Open Access Research article

Dynamics of circulating endothelial cells and endothelial progenitor cells in breast cancer patients receiving cytotoxic chemotherapy

Yu-Hsuan Kuo12, Ching-Hung Lin2, Wen-Yi Shau3, Te-Jung Chen24, Shih-Hung Yang2, Shu-Min Huang2, Chun Hsu2, Yen-Shen Lu2* and Ann-Lii Cheng25*

Author Affiliations

1 Department of Oncology, Chi-Mei Hospital, Tainan, Taiwan

2 Department of Oncology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, 10002, Taiwan

3 Center For Drug Evaluation, Division of Health Technology Assessment, Taipei, Taiwan

4 Oncology Translational Research Center, TTY Biopharm Company Limited, Taipei, Taiwan

5 Departments of Internal Medicine and Oncology, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei, 10002, Taiwan

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BMC Cancer 2012, 12:620  doi:10.1186/1471-2407-12-620

Published: 26 December 2012

Additional files

Additional file 1: Figure S1.:

CEC and CEP test were preformed within 24 hours of collection of blood samples. The gating strategy is described below. Exclude debris and red blood cells first. CEC and CEPs are within CD45dim population (P2). CD31/CD146 double positive population (Q2) were defined as CECs (S-Figure 1 and 2). Both CD31/CD133 (Q2-1) and CD146/CD133 (Q2-2) double positive were CEPs (S-Figure 1 and 2). CEPs number presented here were the average of Q2-1 dot number and Q2-2 dot number. For gating viable-CEC, excluding debris and red blood cells first and CEC and CEPs are within CD45dim population (P2). CD146 was CEC maker and 7AAD staining was used to identify the cell viability. Cells in Q2 are apoptotic CECs and in Q4 are viable CECs. (S-Figure 3 and 4) Unstain sample was used as a negative control (S-Figure 1 and 3). CEC/CEP and viable CEP gating was follow the unstain one. (S-Figure 2 and 4).

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Additional file 2: Figure S2.:

CEC and CEP test were preformed within 24 hours of collection of blood samples. The gating strategy is described below. Exclude debris and red blood cells first. CEC and CEPs are within CD45dim population (P2). CD31/CD146 double positive population (Q2) were defined as CECs (S-Figure 1 and 2). Both CD31/CD133 (Q2-1) and CD146/CD133 (Q2-2) double positive were CEPs (S-Figure 1 and 2). CEPs number presented here were the average of Q2-1 dot number and Q2-2 dot number. For gating viable-CEC, excluding debris and red blood cells first and CEC and CEPs are within CD45dim population (P2). CD146 was CEC maker and 7AAD staining was used to identify the cell viability. Cells in Q2 are apoptotic CECs and in Q4 are viable CECs. (S-Figure 3 and 4) Unstain sample was used as a negative control (S-Figure 1 and 3). CEC/CEP and viable CEP gating was follow the unstain one. (S-Figure 2 and 4).

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Additional file 3: Figure S3.:

CEC and CEP test were preformed within 24 hours of collection of blood samples. The gating strategy is described below. Exclude debris and red blood cells first. CEC and CEPs are within CD45dim population (P2). CD31/CD146 double positive population (Q2) were defined as CECs (S-Figure 1 and 2). Both CD31/CD133 (Q2-1) and CD146/CD133 (Q2-2) double positive were CEPs (S-Figure 1 and 2). CEPs number presented here were the average of Q2-1 dot number and Q2-2 dot number. For gating viable-CEC, excluding debris and red blood cells first and CEC and CEPs are within CD45dim population (P2). CD146 was CEC maker and 7AAD staining was used to identify the cell viability. Cells in Q2 are apoptotic CECs and in Q4 are viable CECs. (S-Figure 3 and 4) Unstain sample was used as a negative control (S-Figure 1 and 3). CEC/CEP and viable CEP gating was follow the unstain one. (S-Figure 2 and 4).

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Additional file 4: Figure S4.:

CEC and CEP test were preformed within 24 hours of collection of blood samples. The gating strategy is described below. Exclude debris and red blood cells first. CEC and CEPs are within CD45dim population (P2). CD31/CD146 double positive population (Q2) were defined as CECs (S-Figure 1 and 2). Both CD31/CD133 (Q2-1) and CD146/CD133 (Q2-2) double positive were CEPs (S-Figure 1 and 2). CEPs number presented here were the average of Q2-1 dot number and Q2-2 dot number. For gating viable-CEC, excluding debris and red blood cells first and CEC and CEPs are within CD45dim population (P2). CD146 was CEC maker and 7AAD staining was used to identify the cell viability. Cells in Q2 are apoptotic CECs and in Q4 are viable CECs. (S-Figure 3 and 4) Unstain sample was used as a negative control (S-Figure 1 and 3). CEC/CEP and viable CEP gating was follow the unstain one. (S-Figure 2 and 4).

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Additional file 5: Figure S5.:

Representative flow cytometry dot plot for defining viable CECs and apoptotic CECs, (A) Exclude debris and red blood cells. (B) CEC and CEPs are within CD45dim population (P2). (C)CD146 was CEC maker and 7AAD staining was used to identify the cell viability. Cells in Q2 are apoptotic CECs and in Q4 are viable CECsRepresentative data of dynamic change of CEC, CEP (S-Figure 5a, 5c, 5e and 5 g) and viable-CEC (S-Figure 5b, 5d, 5f and 5 h) levels during second cycle of chemotherapy from one patient. CEC, CEP (S-Figure 5a) and viable-CEC (S-Figure 5b) at the day before chemotherapy (for this patient, taxotere /epirubicin /cyclophosphamide) were shown in S-Figure 5a and 5b. Patient’s CEC, CEP and viable-CEC levels were dropping at day 4 and day 7 (S-Figure 5c, 5d, 5e and 5f) after chemotherapy. Three weeks after chemotherapy, CEC and CEP levels were increased again(S-Figure 5e) and most of the CECs were viable (S-Figure 5f).

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Additional file 6: Figure S6.:

Standardized trend of CEC, V-CEC, CEP, as a function of chemotherapy the day before tumor resection (A-D), or after tumor resection.(E-G). The CEC and CEP kinetics consistently showed similar wave pattern and had no obvious differences between patients with and without tumor bearing.

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Additional file 7: Figure S7.:

Standardized trend of CEC, V-CEC, CEP, as a function of different chemotherapy regimens. (A) Data from a patient who received adjuvant docetaxel, cisplatin, and herceptin. (B) Data from a patient who received adjuvant cyclophosphamide, epirubicin, and Fluorouracil. (C) Data from a patient who received adjuvant docetaxel, epirubicin, and cyclophophamide. (D) Data from a patient who received neoadjuvant docetaxel, Epirubicin, and cyclophophamide. (E) Data from a patient who received neoadjuvant vinorelbine and infusion fluorouracil. Their CEC and CEP kinetics consistently show similar wave pattern. It suggests that dynamic changes of CEC and CEP induced by chemotherapy may have more significant effect than using different drugs.

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