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Potdar et al. Circulating tumor cells in liquid biopsies

CTC isolation and enumeration. [16] Comparison of Moreover, the values obtained by enumeration of CTCs
the OncoQuick density gradient method with Ficoll- correlated well with those from previous reports. [13,14]
Hypaque density gradients for separation of tumor
®
cells from bone marrow and peripheral blood aspirates The CELLSEARCH system, is primarily selective for
[34]
indicated that CTCs can be easily aspirated by both EpCAM- and cytokeratin-positive CTCs; however,
methods and analyzed further to determine their Lianidou et al. demonstrated that CTCs which have
[15]
presence and quantity. In the present study, we used undergone EMT exhibit down-regulation of epithelial
[30]
the simple and cost-effective Ficoll-Hypaque density markers, including EpCAM and K19 and hence such
gradient method to separate PBMCs from whole cells will be missed by this technology. Similarly, some
plasma of cancer patients. These cells were then cases of HER2-positive metastatic breast cancer have
cultured to isolate CTCs, which were identified by their EpCAM-negative CTCs. Hence, additional markers
morphology (i.e. large oval shape, with large nuclei should be considered in the development of methods
and peripheral spikes) [Figure 1]. Similar morphology to isolate and enumerate CTCs in metastatic cancer.
was first reported by Gascoyne et al. It is likely that Our study also demonstrated that KRT19 and CADH1
[31]
the spike-like processes allow CTCs to adhere to the were not expressed in liquid biopsies from any breast
surface of basement membranes during the process of cancer patients [Figure 5]. Moreover, the observed
invasion. We confirmed the phenotype of these cells absence of CADH1 expression indicated that CTCs
by studying cancer-associated genes in isolated CTCs obtained from all cancer patients were in EMT. As the
and from liquid biopsies [Figures 3 and 4]. method we used was based on counting intact cultured
CTCs by phase contrast microscopy, the possibility of
The presence of CTCs in an advanced metastatic missing CTCs was small.
cancer patient was first reported in 1869 by Ashworth;
[32]
however, limited information is available regarding the The soft agar colony formation assay is a common
numbers of CTCs in the blood of patients with different method to monitor anchorage-independent growth
stages and types of cancer, making proficient isolation in semi-solid agar during 3-4 week incubation by
[33]
of CTCs in a viable and intact state challenging. The manual counting of colonies under phase contrast
CELLSEARCH system is the only method approved microscopy. This method is considered one of the most
®
[34]
by the US FDA for clinical use in metastatic breast, accurate and sensitive in vitro assays for detection of
colorectal, and prostate cancer. [13,17,35] Patients with > malignant transformation of cells and confirmation
80 CTCs in 7.5 mL of blood were reported to die within of the phenotype of tumor cells with metastatic
[36]
one year of diagnosis from metastatic disease, and potential. In this study, we found that all PBMCs
[16]
CTCs continue to be detected after the first round of isolated from metastatic cancer patients resulted in
chemotherapy in some breast cancer patients with significant numbers of colonies on soft agar, indicating
rapid progression of the disease to metastasis; it their high tumorigenic potential. There were rings
[17]
has been suggested that, in such cases, it may be around each colony [Figure 2], indicating the activity
preferable to opt for an alternative treatment with novel of secretory proteases from these cells, which are
therapeutic agents, rather than continuing with the primarily responsible for the degradation of basement
same chemotherapy. membranes during invasion and metastasis. [37]

In the present study, we established a simple method Molecular profiling of tumor cells has become
of isolation and enumeration of CTCs from metastatic important for understanding genes involved in
cancer patients. PBMCs were cultured in 65-mm cancer development, and provides insights into the
[38]
dishes and numbers of CTC-like cells were counted selection of tumor cells and resistance mechanisms
manually under a phase contrast microscope. We in patients undergoing systemic therapies; therefore,
observed that there were more CTCs in patients with molecular profiling of liquid biopsies or isolated
final-stage, compared with early-stage, metastatic CTCs, to determine the major genes involved in the
cancer. We found approximately 50-160 CTCs in the processes of invasion and metastasis, was a primary
majority of cancer patients, representing 0.01-0.04% aim of this study. We performed molecular profiling
of overall PBMCs. All three breast cancer patients and of isolated CTCs as well as liquid biopsies obtained
one prostate cancer patient were determined to be from metastatic cancer patients. The results indicated
in the late stages of metastatic disease, according to that CTCs and liquid biopsies from all cancer patients
CTC enumeration, which was confirmed by molecular expressed KRT18, PROM1, CD44, CXCR4, and
profiling. Hence, our results indicate that enumeration NOTCH2 genes [Figures 3 and 4], demonstrating
and molecular profiling of CTCs can distinguish early- that the malignant phenotypes of CTCs and liquid
and later-stage disease in metastatic cancer patients. biopsies can be evaluated by molecular profiling. We
12 Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ January 23, 2017