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Potdar et al. Circulating tumor cells in liquid biopsies

determining the changing course of disease in a timely aliquots of PBMC-rich plasma were mixed with 300 µL
manner and have potential to determine the metastatic of 1 × PBS and this mixture was layered onto 300 µL
state of breast cancer. of Ficoll-Hypaque solution. We prepared two tubes
each for cancer patients and healthy individuals for
CTCs are present in very low numbers in whole blood our culture study. Tubes were centrifuged at 3,000 g
and are difficult to identify and characterize. The for 30 min to obtain a middle layer containing PBMCs.
primary aims of the present study were to establish The PBMCs isolated from the two tubes were pooled
a simple protocol for the isolation, identification, and together, washed with 1 × PBS, and suspended in 4 mL of
enumeration of CTCs from various metastatic cancer RPMI growth media, supplemented with 10% FBS, 1%
patients and to molecularly profile genes involved PenStrep, L-glutamine, and vitamin C. Tissue culture
in the processes of invasion and metastasis, and to dishes (65 mm) containing PBMCs were incubated in
evaluate the potential clinical utility of liquid biopsies in a 5% CO incubator at 37 °C. After 24 h, non-adherent
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the treatment of advanced stage cancer. cells were removed and the remaining adherent cells
fed with 4 mL of DMEM growth medium supplemented
METHODS with 10% FBS + PenStrep + L-glutamine, which were
changed twice a week. Cultures were observed and
Materials photographed every day for 30 days under a phase
Low-glucose Dulbecco’s modified Eagle’s Medium contrast microscope (model AXiovert 40CFL from
(DMEM), penicillin/streptomycin (PenStrep), Carl Zeiss), equipped with TS view software (Tucsen
phosphate-buffered saline (PBS), trypsin EDTA, Imaging, Fuzhou, and PR China) and images were
erythrosin B, and colchicine, were purchased from captured and analyzed to determine and record the
HiMedia (Mumbai, India); fetal bovine serum (FBS) morphology of adherent cells. Adherent cells that
from GIBCO BRL (Carboside, MA); Trizol reagent, appeared CTC-like by phase contrast microscopy were
cDNA preparation kits, and agarose from Invitrogen counted manually under a phase contrast microscope
(Carlsbad, CA, USA); and histopaque and primers for to calculate the number of cells present in each
KRT18, KRT19, PROM1, CD44, CXCR4, NOTCH2, metastatic cancer patient and were then fixed in 50%
VEGFA, MMP1, MMP2, MMP9, ICAM1, CDH1, methanol, stained with Giemsa, and examined by light
KCNH2, and ACTB from Sigma Chemicals, USA. microscopy (AXiovert 40CFL, Carl Zeiss) to determine
their general morphological features.
Sample collection
A total of eight metastatic cancer patients recruited Anchorage-independent soft agar assay
by oncology clinic of Jaslok Hospital and Research PBMCs were isolated from metastatic cancer patients
Centre, Mumbai India (three breasts, two ovarian, and healthy individuals and their tumorigenic potential
two prostate, and one nasopharyngeal cancer) and determined using soft agar assays. A total of 3 × 10
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five healthy individuals were included in this study. PBMCs per individual were layered on 0.4% soft agar
All patients had stage IV disease, with invasive and in DMEM growth medium in 65 mm dishes. The plates
metastatic cancer. All tumors were histopathologically were then incubated at 37 °C with 5% CO for 2 weeks.
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proven to have metastatic potential. Fresh blood Emerging colonies were observed under a phase
samples (10 mL) were collected from each metastatic contrast microscope and photographed.
cancer patient and healthy control individual in sterile
EDTA vacutainers with proper consent from the Molecular markers in PBMCs (liquid biopsies)
patients, according to the ethical committee guidelines and cultured CTCs
of Jaslok Hospital and Research Center, Mumbai, Total RNA was extracted from all patient’s PBMCs
India, and sent to the tissue culture laboratory of the and isolated cultured CTCs from Ovarian, prostate
Molecular Medicine and Biology Department. and CNS cancer patients using the Trizol method.
RNA was then reverse transcribed to cDNA using the
Culture of peripheral blood mononuclear cells Applied Biosystems High Capacity cDNA Kit (Applied
for isolation of CTCs Biosystems, USA). PCR was carried out using specific
Plasma rich in peripheral blood mononuclear cells forward and reverse primers with defined annealing
(PBMCs) was separated from blood samples from temperatures [Table 1]. For all genes, PCR reactions
cancer patients and healthy individuals by allowing were performed at 95 °C for 5 min; followed by 40
blood to stand for approximately 1 h. We obtained cycles at 95 °C for 30 s, at the respective annealing
approximately 2 mL of PBMC-rich plasma from 10 mL of temperatures [Table 1] for 30 s, and at 72 °C for 30 s;
blood. Next, PBMCs were isolated by Ficoll-Hypaque with a final extension at 72 °C for 7 min. Amplicon sizes
gradient centrifugation as follows. Briefly, 300 µL were checked by 2% agarose gel electrophoresis,
8 Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ January 23, 2017