10. We have found that 10
7 cells are suitable for efficient isolation.
A greater or lesser number of cells can be used, but this may
require optimization (e.g., additional washes, more volume,
etc.).
11. Step 9 in Subheading 3.3 is optional for confirmation purposes. Generally, flow cytometry is used to confirm purity, but
other methods can be used (i.e., western blotting, microscopy,
etc.) (Fig. 2b).
12. The LD column can be removed from the separator to collect
the ECAD-intermediate population if desired.
13. Although the unstained control contains both antibody colors,
for clarity we typically run single color controls as well as the
dual color.
References
1. Yuan S, Norgard RJ, Stanger BZ (2019) Cellular plasticity in cancer. Cancer Discov 9
(7):837–851.
https://doi.org/10.1158/
2159-8290.CD-19-0015
2. Dongre A, Weinberg RA (2019) New insights
into the mechanisms of epithelial–mesenchymal transition and implications for cancer. Nat
Rev Mol Cell Biol 20(2):69–84
3. Lamouille S, Xu J, Derynck R (2014) Molecular mechanisms of epithelial-mesenchymal
transition. Nat Rev Mol Cell Biol
15:178–196.
https://doi.org/10.1038/
nrm3758
4. Pastushenko I, Blanpain C (2019) EMT transition states during tumor progression and
metastasis. Trends Cell Biol 29(3):212–226
5. Nieto MA, Huang RYYJ, Jackson RAA, Thiery
JPP (2016) EMT: 2016. Cell 166:21–45
6. Aiello NM, Maddipati R, Norgard RJ et al
(2018) EMT subtype influences epithelial plasticity and mode of cell migration. Dev Cell
45:681–695.e4. https://doi.org/10.1016/j.
devcel.2018.05.027
7. Maddipati R, Stanger BZ (2015) Pancreatic
cancer metastases harbor evidence of polyclonality. Cancer Discov 5:1086–1097. https://
doi.org/10.1158/2159-8290.CD-15-0120
8. Jolly MK, Somarelli JA, Sheth M et al (2019)
Hybrid epithelial/mesenchymal phenotypes
promote metastasis and therapy resistance
across
carcinomas.
Pharmacol
Ther
194:161–184
9. Shibue T, Weinberg RA (2017) EMT, CSCs,
and drug resistance: the mechanistic link and
clinical implications. Nat Rev Clin Oncol 14
(10):611–629
10. Tarin D (2005) The fallacy of epithelial mesenchymal transition in neoplasia. Cancer Res
65:5996–6000
11. Rhim AD, Mirek ET, Aiello NM et al (2012)
EMT and dissemination precede pancreatic
tumor formation. Cell 148:349–361. https://
doi.org/10.1016/j.cell.2011.11.025
326
Robert J. Norgard and Ben Z. Stanger
7 cells are suitable for efficient isolation.
A greater or lesser number of cells can be used, but this may
require optimization (e.g., additional washes, more volume,
etc.).
11. Step 9 in Subheading 3.3 is optional for confirmation purposes. Generally, flow cytometry is used to confirm purity, but
other methods can be used (i.e., western blotting, microscopy,
etc.) (Fig. 2b).
12. The LD column can be removed from the separator to collect
the ECAD-intermediate population if desired.
13. Although the unstained control contains both antibody colors,
for clarity we typically run single color controls as well as the
dual color.
References
1. Yuan S, Norgard RJ, Stanger BZ (2019) Cellular plasticity in cancer. Cancer Discov 9
(7):837–851.
https://doi.org/10.1158/
2159-8290.CD-19-0015
2. Dongre A, Weinberg RA (2019) New insights
into the mechanisms of epithelial–mesenchymal transition and implications for cancer. Nat
Rev Mol Cell Biol 20(2):69–84
3. Lamouille S, Xu J, Derynck R (2014) Molecular mechanisms of epithelial-mesenchymal
transition. Nat Rev Mol Cell Biol
15:178–196.
https://doi.org/10.1038/
nrm3758
4. Pastushenko I, Blanpain C (2019) EMT transition states during tumor progression and
metastasis. Trends Cell Biol 29(3):212–226
5. Nieto MA, Huang RYYJ, Jackson RAA, Thiery
JPP (2016) EMT: 2016. Cell 166:21–45
6. Aiello NM, Maddipati R, Norgard RJ et al
(2018) EMT subtype influences epithelial plasticity and mode of cell migration. Dev Cell
45:681–695.e4. https://doi.org/10.1016/j.
devcel.2018.05.027
7. Maddipati R, Stanger BZ (2015) Pancreatic
cancer metastases harbor evidence of polyclonality. Cancer Discov 5:1086–1097. https://
doi.org/10.1158/2159-8290.CD-15-0120
8. Jolly MK, Somarelli JA, Sheth M et al (2019)
Hybrid epithelial/mesenchymal phenotypes
promote metastasis and therapy resistance
across
carcinomas.
Pharmacol
Ther
194:161–184
9. Shibue T, Weinberg RA (2017) EMT, CSCs,
and drug resistance: the mechanistic link and
clinical implications. Nat Rev Clin Oncol 14
(10):611–629
10. Tarin D (2005) The fallacy of epithelial mesenchymal transition in neoplasia. Cancer Res
65:5996–6000
11. Rhim AD, Mirek ET, Aiello NM et al (2012)
EMT and dissemination precede pancreatic
tumor formation. Cell 148:349–361. https://
doi.org/10.1016/j.cell.2011.11.025
326
Robert J. Norgard and Ben Z. Stanger