R. Bürglin T. et al.Distinct Contributions of Partial and Full EMT to Breast Cancer Malignancy.Dev. Cell. 2021; 56: 3203-3221Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). These conflicting data stress that in vivo tracing results should be interpreted with caution, as the sensitivity and specificity of lineage tracing is likely influenced by the selective labeling of the examined cancer cells, used EMT markers, variable kinetics of tumor progression in different genetic backgrounds, and the duration of tamoxifen treatment for tracing. Forthcoming, carefully controlled comparative tracing studies will have to assess at which stages of the EMT spectrum N-cadherin is expressed and whether this differs between distinct cancer related EMT programs.Importantly, the data reported by Lüönd et al. shift Tenascin C to the frontline as a robust candidate marker of early EMT. Exploiting this marker may enable development of even more sensitive lineage tracing systems in the future. Such systems could reveal in detail the contribution of different partial EMT-associated phenotypes, not only in different stages of cancer progression but also in developmental, physiological, and different pathological processes. Main textEpithelial-mesenchymal transition (EMT) is a well-characterized, progressive transition from an epithelial to a mesenchymal phenotype. Mesenchymal-like cells generated by EMT may regain their epithelial phenotype by undergoing the reverse process: mesenchymal-epithelial transition (MET) (Figure 1A; 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 (2567) Google Scholar). While EMT and MET have long been considered binary switches, it is now clear that epithelial cells may linger in a series of metastable, intermediate states that express both epithelial and mesenchymal markers. A 2020 consensus statement by EMT researchers favored the term epithelial mesenchymal plasticity to describe these dynamic cell states (Yang et al., 2020Yang J. Antin P. Berx G. Blanpain C. Brabletz T. Bronner M. Campbell K. Cano A. Casanova J. Christofori G. et al.EMT International Association (TEMTIA)Guidelines and definitions for research on epithelial-mesenchymal transition.Nat. Rev. Mol. Cell Biol. 2020; 21: 341-352Crossref PubMed Scopus (586) Google Scholar). Recently, single cell analysis confirmed this spectrum of epithelial mesenchymal states in circulating tumor cells and carcinomas. In absence of reliable post-EMT markers, classical epithelial and mesenchymal markers are used to match epithelial-mesenchymal traits: E-cadherin, EpCAM, Snail, ZEB1, N-cadherin, fibronectin, FSP-1, or Vimentin (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 (2567) Google Scholar; Yang et al., 2020Yang J. Antin P. Berx G. Blanpain C. Brabletz T. Bronner M. Campbell K. Cano A. Casanova J. Christofori G. et al.EMT International Association (TEMTIA)Guidelines and definitions for research on epithelial-mesenchymal transition.Nat. Rev. Mol. Cell Biol. 2020; 21: 341-352Crossref PubMed Scopus (586) Google Scholar). These markers label either the initial epithelial state or the terminal mesenchymal state. Interestingly, Tenascin C (TNC), a highly conserved extracellular matrix protein, was recently identified as a potential early and partial EMT marker (Saxena et al., 2018Saxena M. Kalathur R.K.R. Neutzner M. Christofori G. PyMT-1099, a versatile murine cell model for EMT in breast cancer.Sci. Rep. 2018; 8: 12123Crossref PubMed Scopus (11) Google Scholar).EMT is essential during embryogenesis and has been implicated in tissue regeneration and degeneration, cancer progression, and therapy resistance. Based on EMT-driven attenuation of cell adhesion and enhanced cellular motility in embryonic tissues and in vitro cancer cell lines, EMT has been proposed as a driver of metastasis formation. MET, on the other hand, is believed to contribute to the metastatic outgrowth of disseminated cancer cells (Goossens et al., 2017Goossens S. Vandamme N. Van Vlierberghe P. Berx G. EMT transcription factors in cancer development re-evaluated: Beyond EMT and MET.Biochimica et biophysica acta. Reviews on cancer. 2017; 1868: 584-591Crossref PubMed Scopus (163) Google Scholar; 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 (2567) Google Scholar). Lately, however, the effective in vivo contribution of binary EMT/MET switches in metastasis has been questioned (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 (1235) 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 (205) Google Scholar). Although intermediate EMT states have been proven highly metastatic in experimental metastasis models (Pastushenko et al., 2018Pastushenko I. Brisebarre A. Sifrim A. Fioramonti M. Revenco T. Boumahdi S. Van Keymeulen A. Brown D. Moers V. Lemaire S. et al.Identification of the tumour transition states occurring during EMT.Nature. 