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as shown in Figure 6. for inhibition of growth of metastatic esophageal cancer.

Chemokines in CAF- and Ch-Np-treated CAF Cancer cells are resistant to contact inhibition, a common
CXCR4, CXCR7, and CCR5 are chemokine receptors phenomenon in normal cells. In current study CAF cells
[10]
mainly involved in the process of metastasis where Sdf- showed random overlapping cell growth in the control
1α is a ligand to CXCR4. Their expressions were studied plates, whereas in Ch-Np-treated cells they were aligned
in CAF and Ch-Np-treated CAF. Figure 7 shows complete in parallel fashion and showed clear cut monolayer cells,
down-regulation of CXCR4 and CCR5 in Ch-Np treated as if they were having normal phenotypic growth. The
CAF cells. Also, there was slight expression observed results further showed that these cells seem to have better
in CXCR7 and Sdf-1α in Ch-Np treated CAF cells as contact inhibition than control CAF cells, which had
compared to control CAF cells, as shown in Figure 7. metastatic potential. This might be the reason for the
[11]
Control CAF cells expressed all these genes normally random growth of CAF since they were isolated from
[Figure 7]. metastatic PBMC cells of the esophageal cancer patient.
These phenotypic changes were confirmed by molecular
Evaluation of metastatic potency of CAF cells markers studied in this project.
by scratch assay
Scratch assay is mainly useful to evaluate migration Several studies have shown that Oct4, Nanog, and Sox2
potencies of metastatic cells. As CAFs were isolated from are excellent pluripotency markers in cancer cells. [12,13]
a metastatic patient, the scratch assay was used to study In the present study mRNA expression of Oct4, Nanog,
the metastatic potency in control CAF cells and Ch-Np- and Sox2 in control CAF and Ch-Np-treated CAF were
treated CAF. studied. Prominent expression of this pluripotency
marker in control CAF was found. According to one
This effect was studied at two time points, i.e, 24 and study, Oct4 played an important role in promoting
[14]
48 h after Ch-Np treatment, as shown in Figure 8. It was carcinogenesis and also in preventing cancer cells from
observed that there was good migration of these cells in undergoing apoptosis. Another group found that
[15]
a scratch area in control CAF dishes, even at 24 h. In expression of Oct4 and Sox2 was altered in ESCC and
48 h many cells were seen in scratch area in control CAF together they impart a poor prognosis in the disease. In
[Figure 8a, 8b, and 8c], whereas CAF cells treated with the present study down-regulation of Oct-4 and Sox2 in
Ch-Np showed few cells migration after 24 h and 48 h, CAF after Ch-Np treatment was observed. Thus, clinical
as shown in Figure 8d, 8e, and 8f. These observations inhibition in expression of these genes may give hope a
indicated that Ch-Np treatment affects cell motility after better outcome in ESCC.
48 h of treatment.
In order to understand the characteristics of CAFs,
DISCUSSION the expression of keratin18, an epithelial marker in
cancer cells, was studied. Keratin18 is currently mainly
CAFs are some of the most important stromal cells studied to understand prognosis in cancer patients.
[16]
involved in tumor initiation, progression, and its Recently it was [17] noticed that there was up-regulation
metastasis in ESCC. It is important to attempt to of keratin18 in breast cancer patients. This coincides
[7]
target these cells along with their cancer cells when with the observation in the current study where there
developing a drug against the cancer. Recent trends in was a higher expression of Keratin18 in CAF cells.
drug development involve the use of nanoparticles which Furthermore, in the current study there was a complete
are efficient in drug delivery. Chitosan nanoparticle down-regulation of keratin18 after treatment of Ch-Np,
is one such nanoparticle which is being explored in implying the acquisition of a normal cell phenotype
various cancers and other diseases. Our study focused and loss of tumor progression capability. Vimentin was
[8]
on evaluating the anti-metastatic effect of Ch-Np on also studied in the present study and its expression was
CAF isolated from the peripheral blood of a patient with noticed on CAFs. This is accordance with a previous
metastatic esophageal cancer. study. [18] Over-expression of Vimentin in cancer cells has
been associated with increased invasion and metastasis in
CAFs have shown extensive growth proliferation and tumor. According to one study [19] inhibition of vimentin
multiply at a doubling time of 25 ± 0.38 h. However expression reduced cancer cell migration. Down-
Ch-Np-treated cells have inhibited the growth of these regulation in expression of vimentin was observed in the
cells, indicating an inhibitory effect of Ch-Np on CAFs. Ch-Np-treated CAF, suggesting that Ch-Np can reduce
Studies have shown that Ch-Np inhibited the growth of cell migration and, ultimately, metastasis. Metastasis is
breast cancer cells in vitro. Similarly, another group an important characteristic of cancers which is governed
[9]
found that Ch-Np effectively inhibited proliferation of a by genes such as VEGF, MMP1, MMP9, and E-Cadherin.
[8]
human gastric carcinoma cell line, indicating its potential MMP1 and MMP9 have been found to be associated with
beneficial activity against human gastric cancer. The cancer cell metastasis in ESCC. [20] Down-regulation in
present study also indicated the potential use of Ch-Np these two important genes, MMP1 and MMP9, observed
Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ July 29, 2016 ¦ 265