Heterogeneous Manifestations of Epithelial–Mesenchymal Plasticity of Circulating Tumor Cells in Breast Cancer Patients
Abstract
:1. Introduction
2. Results
2.1. Frequency Analysis of CTCs with Co-Expression of Epithelial Markers Depending on Snail, N-Cadherinn, and Vimentin Expression
2.2. Quantitative Analysis of CTC Subsets with Heterogeneous EMT Manifestations
2.3. Interpersonal Heterogeneity of the CTC Subsets
2.4. The Phenotypes of Tumor Cells in the Primary Tumor and in the Peripheral Blood
2.5. Association of CTC Phenotypes with Clinicopathological Characteristics of Breast Cancer
3. Discussion
4. Materials and Methods
4.1. Patients
4.2. CTC Enrichment and Flow Cytometry
4.3. Multiplex Immunofluorescence (mIF) Staining
4.4. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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EpCAM+ CK7− | EpCAM+ CK7+ | EpCAM− CK7+ | ||
---|---|---|---|---|
1 | 2 | 3 | ||
Snail– N-cadherin– Vimentin– | a | 7.05 (0.83–17.01) | 0.00 (0.00–0.00) p1–2 = 0.000 | 1.66 (0.00–5.39) p1–3 = 0.007 p2–3 = 0.000 |
Snail– N-cadherin– Vimentin+ | b | 0.83 (0.00–1.66) p = 0.000 | 0.00 (0.00–0.00) p1–2 = 0.000 p = 0.115 | 3.73 (0.83–7.07) p1–3 = 0.001 p2–3 = 0.000 p = 0.126 |
Snail+ N-cadherin– Vimentin– | c | 0.00 (0.00–0.83) | 0.00 (0.00–0.00) p1–2 = 0.005 | 0.00 (0.00–0.00) p1–3 = 0.091 p2–3 = 0.043 |
Snail+ N-cadherin– Vimentin+ | d | 0.00 (0.00–0.00) p = 0.012 | 0.00 (0.00–0.00) p1–2 = 0.117 p - no data | 0.41 (0.00–1.66) p1–3 = 0.002 p2–3 = 0.001 p = 0.021 |
Snail– N-cadherin+ Vimentin– | e | 0.00 (0.00–0.83) | 0.00 (0.00–0.00) p1–2 = 0.008 | 0.00 (0.00–0.00) p1–3 = 0.007 p2–3 = 0.248 |
Snail– N-cadherin+ Vimentin+ | f | 0.00 (0.00–0.83) p = 0.139 | 0.00 (0.00–0.83) p1–2 = 0.529 p = 0.010 | 1.24 (0.00–4.56) p1–3 = 0.015 p2–3 = 0.001 p = 0.000 |
Snail+ N-cadherin+ Vimentin– | g | 0.00 (0.00–0.83) | 0.00 (0.00–0.00) p1–2 = 0.019 | 0.00 (0.00–0.00) p1–3 = 0.003 p2–3 = 0.201 |
Snail+ N-cadherin+ Vimentin+ | h | 0.00 (0.00–0.41) p = 0.352 | 0.00 (0.00–1.66) p1–2 = 0.151 p = 0.002 | 0.00 (0.00–1.24) p1–3 = 0.130 p2–3 = 0.733 p = 0.005 |
CTC Phenotypes | Tumor Size, sm | |
---|---|---|
<2 | 2–5 | |
CD45–EpCAM+CK7–Snail–N-cadherin–Vimentin– | 0.00 (0.00–3.32) | 12.45 (1.66–17.43) p = 0.038 |
CD45–EpCAM+CK7–Snail–N-cadherin–Vimentin+ | 0.00 (0.00–0.00) | 0.83 (0.83–2.49) p = 0.024 |
CD45–EpCAM–CK7+Snail–N-cadherin–Vimentin– | 0.00 (0.00–0.00) | 1.66 (0.83–9.96) p = 0.038 |
Lymph Node Metastasis | ||
---|---|---|
No | Yes | |
CD45–EpCAM–CK7+Snail–N-cadherin+Vimentin+ | 46.7 (14/30) | 66.7 (6/9) p = 0.45 |
CD45–EpCAM–CK7+Snail+N-cadherin+Vimentin+ | 16.7 (5/30) | 55.6 (5/9) p = 0.03 |
CD45–EpCAM+CK7+Snail–N-cadherin+Vimentin+ | 30.0 (9/30) | 33.3 (3/9) p = 1.00 |
CD45–EpCAM+CK7+Snail+N-cadherin+Vimentin+ | 33.3 (10/30) | 33.3 (3/9) p = 1.00 |
CD45–EpCAM–CK7+Snail–N-cadherin+Vimentin– | 10.0 (3/30) | 22.2 (2/9) p = 0.63 |
CD45–EpCAM+CK7+Snail–N-cadherin+Vimentin– | 6.7 (2/30) | 11.1 (1/9) p = 0.55 |
