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Page 2 of 9 Zhang et al. J Cancer Metastasis Treat 2019;5:56 I http://dx.doi.org/10.20517/2394-4722.2018.112

cells outpace their blood supply and become nutrient and oxygen deprived. This results in necrotic cell death
at the tumor’s core and this releases pro- inflammatory cytokines, such as IL-1.

Cytokines act as cell regulators of many different biological processes including cell growth, differentiation,
metabolism, immunity and inflammation. Cytokines can enhance cancer cell growth by modulating the
cellular microenvironment or by affecting the cells directly. Pro- inflammatory cytokines can be oncogenic
and thereby inducing elevated levels of pro- invasive factors such as metalloproteinase-2 (MMP-2) and
epithelial growth factor.

Pro-inflammatory cytokines including TNF-a, IL-1b, IL-6 and IL-8 are regulated by the transcription factor
NF-kB, which is involved in activating genes in neoplastic transformation . Highly glycosylated intestinal
[1,4]
mucins, such as MUC2 and MUC4, are regulated through NF-kB and by the gp130/STAT3 pathways,
respectively .
[5,6]
Sialic acid (SA), or neuraminic acid, is the outermost sugar molecule of glycans, thus forming the outer
surface of cells by being attached to proteins or lipids bound to the plasma membrane. SA is very important
for the function of living organisms since it is involved in cell processes like proliferation, differentiation,
angiogenesis, invasiveness and metastasis . The over-expression of SA creates a negative charge on the cell
[7,8]
surface, which is important in cell-cell and cell-matrix communication. An increased level of SA on the cell
membrane, which have been reported on malignant and metastatic cancer cells, makes cells repel each other
leading to an increased motility . In addition, it has been proven that aberrant expression of in particular
[9]
the a2,3-SA variants are related to tumor adhesion and invasion [10,11] .

Today, there is a lack of tools for specific targeting to glycans with high affinity. Lectins and glycan-specific
antibodies have been used by many research groups to detect altered glycosylation, but the current tools
do not perform with high specificity or affinity . We have developed fluorescent nanoprobes to make
[12]
convenient targeting and imaging of cell surface SA possible . Based on other glycan specific receptors, we
[13]
have developed SA-imprinted molecularly polymers (SA-MIPs) by using silica core particles and implemented
NBD-fluorophores, which have favorable spectroscopic properties [13,14] .

In this study, two different cancer cell lines, MCF-7 and RAW 264.7, were stimulated with recombinant
IL-4, IL-6, IL-8 and a cocktail of cytokines obtained by stimulating Jurkat T leukemia cells with
phytohemagglutinine (PHA). The resulting expression of SA on the membrane of the stimulated cancer
cells was analyzed with flow cytometry using both lectins and the SA-MIPs. One of the cell lines showed
an increased binding of the SA-MIPs after treatment with recombinant cytokines and with the cytokine
cocktail from PHA-stimulated Jurkat cells.

MATERIAL AND METHODS
Cell culture
The cell lines MCF-7 (ATCC HTB-22), RAW 264.7 (ATCCTIB-71) and Jurkat (ATCCTIB-152) were obtained
from the American Type Culture Collection (ATCC/LGC Standards, Teddington, UK). MCF-7 and Jurkat
cells were cultured in RPMI-1640 medium (Invitrogen, San Diego, CA, USA) supplemented with 10% fetal
bovine serum (FBS, Invitrogen) and 50 µg/mL gentamycin (Invitrogen) (complete medium). RAW 264.7 cells
were cultured in RPMI-1640 medium supplemented with 10% FBS and penicillin-streptomycin (Invitrogen).
The cell lines were incubated in 37 °C with 5% CO2 in 100 % humidity.

Stimulation of Jurkat cells
5 × 10 Jurkat cells in 5 mL of were stimulated in complete medium with the addition of 10 ug/mL of PHA
6
(Sigma Aldrich, St Louis, USA) for 72 h. The cells were harvested and centrifuged at 300 xg for 10 min. The
supernatant was kept at 4 °C until used for cell experiments.