In this study, we identified CTCs using the previously reported CanPatrol CTC enrichment technique from peripheral blood samples of 126 patients with colorectal cancer (CRC) and found that CTCs could be classified into three subpopulations based on expression of epithelial cell adhesion molecule (EpCAM) (E-CTCs), the mesenchymal cell marker vimentin (M-CTCs), or both EpCAM and vimentin (biphenotypic E/M-CTCs). Circulating tumor microemboli (CTMs) were also identified in peripheral blood samples. Meanwhile, E-CTCs, M-CTCs, E/M-CTCs, and CTMs were detected in 76.98%, 42.06%, 56.35%, and 36.51% of the 126 patients, respectively. Interestingly, the presence of CTMs and each CTC subpopulation was significantly associated with blood lymphocyte counts and tumor-node-metastasis stage (
Colorectal cancer (CRC) is one of the most common types of tumor in industrialized countries. The primary cause of all cancer-related deaths worldwide is metastasis to distant organs. Circulating tumor cells (CTCs), which can be collected by liquid biopsy, are considered to be responsible for metastasis, and they have been widely studied over the past 5 years as potential prognostic markers for various malignancies, including CRC [
Epithelial-to-mesenchymal transition (EMT) and the concomitant acquisition of invasive potential play an important role in the biological progression of metastasis. Recent work has suggested that EMT phenotypes in CTCs may be responsible for metastasis, raising interest in the correlation between EMT-CTC subpopulations and features of metastatic cancer [
In the present study, we identified CTCs based on EMT phenotypes using the previously reported CanPatrol CTC enrichment technique from peripheral blood samples of patients with CRC and investigated the clinical significance of CTCs with different EMT phenotypes in CRC by examining the relationships between clinicopathological parameters and the relative abundance of three circulating EMT-CTC subpopulations.
This study examined blood samples collected from 126 patients who were diagnosed with CRC between July 2014 and June 2016 at the Affiliated Hospital of Binzhou Medical University (Binzhou, China). All cancer diagnoses were confirmed by histopathological analysis. The study protocol and patient consent forms were approved by the Ethics Review Committee of the Affiliated Hospital of Binzhou Medical University, and all patients signed the consent forms before inclusion in the study. The first 2 ml peripheral blood collected was discarded to avoid potential skin cell contamination from the venipuncture, and 7.5 ml blood was collected into a 10 ml tube containing 2.5 ml of EDTA anticoagulant. All blood samples were processed within 4 h of collection. Patients did not undergo surgery or received any other treatment prior to sample collection.
To avoid bias, the physicians and research scientists who collected samples, collected and/or analyzed data, or evaluated the CTC subtypes were blinded to the patient clinical characteristics. A CTC count of >2/7.5 ml blood was considered to be a CTC-positive result.
CTCs were identified using the previously reported CanPatrol CTC enrichment technique [
RNA ISH, a technique based on branched DNA (bDNA) signal amplification, was performed as described by Wu et al. [
Statistical analysis was performed using SPSS 21.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, CA, USA) software. Associations between CTCs and clinical parameters were evaluated using a two-sided
CTCs were identified according to differential expression of the epithelial marker EpCAM, the mesenchymal marker vimentin, and the leukocyte marker CD45, following enrichment of peripheral blood cells using a filter-based method. The cells were classified as E-CTCs, E/M-CTCs, and M-CTCs. Circulating tumor microemboli (CTMs) were also detected in the peripheral blood samples (Figure
Detection of CTCs and CTMs in the peripheral blood of patients with colorectal cancer. (a) EpCAM expression (red fluorescence) in epithelial CTCs (E-CTCs). (b) EpCAM and vimentin expression (red and green fluorescence, resp.) in epithelial/mesenchymal CTCs (E/M-CTCs). (c) Vimentin expression (green fluorescence) in mesenchymal CTCs (M-CTCs). (d) CTMs, consisting of E/M-CTCs. Magnification, ×100. CTC, circulating tumor cell; CTMs, circulating tumor microemboli; EpCAM, epithelial cell adhesion molecule.
The clinicopathological characteristics of the 126 patients with CRC included in this study are summarized in Table
Clinical characteristics of the patients and detection of CTCs.
