Experimental Study Of Breast Cancer Cells Molecular Imaging For GLUT1

Objective:1,To compare Glucose transporter 1 (Glut1) mRNA and protein expressed in breast cancer MDA-MB-231 and MCF-7 cells for revealing the relationship between grade malignancy and Glutl expression in breast cancer,and select the most significant cell line of Glutl overexpression in breast cancer as an experimental model.2,To assess the feasibility of fluorescent 2-deoxyglucose analog,2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl) amino]-2-deoxyglucose (2-NBDG) was targetedly taken up by breast cancer cells that overexpress Glutl, which confirmed that 2-DG molecule could target tumor cell with Glut1 expression.3,To explore the feasibility of USPIO labeled with 2-DG construction for preparation tumor targeted molecular magnetic resonance imaging probe 2-deoxyglucose-ultra-small super-paramagnetic iron oxide (2-DG-USPIO).4,To explore the effect of the molecular magnetic resonance imaging probe 2-DG-USPIO targeting breast cancer cells with Glut1 overexpression.Methods:1,Firstly, mRNA and protein expression of Glutl in breast cancer MDA-MB-231 cells were detected via RT-PCR and immunohistochemistry respectively,then protein expression of Glutl between breast cancer MDA-MB-231 cells and MCF-7 cells were compared via Western blot.2,MDA-MB-231 cells were grown in 6-well plates for analysis of 2-NBDG uptake via fluorescence microscopy imaging and flow cytometer. And the control was addion D glucose in the medium of 2-NBDG for competitive inhibition.The amount differences of absorption of 2-NBDG between MDA-MB-231 and MCF-7 cells were compared by flow cytometry.3,The molecular magnetic resonance imaging probe was prepared through 2-DG conjugated to USPIO using chemical method. The agent were tested by transmission electron microscope and infra-red spectrum.4,Human breast cancer cells (MDA-MB-231) were incubated with 2-DG-USPIO or USPIO for 10 minute to 2 hour respectively. Accumulation in the cells was evaluated using Prussian blue staining and magnetic resonance (MR) imaging, further intracellular iron absorption observed by electron microscopy.Results:1,MDA-MB-231 cells and MCF-7 cells overexpress Glut1 mRNA and protein.MDA-MB-231 cells express Glutl protein(0.946±0.007) higher than MCF-7 cells express (0.833±0.010).2,Fluorescence microscopy imaging and flow cytometer analysis displayed MDA-MB-231 cells could uptake 2-NBDG. Addition of 50 mM D-glucose to the media reduced 2-NBDG uptake by 46%. Furthermore, fluorescence intensity of MDA-MB-231 cells (25.10±0.57) is higher than MCF-7 cells'(10.12±0.62) when incubated by 2-NBDG for 20 minute.3,The result of transmission electron microscope displayed 2-DG-USPIO nanoparticles were spherical. The diameters of particles were 10 nm. The result of infra-red spectrum proved that 2-DG was conjugated to the surfaces of USPIO. 4,After 10 mins, a large number of blue granules was observed in the cytoplasm of MDA-MB-231 incubated by 2-DG-USPIO, whereas there was no significant blue granules in cells incubated by USPIO.The signal of MDA-MB-231 cells after 10-mins incubation with 2-DG-USPIO was clearly lower than the signal of MDA-MB-231 cells incubated with plain USPIO in clinical 1.5-T MR T2-weighted image.Conclusion:1,Glutl expression is closely related to malignant grade in breast cancer cells, the higher cells malignant grade was,the higher Glutl protein expressed,so highly malignant breast cancer MDA-MB-231 cells was chosed as the tumor cells with high metabolic experimental model.2,The preliminary data clearly demonstrate 2-NBDG was taken up and accumulated in breast cancer cells with overexpressing Glutl,and may as a optic probe for glucose uptake in hypermetabolism malignant cells, And indicate the 2-DG molecule can be targetedly uptaken by tumor cells with Glutl overexpression.3,2-DG was labelled to the surface of USPIO coated with DMSA successfully. The molecular probe 2-DG-USPIO could be targetedly uptaken by breast cancer MDA-MB-231 cells with Glutl overexpression.