CD45–EpCAM–CK7+Snail+N-cadherin+Vimentin– | 3.3 (1/30) | 11.1 (1/9) p = 0.41 |
CD45–EpCAM+CK7+Snail+N-cadherin+Vimentin– | 3.3 (1/30) | 33.3 (3/9) p = 0.03 |
CD45–EpCAM+CK7–Snail–N-cadherin–Vimentin– | 50.0 (15/30) | 77.8 (7/9) p = 0.25 |
CD45–EpCAM+CK7–Snail+N-cadherin–Vimentin– | 23.3 (7/30) | 33.3 (3/9) p = 0.68 |
CD45–EpCAM+CK7–Snail–N-cadherin–Vimentin+ | 46.7 (14/30) | 55.5 (5/9) p = 0.71 |
CD45–EpCAM+CK7–Snail+N-cadherin–Vimentin+ | 6.7 (2/30) | 22.2 (2/9) p = 0.22 |
CD45–EpCAM–CK7+Snail–N-cadherin–Vimentin+ | 56.7 (17/30) | 88.9 (8/9) p = 0.12 |
CD45–EpCAM–CK7+Snail–N-cadherin–Vimentin– | 40.0 (12/30) | 77.8 (7/9) p = 0.06 |
CD45–EpCAM–CK7+Snail+N-cadherin–Vimentin+ | 30.0 (9/30) | 55.5 (5/9) p = 0.23 |
CD45–EpCAM–CK7+Snail+N-cadherin–Vimentin– | 13.3 (4/30) | 11.1 (1/9) p = 1.00 |
CD45–EpCAM+CK7+Snail–N-cadherin–Vimentin+ | 10.0 (3/30) | 0.0 (0/9) p = 1.00 |
CD45–EpCAM+CK7+Snail–N-cadherin–Vimentin– | 16.7 (5/30) | 11.1 (1/9) p = 1.00 |
CD45–EpCAM+CK7+Snail+N-cadherin–Vimentin+ | 3.3 (1/30) | 0.0 (0/9) p = 1.00 |
CD45–EpCAM+CK7+Snail+N-cadherin–Vimentin– | 0.0 (0/30) | 0.0 (0/9) p-no data |
CD45–EpCAM+CK7–Snail–N-cadherin+Vimentin– | 26.7 (8/30) | 33.3 (3/9) p = 0.69 |
CD45–EpCAM+CK7–Snail+N-cadherin+Vimentin+ | 10.0 (3/30) | 44.4 (4/9) p = 0.03 |
CD45–EpCAM+CK7–Snail+N-cadherin+Vimentin– | 20.0 (6/30) | 55.5 (5/9) p = 0.08 |
CD45–EpCAM+CK7–Snail–N-cadherin+Vimentin+ | 23.3 (7/20) | 55.5 (5/9) p = 0.10 |
Lymph Node Metastasis | ||
---|---|---|
No | Yes | |
CD45–EpCAM–CK7+Snail–N-cadherin+Vimentin+ | 1.24 (0.00–3.32) | 1.24 (0.41–6.22) p = 0.86 |
CD45–EpCAM–CK7+Snail+N-cadherin+Vimentin+ | 0.00 (0.00–0.41) | 0.83 (0.00–2.07) p = 0.19 |
CD45–EpCAM+CK7+Snail–N-cadherin+Vimentin+ | 0.00 (0.00–0.83) | 0.00 (0.00–0.83) p = 0.67 |
CD45–EpCAM+CK7+Snail+N-cadherin+Vimentin+ | 0.41(0.00–1.24) | 0.00 (0.00–2.90) p = 0.98 |
CD45–EpCAM–CK7+Snail–N-cadherin+Vimentin– | 0.00 (0.00–0.00) | 0.00 (0.00–0.41) p = 0.82 |
CD45–EpCAM+CK7+Snail–N-cadherin+Vimentin– | 0.00 (0.00–0.00) | 0.00 (0.00–0.00) p = 0.90 |
CD45–EpCAM–CK7+Snail+N-cadherin+Vimentin– | 0.00 (0.00–0.00) | 0.00 (0.00–0.00) p = 0.78 |
CD45–EpCAM+CK7+Snail+N-cadherin+Vimentin– | 0.00 (0.00–0.00) | 0.00 (0.00–0.83) p = 0.21 |
CD45–EpCAM+CK7–Snail–N-cadherin–Vimentin– | 9.54 (0.41–17.43) | 6.22 (2.49–11.20) p = 0.63 |
CD45–EpCAM+CK7–Snail+N-cadherin–Vimentin– | 0.00 (0.00–0.83) | 0.00 (0.00–4.15) p = 0.78 |
CD45–EpCAM+CK7–Snail–N-cadherin–Vimentin+ | 0.83 (0.00–2.90) | 0.83 (0.00–1.24) p = 0.38 |
CD45–EpCAM+CK7–Snail+N-cadherin–Vimentin+ | 0.00 (0.00–0.00) | 0.00 (0.00–0.41) p = 0.60 |
CD45–EpCAM–CK7+Snail–N-cadherin–Vimentin+ | 3.32 (0.83–7.07) | 4.15 (1.66–6.22) p = 0.82 |
CD45–EpCAM–CK7+Snail–N-cadherin–Vimentin– | 1.66 (0.00–7.05) | 1.66 (0.83–2.90) p = 0.94 |
CD45–EpCAM–CK7+Snail+N-cadherin–Vimentin+ | 0.00 (0.00–1.24) | 0.83 (0.00–1.66) p = 0.60 |
CD45–EpCAM–CK7+Snail+N-cadherin–Vimentin– | 0.00 (0.00–0.00) | 0.00 (0.00–0.00) p = 0.70 |