2018; 556: 463-468Crossref PubMed Scopus (694) Google Scholar), solid evidence on the role of partial EMT in spontaneous metastasis is limited.In this issue of Developmental Cell, Lüönd et al., 2021Lüönd F. Sugiyama N. Bill R. Bornes L. Hager C. Tang F. Santacroce N. Beisel C. Ivanek R. Bürglin T. et al.Distinct Contributions of Partial and Full EMT to Breast Cancer Malignancy.Dev. Cell. 2021; 56: 3203-3221Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar described an in vivo fate mapping of partial and late EMT states during cancer metastasis and chemotherapy in a mammary cancer mouse model. To mark with high precision mammary gland cancer cells that have undergone EMT, the MMTV-PyMT model was equipped with an innovative orthogonal dual recombinase lineage tracing system (Figure 1B). In this sensor system, expression of a red fluorescent protein (mCherry) and green fluorescent protein (GFP) is prevented by a set of stop cassettes. The MMTV promotor is used to control expression of a murine-optimized Flippase (Flpo). Flpo causes recombination between FRT sites, resulting in expression of mCherry in mammary epithelial cells and cancer cells derived thereof. To irreversibly label cells which have undergone partial or full EMT, a Cre-recombinase fused to the estrogen receptor (CreERT2) was placed under control of the Tenascin C promotor (Tnc-CreERT2 mice), active during early EMT, or the N-cadherin promotor (Cdh2-CreERT2 mice), active only after full EMT. As such, cells reaching partial or full EMT will express the CreERT2 recombinase. In the presence of tamoxifen, functional CreERT2 causes recombination of the LoxP sites, switching mCherry for GFP. As a result, cells which have reached a partial or full EMT state during tamoxifen treatment will permanently express GFP, irrespective of their current differentiation state. Importantly, recombination by CreERT2 in mCherry-negative cells will not cause GFP expression. Therefore, cells that express N-cadherin or Tenascin C—but which are not derived from the mammary epithelium, like mesenchymal stromal cells—will remain unlabeled. Furthermore, because the system is inducible, timely tamoxifen treatment allows assessment of the kinetics and reversibility of early and late EMT-states. Using such a system, the authors found that in primary tumors, cells infrequently undergo partial EMT. However, the majority of partial EMT cells revert to an epithelial state by MET within 7 days. In contrast, mammary cancer cells only very rarely reach a full EMT state. Once reached, however, these cells retain their mesenchymal phenotype.To assess if different EMT states contribute to chemoresistance, mice were treated with paclitaxel, cyclophosphamide, or doxorubicin. While primary tumors seemed to be enriched for both partial and full EMT cells following treatment with chemotherapeutics, metastases were enriched in full EMT cells only. Based on these data, the authors claim that partial—but especially full—EMT contributes to chemoresistance which is consistent with earlier publications (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 (1235) Google Scholar).Compared to the primary tumors, lung metastases were significantly enriched in cells which had undergone partial EMT. Compellingly, elegant ex vivo imaging identified that Tenascin C-positive partial EMT cells act as highly migratory leader cells, promoting collective invasion (Figure 1B). Furthermore, in vivo transplantation of aggregates of epithelial and partial EMT cells resulted in significantly more metastases compared to aggregates consisting of any single population. Lastly, ablation of partial EMT cells reduced the metastatic burden. Together, the experimental data in this mammary cancer model strongly supports that cells undergoing partial EMT cooperate with fully differentiated epithelial cells to promote metastatic tumor progression. In contrast, cells undergoing full EMT did not noticeably contribute to the metastatic process.Currently, several attempts have been made to map EMT in vivo during cancer progression in the PyMT mammary cancer model (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 (1235) Google Scholar; Li et al., 2020Li Y. Lv Z. Zhang S. Wang Z. He L. Tang M. Pu W. Zhao H. Zhang Z. Shi Q. et al.Genetic Fate Mapping of Transient Cell Fate Reveals N-Cadherin Activity and Function in Tumor Metastasis.Dev. Cell. 2020; 54: 593-607.e5Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar; Lüönd et al., 2021Lüönd F. Sugiyama N. Bill R. Bornes L. Hager C. Tang F. Santacroce N. Beisel C. Ivanek R. Bürglin T. et al.Distinct Contributions of Partial and Full EMT to Breast Cancer Malignancy.Dev. Cell. 2021; 56: 3203-3221Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The