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Potdar et al. Circulating tumor cells in liquid biopsies

containing PBMCs isolated from all metastatic [Figure 3]. CTCs from CNS cancer exhibited expression
cancer patients [Figure 2B]. Hence PBMC samples of KRT18 and NOTCH2 and absence of expression of
from metastatic cancer patients clearly contained a PROM1, CD44, and CXCR4, whereas prostate cancer
tumorigenic population, whereas those from healthy CTCs expressed all five genes [Figure 3]. Overall our
individuals did not. We also observed the formation of results indicated that the molecular profiles of CTCs
circular rings around each colony, indicating that the varied according to the type of cancer; therefore, we
cells secreted proteolytic enzymes [Figure 2B]. This suggest that individual profiling of metastatic cancer
assay only indicates the basic cancer phenotype of patients will be essential for management of therapy
cells; tumorigenic potential can be confirmed by “in vivo in these patients.
transformation assays” using nude mice, or by PCR
determining their molecular phenotypes. We confirmed Molecular markers in PBMCs from metastatic
the tumorigenicity of the cells by molecular analysis. cancer patients (liquid biopsies)
We also examined molecular markers present in
Molecular markers in isolated cultured CTCs whole plasma PBMCs (liquid biopsies), from patients
To determine the molecular cancer phenotypes of with metastatic breast (n = 3), ovarian (n = 1),
isolated CTCs, we harvested cells after 30 days of nasopharyngeal (n = 1), and prostate (n = 1) cancer. We
analyzed expression of 11 genes related to metastasis:
culture of PBMCs from metastatic cancer patients and KRT18, CD44, PROM1, CXCR4, NOTCH2, MMP1,
determined the expression of specific mRNAs involved MMP2, MMP9, KCNH2, ICAM1 and CADH1. KRT18,
in metastasis in these cells by RT-PCR. We studied CD44, PROM1, CXCR4, and NOTCH2 were expressed
the expression of only five specific genes: KRT18, by PBMCs from all patients [Figure 4] indicating that
PROM1, CD44, CXCR4, and NOTCH2, in addition to they were present in all cancer patients and have roles
the ACTB housekeeping gene in these samples, due to in the metastatic process.
the limited quantities of mRNA available. We obtained
CTCs from ovarian, prostate, and central nervous The expression of KRT19, MMP1, MMP2, MMP9,
system (CNS) cancers for this analysis. We found ICAM, and CDH1 was studied in samples from three
that ovarian cancer CTCs expressed KRT18, PROM1, breast cancer patients, one ovarian cancer patient,
and CD44; however, CXCR4 and NOTCH2 were not one nasopharyngeal cancer patient, and one prostate
expressed in CTCs obtained from this cancer type cancer patient, and the results are shown in Figure 5.

Figure 1: Morphology of circulating tumor cells (×20). A and B are phase contrast microscopy and C is Giemsa stained

Figure 2: Anchorage independent growth assay (×40). (A) Normal PBMCs; and (B) metastatic cancer PBMCs. Each colony shows
proteolytic enzyme ring (arrows). PBMCs: peripheral blood mononuclear cells
10 Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ January 23, 2017