CD45–EpCAM+CK7+Snail–N-cadherin–Vimentin+ | 0.00 (0.00–0.00) | 0.00 (0.00–0.00) p = 0.56 |
CD45–EpCAM+CK7+Snail–N-cadherin–Vimentin– | 0.00 (0.00–0.41) | 0.00 (0.00–0.00) p = 0.60 |
CD45–EpCAM+CK7+Snail+N-cadherin–Vimentin+ | 0.00 (0.00–0.00) | 0.00 (0.00–0.00) p = 0.86 |
CD45–EpCAM+CK7+Snail+N-cadherin–Vimentin– | 0.00 (0.00–0.00) | 0.00 (0.00–0.00) p-no data |
CD45–EpCAM+CK7–Snail–N-cadherin+Vimentin– | 0.00 (0.00–1.24) | 0.00 (0.00–0.83) p = 0.63 |
CD45–EpCAM+CK7–Snail+N-cadherin+Vimentin+ | 0.00 (0.00–0.00) | 0.41 (0.00–1.66) p = 0.19 |
CD45–EpCAM+CK7–Snail+N-cadherin+Vimentin– | 0.00 (0.00–0.83) | 0.83 (0.00–1.24) p = 0.21 |
CD45–EpCAM+CK7–Snail–N-cadherin+Vimentin+ | 0.00 (0.00–0.83) | 1.24 (0.00–1.66) p = 0.18 |
Parameter | Frequency, % (n) | |
---|---|---|
Age | <35 | 5.1 (2/39) |
35–50 | 30.8 (12/39) | |
>50 | 64.1 (25/39) | |
Menstrual function | Premenopausal | 33.3 (13/39) |
Postmenopausal | 66.7 (26/39) | |
Tumour size | <20 mm | 23.1 (9/39) |
20–50 mm | 71.8 (28/39) | |
No data | 5.1 (2/39) | |
Stage | I | 28.2 (11/39) |
IIA | 53.8 (21/39) | |
IIB | 18.0 (7/39) | |
Molecular subtype | Luminal A | 33.3 (13/39) |
Luminal B | 59.0 (23/39) | |
Triple negative | 5.1 (2/39) | |
Her2-positive | 2.6 (1/39) | |
Tumour grade | 1 | 23.1 (9/39) |
2 | 69.2 (27/39) | |
3 | 7.7 (3/39) | |
Estrogen receptor | positive | 92.3 (36/39) |
negative | 7.7 (3/39) | |
Progesteron receptor | positive | 66.7 (26/39) |
negative | 33.3 (13/39) | |
HER2/neu | positive | 28.2 (11/39) |
negative | 71.8 (28/39) | |
Ki67 expression | <20% | 46.2 (18/39) |
>20% | 53.8 (21/39) | |
Lymph node metastasis | Yes | 23.1 (9/39) |
No | 76.9 (30/39) | |
Distant metastasis | Yes | 0.0 (0/39) |
No | 100.0 (39/39) | |
Intraoperative radiation therapy | No | 20.5 (8/39) |
Yes | 69.2 (27/39) | |
No data | 10.3 (4/39) |
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Tashireva, L.A.; Savelieva, O.E.; Grigoryeva, E.S.; Nikitin, Y.V.; Denisov, E.V.; Vtorushin, S.V.; Zavyalova, M.V.; Cherdyntseva, N.V.; Perelmuter, V.M. Heterogeneous Manifestations of Epithelial–Mesenchymal Plasticity of Circulating Tumor Cells in Breast Cancer Patients. Int. J. Mol. Sci. 2021, 22, 2504. https://doi.org/10.3390/ijms22052504
Tashireva LA, Savelieva OE, Grigoryeva ES, Nikitin YV, Denisov EV, Vtorushin SV, Zavyalova MV, Cherdyntseva NV, Perelmuter VM. Heterogeneous Manifestations of Epithelial–Mesenchymal Plasticity of Circulating Tumor Cells in Breast Cancer Patients. International Journal of Molecular Sciences. 2021; 22(5):2504. https://doi.org/10.3390/ijms22052504
Chicago/Turabian StyleTashireva, Liubov A., Olga E. Savelieva, Evgeniya S. Grigoryeva, Yuri V. Nikitin, Evgeny V. Denisov, Sergey V. Vtorushin, Marina V. Zavyalova, Nadezhda V. Cherdyntseva, and Vladimir M. Perelmuter. 2021. "Heterogeneous Manifestations of Epithelial–Mesenchymal Plasticity of Circulating Tumor Cells in Breast Cancer Patients" International Journal of Molecular Sciences 22, no. 5: 2504. https://doi.org/10.3390/ijms22052504