use of these different complex genetic tracing tools has resulted in discrepancies regarding the role of EMT in metastasis formation. FSP-1-based lineage tracing showed that mesenchymal-like mammary cancer cells do not contribute to pulmonary metastases in the PyMT model (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 (1235) Google Scholar). However, this conclusion was recently disputed using an in vivo real-time EMT sensor, which showed that FSP1 is not expressed in the majority of the PyMT cancer cells (Bornes et al., 2019Bornes L. van Scheppingen R.H. Beerling E. Schelfhorst T. Ellenbroek S.I.J. Seinstra D. van Rheenen J. Fsp1-Mediated Lineage Tracing Fails to Detect the Majority of Disseminating Cells Undergoing EMT.Cell Rep. 2019; 29: 2565-2569.e3Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). In 2020, Li et al. reported that continuous N-cadherin mediated tracing of c-KIT-positive luminal cells fate mapped the majority of pulmonary PyMT metastasis (Li et al., 2020Li Y. Lv Z. Zhang S. Wang Z. He L. Tang M. Pu W. Zhao H. Zhang Z. Shi Q. et al.Genetic Fate Mapping of Transient Cell Fate Reveals N-Cadherin Activity and Function in Tumor Metastasis.Dev. Cell. 2020; 54: 593-607.e5Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). This in sharp contrast to the results of Lüönd et al. that show complete absence of metastases labeled by N-cadherin-mediated tracing (Lüönd et al., 2021Lüönd F. Sugiyama N. Bill R. Bornes L. Hager C. Tang F. Santacroce N. Beisel C. Ivanek R. Bürglin T. et al.Distinct Contributions of Partial and Full EMT to Breast Cancer Malignancy.Dev. Cell. 2021; 56: 3203-3221Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). These conflicting data stress that in vivo tracing results should be interpreted with caution, as the sensitivity and specificity of lineage tracing is likely influenced by the selective labeling of the examined cancer cells, used EMT markers, variable kinetics of tumor progression in different genetic backgrounds, and the duration of tamoxifen treatment for tracing. Forthcoming, carefully controlled comparative tracing studies will have to assess at which stages of the EMT spectrum N-cadherin is expressed and whether this differs between distinct cancer related EMT programs.Importantly, the data reported by Lüönd et al. shift Tenascin C to the frontline as a robust candidate marker of early EMT. Exploiting this marker may enable development of even more sensitive lineage tracing systems in the future. Such systems could reveal in detail the contribution of different partial EMT-associated phenotypes, not only in different stages of cancer progression but also in developmental, physiological, and different pathological processes. Epithelial-mesenchymal transition (EMT) is a well-characterized, progressive transition from an epithelial to a mesenchymal phenotype. Mesenchymal-like cells generated by EMT may regain their epithelial phenotype by undergoing the reverse process: mesenchymal-epithelial transition (MET) (Figure 1A; 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 (2567) Google Scholar). While EMT and MET have long been considered binary switches, it is now clear that epithelial cells may linger in a series of metastable, intermediate states that express both epithelial and mesenchymal markers. A 2020 consensus statement by EMT researchers favored the term epithelial mesenchymal plasticity to describe these dynamic cell states (Yang et al., 2020Yang J. Antin P. Berx G. Blanpain C. Brabletz T. Bronner M. Campbell K. Cano A. Casanova J. Christofori G. et al.EMT International Association (TEMTIA)Guidelines and definitions for research on epithelial-mesenchymal transition.Nat. Rev. Mol. Cell Biol. 2020; 21: 341-352Crossref PubMed Scopus (586) Google Scholar). Recently, single cell analysis confirmed this spectrum of epithelial mesenchymal states in circulating tumor cells and carcinomas. In absence of reliable post-EMT markers, classical epithelial and mesenchymal markers are used to match epithelial-mesenchymal traits: E-cadherin, EpCAM, Snail, ZEB1, N-cadherin, fibronectin, FSP-1, or Vimentin (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 (2567) Google Scholar; Yang et al., 2020Yang J. Antin P. Berx G. Blanpain C. Brabletz T. Bronner M. Campbell K. Cano A. Casanova J. Christofori G. et al.EMT International Association (TEMTIA)Guidelines and definitions for research on epithelial-mesenchymal transition.Nat. Rev. Mol. Cell Biol. 2020; 21: 341-352Crossref PubMed Scopus (586) Google Scholar). These markers label either the initial epithelial state or the terminal mesenchymal state. Interestingly, Tenascin C (TNC), a highly conserved extracellular matrix protein, was recently identified as a potential early and partial EMT marker (Saxena