Parameter | Number of patients | Percentage (%) |
---|---|---|
Total | 126 | |
|
||
Male | 68 | 53.97 |
Female | 58 | 46.03 |
|
||
<60 | 60 | 47.62 |
>60 | 66 | 52.38 |
|
||
Poor | 27 | 21.43 |
Well and moderate | 99 | 78.57 |
|
||
Yes | 70 | 55.56 |
No | 56 | 44.44 |
|
||
I | 17 | 13.49 |
II | 39 | 30.95 |
III | 41 | 32.54 |
IV | 26 | 20.63 |
|
||
≤3.5 ng/ml | 45 | 35.71 |
>3.5 ng/ml | 81 | 64.29 |
|
||
≤27 U/ml | 76 | 60.32 |
>27 U/ml | 50 | 39.68 |
|
||
Negative | 29 | 23.02 |
E | 97 | 76.98 |
E/M | 71 | 56.35 |
M | 53 | 42.06 |
CTM | 46 | 36.51 |
Among the clinicopathological features analyzed, lymph node metastasis and tumor-node-metastasis (TNM) stage were significantly associated with the presence of CTCs. As shown in Table
Correlation between CTC detection rate and clinical characteristics.
Parameter | Number of patients with CTCs |
|
|
|
|
---|---|---|---|---|---|
Negative | Positive | ||||
|
0.684 | — | 0.17 | ||
Male | 15 | 46 | |||
Female | 14 | 51 | |||
|
0.779 | — | 0.08 | ||
≤60 | 12 | 43 | |||
>60 | 17 | 54 | |||
|
0.319 | — | 1.00 | ||
Poor | 11 | 47 | |||
Well and moderate | 18 | 50 | |||
|
0.0001 | 0.33 | 13.50 | ||
No | 20 | 30 | |||
Yes | 9 | 67 | |||
|
0.0001 | 0.45 | 30.54 | ||
I | 17 | 13 | |||
II | 8 | 21 | |||
III | 2 | 37 | |||
IV | 2 | 26 | |||
|
0.05 | 0.18 | 3.848 | ||
≤3.5 ng/ml | 16 | 72 | |||
>3.5 ng/ml | 13 | 25 | |||
|
0.108 | — | 2.59 | ||
≤27 U/ml | 14 | 31 | |||
>27 U/ml | 15 | 66 |
We also quantified the immune cells in the peripheral blood of patients with CRC and analyzed the associations between immune cell parameters and the presence of CTCs (Figure
Immune cells in the peripheral blood of patients with colorectal cancer. (a) Difference in WBC, neutrophil, lymphocyte, and monocyte counts in CTC-positive and CTC-negative patients. (b) Neutrophil-to-lymphocyte ratio in CTC-positive and CTC-negative patients.
The associations between the presence of CTC subpopulations of each EMT phenotype and clinicopathological features were analyzed. As shown in Figure
Distribution of CTC subpopulations in patients with CRC. (a) Distribution of CTC subpopulations in patients with or without metastasis. (b) Distribution of CTC subpopulations in patients at different stages of CRC. CTC, circulating tumor cell; CRC, colorectal cancer.
We also analyzed the association between each CTC subpopulation and the WBC count. As shown in Figure
Quantification of WBCs in patients with colorectal cancer. (a) Total WBC, (b) lymphocytes, (c) neutrophils, (d) monocytes, and the NLR in patients lacking CTCs or positive for E-CTCs, M-CTCs, or E/M-CTCs.
CTCs derived from epithelial tumors often show highly heterogeneous expression or complete loss of epithelial markers, hampering the detection of CTCs by conventional methods based on antibody-mediated capture or cytokeratin staining [
Although millions of tumor cells are shed from a primary tumor into the bloodstream during metastasis, only a few cells survive to form new lesions [
Nevertheless, there are some limitations to the present study. First, additional CTC detection methods should be investigated to determine whether our results were influenced by the detection method. Secondly, the number of patients in our study was small and the results should be interpreted with caution, and the role of the EMT in CTC immune evasion should be confirmed by experiments
In conclusion, our results indicate that the M-CTC subpopulation or CTMs might play more important roles than other CTC subpopulations in tumor metastasis. In addition, EMT may be involved in CTC avoidance of lymphocyte-mediated clearance.
The authors declare that they have no conflicts of interest.
Fengjie Wu and Jun Zhu collected the clinical data and performed the histological analysis. Yongjiang Mao and Xiaomei Li performed the statistical analysis of case data and imaging data. Baoguang Hu conceived the study and wrote the manuscript. Dianliang Zhang revised the manuscript and gave the final approval.
The authors would like to thank Professor Yuming Li (Department of Gastrointestinal Surgery of the Affiliated Hospital of Binzhou Medical University, Binzhou, China) for his kind assistance with this study. This work was supported by grants from the Project of Medical and Health Technology Development Program of Shandong Province (Grant no. 2015WS0483) and the Scientific Research Starting Foundation of Binzhou Medical University (Grant no. BY2014KYQD37).