Determination of cytokine regulated glycan expression by using molecularly imprinted polymers targeting sialic acid
Abstract
Cancer cells often have an increased amount of glycans, such as sialic acid (SA), on the cell surface, which normally play an important role in cell growth, proliferation and differentiation. In this study, SA expression is determined by fluorescent nanoprobes, molecularly imprinted polymers, SA-MIPs. The nanoprobes are synthesized with an imprinting approach to produce tailor-made fluorescent core-shell particles with high affinity for cell surface SA. Inflammation and cytokine production are well known tumor promoters, modulating the cellular microenvironment, including an aberrant cell surface glycan pattern. The recombinant cytokines IL-4, IL-6, IL-8 and a cocktail of cytokines collected from stimulated T leukemia Jurkat cells were used to induce in vitro inflammation in two cell lines, and thereafter analyzed with the use of SA-MIPs and flow cytometry. One of the cell lines showed a different binding pattern of SA-MIPs after treatment with recombinant cytokines and the cytokine cocktail. This study shows that SA-MIPs can be an important tool in the investigation of overexpressed glycans in the tumor microenvironment.Introduction
Abnormal cell growth can be initiated through inflammation[1]. Cancer cells are able to reshape the microenvironment by expression of tumor-promoting chemokines and cytokines[2,3].
Fast growing cancer 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-α, IL-1β, IL-6 and IL-8 are regulated by the
transcription factor NF-kB, which is involved in activating genes in
neoplastic transformation[1,4].
Highly glycosylated intestinal mucins, such as MUC2 and MUC4, are
regulated through NF-κB 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[7,8].
The over-expression of SA creates a negative charge on the cell
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[9].
In addition, it has been proven that aberrant expression of in
particular the α2,3-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[12]. We have developed fluorescent nanoprobes to make convenient targeting and imaging of cell surface SA possible[13].
Based on other glycan specific receptors, we 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.
Min kommentar
I denna studie (som ni får läsa i sin helhet via länken ovanför eller här) har man utgått från 2 kända cell-linjer.
MCF-7 (Michigan Cancer Foundation-7) som är en bröstcancer med ursprung 1970 i USA, Michigan från 69 åriga nunnan Frances Mallon. Denna bröstcancer linje är den första forskarna lyckades odla efter en bröstcancerpatient avlidit och används flitigt i dagens forskning. Frances Mallon har efter sin död bidragit till många forskares ökade kunskaper och förståelse för hur en bröstcancer utvecklas.
Så även i denna studie där Professor Gjörloff-Wingren et al använt den tillsammans med GlycoImaging´s egenutvecklade MIP,s. (Molecularly Imprinted Polymers)
Den andra cell-linjen är RAW 264.7 (som ni får googla fram själva) är en typ av vita blodkroppsceller som ingår i kroppens immunförsvar. Denna cellinje är känd för att snabbt kunna respondera (visa skillnad/resultat) och återspegla dess förmodade resultat på andra kroppsceller (som jag tolkar det).
Det man visar i studien är att MIPs (tillsammans med en cocktail av andra medel) binder/fäster vid ena cell-linjen.
Man beskriver det som : en signifikant ökad bindning av MIPs.
Utan att kunna belägga hur "bra" eller viktig denna studie med dess resultat är (undertecknad är blott en enkel lekman) kan vi ändå konstatera att aktiviteter kopplade till GlycoImaging projektet har ökat avsevärt den senaste tiden. Bloggen ser fler och fler pusselbitar läggas. Läs gärna förra veckans Glyco-inlägg igen.
Mvh the99
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