et al., 2018Saxena M. Kalathur R.K.R. Neutzner M. Christofori G. PyMT-1099, a versatile murine cell model for EMT in breast cancer.Sci. Rep. 2018; 8: 12123Crossref PubMed Scopus (11) Google Scholar). EMT is essential during embryogenesis and has been implicated in tissue regeneration and degeneration, cancer progression, and therapy resistance. Based on EMT-driven attenuation of cell adhesion and enhanced cellular motility in embryonic tissues and in vitro cancer cell lines, EMT has been proposed as a driver of metastasis formation. MET, on the other hand, is believed to contribute to the metastatic outgrowth of disseminated cancer cells (Goossens et al., 2017Goossens S. Vandamme N. Van Vlierberghe P. Berx G. EMT transcription factors in cancer development re-evaluated: Beyond EMT and MET.Biochimica et biophysica acta. Reviews on cancer. 2017; 1868: 584-591Crossref PubMed Scopus (163) Google Scholar; 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 (2567) Google Scholar). Lately, however, the effective in vivo contribution of binary EMT/MET switches in metastasis has been questioned (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 (1235) 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 (205) Google Scholar). Although intermediate EMT states have been proven highly metastatic in experimental metastasis models (Pastushenko et al., 2018Pastushenko I. Brisebarre A. Sifrim A. Fioramonti M. Revenco T. Boumahdi S. Van Keymeulen A. Brown D. Moers V. Lemaire S. et al.Identification of the tumour transition states occurring during EMT.Nature. 2018; 556: 463-468Crossref PubMed Scopus (694) Google Scholar), solid evidence on the role of partial EMT in spontaneous metastasis is limited. In this issue of Developmental Cell, Lüönd et al., 2021Lüönd F. Sugiyama N. Bill R. Bornes L. Hager C. Tang F. Santacroce N. Beisel C. Ivanek R. Bürglin T. et al.Distinct Contributions of Partial and Full EMT to Breast Cancer Malignancy.Dev. Cell. 2021; 56: 3203-3221Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar described an in vivo fate mapping of partial and late EMT states during cancer metastasis and chemotherapy in a mammary cancer mouse model. To mark with high precision mammary gland cancer cells that have undergone EMT, the MMTV-PyMT model was equipped with an innovative orthogonal dual recombinase lineage tracing system (Figure 1B). In this sensor system, expression of a red fluorescent protein (mCherry) and green fluorescent protein (GFP) is prevented by a set of stop cassettes. The MMTV promotor is used to control expression of a murine-optimized Flippase (Flpo). Flpo causes recombination between FRT sites, resulting in expression of mCherry in mammary epithelial cells and cancer cells derived thereof. To irreversibly label cells which have undergone partial or full EMT, a Cre-recombinase fused to the estrogen receptor (CreERT2) was placed under control of the Tenascin C promotor (Tnc-CreERT2 mice), active during early EMT, or the N-cadherin promotor (Cdh2-CreERT2 mice), active only after full EMT. As such, cells reaching partial or full EMT will express the CreERT2 recombinase. In the presence of tamoxifen, functional CreERT2 causes recombination of the LoxP sites, switching mCherry for GFP. As a result, cells which have reached a partial or full EMT state during tamoxifen treatment will permanently express GFP, irrespective of their current differentiation state. Importantly, recombination by CreERT2 in mCherry-negative cells will not cause GFP expression. Therefore, cells that express N-cadherin or Tenascin C—but which are not derived from the mammary epithelium, like mesenchymal stromal cells—will remain unlabeled. Furthermore, because the system is inducible, timely tamoxifen treatment allows assessment of the kinetics and reversibility of early and late EMT-states. Using such a system, the authors found that in primary tumors, cells infrequently undergo partial EMT. However, the majority of partial EMT cells revert to an epithelial state by MET within 7 days. In contrast, mammary cancer cells only very rarely reach a full EMT state. Once reached, however, these cells retain their mesenchymal phenotype. To assess if different EMT states contribute to chemoresistance, mice were treated with paclitaxel, cyclophosphamide, or doxorubicin. While primary tumors seemed to be enriched for both partial and full EMT cells following treatment with chemotherapeutics, metastases were enriched in full EMT cells only. Based on these data, the authors claim that partial—but especially full—EMT contributes to chemoresistance which is consistent with earlier publications (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 (1235) Google Scholar). Compared to the primary tumors, lung metastases were significantly enriched in cells