Optical Imaging For Three Negative Breast Cancer With An Icam-1-targeting Small Molecule Probe

Objective This work was to study the value of near infrared fluorescent probe y3-cy5.5 in the realization of the three negative breast cancer specific imaging.Our goal is to provide noninvasive,dynamic,and specific means for the early diagnosis of three negative breast cancer.Methods The three negative breast cancer cell line MDA-MB-231 and non-three negative breast cancer cell line MCF-7 were selected as the research object in this study.The expression of ICAM-1 in three negative breast cancer cell line MDA-MB-231 and non-three negative breast cancer cell line MCF-7 was detected by Western blot and flow cytometry.Laser confocal microscopy and flow cytometry was used to examine the intake of y3-FITC by MDA-MB-231 cells.The blocking effect of non-fluorescent labeled y3 to the y3-FITC uptake were observed by the vitro competitive inhibition assay.MDA-MB-231 human breast cancer orthotopic xenograft nude mice models were established.In vivo NIRF imaging was acquired after intravenous injection of y3-Cy5.5(50μM/200μl)immediately and different time intervals.Two hours later,the tumor bearing mice were sacrificed and the organs were imaged.SPSS 19.0 software was used to analyze the data in the experiment.All the quantitative data were expressed as mean ±standard deviation.The t test was used to compare the quantitative data,and the p<0.05 was considered significant.Results1.The results of Western blot showed that the expression of ICAM-1 protein in three negative breast cancer cell line MDA-MB-231 was about 3 fold higher than that of non-three negative breast cancer cell line MCF-7.Flow cytometry indicated the ICAM-1 expression percentage of MDA-MB-231 was(99.12±1)%and the percentage of MCF-7 was(65.1±2.4)%,compared with both,there was significant statistical difference(p<0.01).Western blot and flow cytometry showed that ICAM-1 was highly expressed in three negative breast cancer cell line MDA-MB-231.2.Laser confocal microscopy showed that MDA-MB-231 can specially combine γ3-FITC,and with the increase of the concentration of y3-FITC,the combination of MDA-MB-231 cells was increasing,while MDA-MB-231 had little combination with the control probe y3S-FITC.Flow cytometry results showed that the concentration of y3-FITC was2.5μM,binding percentage of MDA-MB-231 with y3-FITCwas(4.1±0.6)%.When the y3-FITC concentration increased to 5μM,the binding percentage was(53.33±4.31)%.The concentration of y3-FITC is further increased to 20μM,the binding percentage increased to(99±0.9)%.However,regardless of the concentration control probe γ3S-FITC is 2.5μM or 20μM,the MDA-MB-231 cells with y3S-FITC have no obvious binding.Compared to the two groups,the p value was<0.01,which shows that there is statistically significant difference.In addition,the results of flow cytometry showed that the binding of MDA-MB-231 cells with y3-FITC could be blocked by γ3.3.After injecting γ3-cy5.5 probe by intravenous,the near-infrared fluorescence signal in the tumor tissue of three negative breast cancer bearing mice gradually increased with the time,and reached the peak in 2h.However,in the control group,after the injection of the probeγ3S-cy5.5,the near-infrared fluorescence signal in the tumor tissue of the bearing mice did not obviously increase with the time.After two hours,the mice were killed and the isolated organs and tumors were obtained.It can be seen that the fluorescence signal intensity of the experimental group tumor was significantly higher than the control group tumor,but there was no significant difference in the distribution of the experimental group probe and the control probe in the heart,liver,spleen,lung and kidney.At different time points,the target-background ratio of the tumor in the experimental group and the control group was quantitatively analyzed,p<0.01,there was significant difference between the two groups.The target-background ratio of isolated organs and tumor of the experimental group and isolated organ and tumor of the control group was quantitatively analyzed,the experimental group and the control group of isolated organs of the target-background than no statistically significant differences.Target-background ratio of isolated tumors in the experimental group and control group was compared,p<0.01.There is statistically significant difference between the two groups.Conclusion The study suggested that near infrared fluorescent probe y3-cy5.5 can specifically combined with ICAM-1,and it can realize the three negative breast cancer specific imaging.This study provides a noninvasive,dynamic and specific diagnosis method for three negative breast cancer.It not only enriched the evaluation system of three negative breast cancer from the angle of molecular imaging,but also lay the foundation for the targeting mechanism of specific probes and targeted drug development.This will provide a new platform for the treatment of three negative breast cancer.