Page 270 - Read Online

Esophageal cancer is an aggressive cancer, affecting Development and maintenance of esophageal
450,000 patients. In esophageal squamous cell carcinoma CAF
(ESCC) expression of HGF and fibroblast growth factor Peripheral blood from an esophageal squamous cell
(FGF) in CAFs has been found to be related to tumor cell carcinoma (ESCC) patient was taken for extraction of
proliferation. CAF-derived wnt2, an important signaling CAF. Ficol-gradient was performed and separated cells
[4]
molecule, was able to enhance a process called epithelial cultured in RPMI supplemented with 10% FBS, Penstrep,
mesenchymal transition (EMT). This EMT involves loss and glutamate. After 24 h of culture the media were
of intracellular adhesion and polarity by tumor cells of replaced with complete DMEM supplemented with 10%
epithelial origin. These cells can be transformed into FBS, 1% Penstrep, 0.2% Glutamate, and Vit C. CAFs
mesenchymal cells with the capability of migration and were seen after about 34 days of culturing. The cells were
invasion. Protein levels of CAF are also related to a confluent within a week, then stored in -80℃ while some
[5]
poor prognosis of patients with esophageal cancer. Also, were maintained in culture. These cells were labeled as
proteins such as α smooth muscle actin, CD-10, and esophageal CAF. Frozen cells were revived and cultured
periostin have been found to be related to the poor patient in growth medium at passage number 31. Culture dishes
survival. Thus, it is evident that CAFs are important to were incubated at 37℃ with 5% CO and the media
[3]
2
tumor cells in esophageal cancer since they are associated removed on alternate days, followed by washing of cells
with invasion, migration, and a poor prognosis. Many with PBS and supplementing with new media. After
drug trials are carried out in order to develop a successful reaching confluency cells were trypsinized with 1%
treatment strategy against esophageal cancer, but the trypsin and transferred into fresh flask for expansion.
role of CAF has been neglected. Hence, it is essential to
attempt to target their CAF cells in order to prevent tumor Staining of CAF cell line (Giemsa, Alizarin Red,
progression. and Oil Red staining)

Culture plates were washed with PBS, fixed with methanol
In current cancer research nanoparticles are replacing (50%), and incubated for 30 min at 4℃. The plates were
traditional chemotherapeutic drugs because of their then washed with D/W to remove the methanol. Fixed and
specificity, small size, and permeability into cells. washed plates of CAF were stained with Giemsa stain.
Nanoparticles made up of biodegradable material such
as chitosan have appeal since they are cheaper, do not Alizarin Red staining was required for the methanol-fixed
involve toxic chemicals in their preparation, and have low cells, which were stained with 2% Alizarin stain (pH 4.2)
cell cytotoxicity. The chitosan nanoparticle has shown for 30 min. After oil red staining, the fixed and washed
[6]
cells were incubated with 60% isopropanol for 5 min.
therapeutic significance in various cancers, including
breast, gastric, and oral cancers. Chitosan nanoparticles This was followed by removing the isopropanol and then
have not been explored in esophageal cancer and their staining with the 0.3% oil red for 5 min. After staining
effect on CAFs has not yet studied. The current project the plates were washed with D/W to remove excess stain.
was designed to understand the effects of chitosan Phase contrast microscopy
nanoparticle on human peripheral blood-derived CAF by
performing gene expression studies. We have attempted to Inverted Phase contrast Microscope (Carl Zeiss Co.)
demonstrate that chitosan nanoparticles alter expressions was used for studying morphology of the cultured cells.
of genes involved in esophageal tumors and have found The microscope was attached to the computer having TS
that these nanoparticles effectively reduced the metastasis View software for observing and capturing the images.
of CAF cells. These results suggest that using chitosan The cells were monitored regularly with the use of phase
nanoparticles targeting esophageal CAFs could be a contrast microscope and images captured.
potential therapeutic strategy against esophageal cancers.
Chitosan nanoparticle (Ch-Np) preparation
METHODS Ch-Np was prepared using the ionic gelation method.
Low molecular weight chitosan was dissolved in 1%
Materials acetic acid under constant stirring conditions. Zero
Low Molecular weight Chitosan (≥ 75% deacetylation), point one percent sTPP prepared in D/W was added
sodium Tri-polyphosphate (sTPP), Acetic Acid, 1N in the chitosan solution, drop by drop, under constant
NaOH, D/W, Low-glucose Dulbecco’s Modified Eagle stirring. A solution change from clear to turbid was
Medium (DMEM) , Fetal Bovine Serum (FBS), Penicillin taken as confirmation for nanoparticle formation. pH
Streptomycin (PenStrep), L-Glutamine, Vitamin C, of the chitosan solution was adjusted to 7 using 1N
Phosphate Buffer Saline (PBS), Trypsin EDTA, TRIZOL NaOH. Formed nanoparticles suspended in the solution
reagent, cDNA Preparation kit (Applied Biosystem, were separated by centrifuging at 2000 g for 3 min. The
USA), Agarose, Primers for Actin, Keratin18, Vimentin, supernatant was discarded and the nanoparticles were
VEGF, MMP1, MMP9, E-cadherin, CXCR-4, CXCR-7, washed with DMEM and again centrifuged in order to
CCR5, Sdf1α, Oct4, Nanog, SOX-2 were purchased from remove any chemical residue. The nanoparticles were
Sigma Chemicals, USA. then suspended in the media for later use.
260
Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ July 29, 2016 ¦