which had undergone partial EMT. Compellingly, elegant ex vivo imaging identified that Tenascin C-positive partial EMT cells act as highly migratory leader cells, promoting collective invasion (Figure 1B). Furthermore, in vivo transplantation of aggregates of epithelial and partial EMT cells resulted in significantly more metastases compared to aggregates consisting of any single population. Lastly, ablation of partial EMT cells reduced the metastatic burden. Together, the experimental data in this mammary cancer model strongly supports that cells undergoing partial EMT cooperate with fully differentiated epithelial cells to promote metastatic tumor progression. In contrast, cells undergoing full EMT did not noticeably contribute to the metastatic process. Currently, several attempts have been made to map EMT in vivo during cancer progression in the PyMT mammary cancer model (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 (1235) Google Scholar; Li et al., 2020Li Y. Lv Z. Zhang S. Wang Z. He L. Tang M. Pu W. Zhao H. Zhang Z. Shi Q. et al.Genetic Fate Mapping of Transient Cell Fate Reveals N-Cadherin Activity and Function in Tumor Metastasis.Dev. Cell. 2020; 54: 593-607.e5Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar; Lüönd et al., 2021Lüönd F. Sugiyama N. Bill R. Bornes L. Hager C. Tang F. Santacroce N. Beisel C. Ivanek R. Bürglin T. et al.Distinct Contributions of Partial and Full EMT to Breast Cancer Malignancy.Dev. Cell. 2021; 56: 3203-3221Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The use of these different complex genetic tracing tools has resulted in discrepancies regarding the role of EMT in metastasis formation. FSP-1-based lineage tracing showed that mesenchymal-like mammary cancer cells do not contribute to pulmonary metastases in the PyMT model (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 (1235) Google Scholar). However, this conclusion was recently disputed using an in vivo real-time EMT sensor, which showed that FSP1 is not expressed in the majority of the PyMT cancer cells (Bornes et al., 2019Bornes L. van Scheppingen R.H. Beerling E. Schelfhorst T. Ellenbroek S.I.J. Seinstra D. van Rheenen J. Fsp1-Mediated Lineage Tracing Fails to Detect the Majority of Disseminating Cells Undergoing EMT.Cell Rep. 2019; 29: 2565-2569.e3Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). In 2020, Li et al. reported that continuous N-cadherin mediated tracing of c-KIT-positive luminal cells fate mapped the majority of pulmonary PyMT metastasis (Li et al., 2020Li Y. Lv Z. Zhang S. Wang Z. He L. Tang M. Pu W. Zhao H. Zhang Z. Shi Q. et al.Genetic Fate Mapping of Transient Cell Fate Reveals N-Cadherin Activity and Function in Tumor Metastasis.Dev. Cell. 2020; 54: 593-607.e5Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). This in sharp contrast to the results of Lüönd et al. that show complete absence of metastases labeled by N-cadherin-mediated tracing (Lüönd et al., 2021Lüönd F. Sugiyama N. Bill R. Bornes L. Hager C. Tang F. Santacroce N. Beisel C. Ivanek R. Bürglin T. et al.Distinct Contributions of Partial and Full EMT to Breast Cancer Malignancy.Dev. Cell. 2021; 56: 3203-3221Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). These conflicting data stress that in vivo tracing results should be interpreted with caution, as the sensitivity and specificity of lineage tracing is likely influenced by the selective labeling of the examined cancer cells, used EMT markers, variable kinetics of tumor progression in different genetic backgrounds, and the duration of tamoxifen treatment for tracing. Forthcoming, carefully controlled comparative tracing studies will have to assess at which stages of the EMT spectrum N-cadherin is expressed and whether this differs between distinct cancer related EMT programs. Importantly, the data reported by Lüönd et al. shift Tenascin C to the frontline as a robust candidate marker of early EMT. Exploiting this marker may enable development of even more sensitive lineage tracing systems in the future. Such systems could reveal in detail the contribution of different partial EMT-associated phenotypes, not only in different stages of cancer progression but also in developmental, physiological, and different pathological processes. Distinct contributions of partial and full EMT to breast cancer malignancyLüönd et al.Developmental CellNovember 29, 2021In BriefLüönd et al. establish genetic lineage tracing systems to monitor mammary tumor cells undergoing early partial and late full epithelial-mesenchymal transition (EMT). They demonstrate that partial EMT cells, but not full EMT cells, are required for lung metastasis, while both contribute to the development of chemoresistance. Full-Text PDF Open Access
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