| Background With the development of cancer research, the study of biological behavior under physiological environment and the visualization of in vivo tumor site become the focus to promote tumor diagnosis and treatment, but to achieve the aim needs an excellent research method. As a new nano fluorescent material, quantum dots (QDs) provide an effective research method for the tumor in vivo studies according to its unique fluorescent properties. However, this new nano fluorescent material has not been widely recognized, and whether it has outstanding sensitivity, stability and biological toxicity for biomedical applications are still needed to explore. In this study, quantum-dot-based technology was used for labeling tissue sample and cell in vitro of genitourinary tract tumors, and to explore its biomedical application characteristics.Purpose On one hand, comparison of quantum-dot-based labeling technology and immunohistochemistry (IHC) for detecting CD44v6antigen in bladder cancer (BC) and prostate stem cell antigen (PSCA) in prostate cancer (PCa), and to evaluate the sensitivity and stability of quantum-dot-based technology; on the other hand, to detect the biological toxicity of QDs on bladder cancer cell line EJ and prostate cancer cell line PC-3, and on those grounds to investigate the value of QDs as a new type of fluorescent material for biomedical applications.Methods Quantum-dot-based labeling and IHC staining analysis of CD44v6and PSCA expression were performed on80bladder cancer and40prostate cancer tissues, respectively. The intensity and density of labeling were read immediately under microscope after labeling and graded on a scale of0to3+. With the tumor grade and stage data, evaluated the sensitivity of Quantum-dot-based labeling in comparison with IHC staining, and observed of fluorescence durability on positive expression of Quantum-dot-based labeling. And then, the cytotoxic effect of QDs was detected on bladder cancer cell line EJ and prostate cancer cell line PC-3, and examined labeled effect on these live cells after marked by quantum-dot-based specific and nonspecific labeling. In addition, according to unique fluorescent properties of QDs, bi-color labeling was used for detection of different antigen in tissue and cell.Results1. The results of quantum-dot-based labeling and IHC for detecting CD44v6expression in bladder cancer tissues:The data demonstrated that increasing levels of CD44v6expression accompanied lower tumor grade (QDs, r=-0.345, P=0.002; IHC, r=-0.392, P＜0.001) and stage (QDs, r=-0.243, P=0.03; IHC, r=-0.306, P=0.006), and the similar tendency was detected by two methods, and by comparison between the two methods, quantum-dot-based labeling was consistent with IHC in detecting the expression of CD44v6in bladder cancer tissue (K=0.931, P＜0.001).2. The results of quantum-dot-based labeling and IHC for detecting PSCA expression in prostate cancer tissues:The data showed that PSCA expression level with tumor Gleason score (QDs, r=0.732, P＜0.001; IHC, r=0.683, P＜0.001) and stage (QDs, r=0.514, P=0.001; IHC, r=0.432, P=0.005) in prostate cancer tissue was positively correlated and two methods manifested a similar tendency as well. Using Cohen’s kappa statistic, both methods had statistically similar detection rates for the PSCA expression (K=0.874, P＜0.001).3. Optical stability test after quantum-dot-based labeling:The fluorescence of positive samples (CD44v6,51samples; PSCA,39samples) after labeled were observed for1month, and imaged them at10,20and30days. Ten days after the experiment, fluorescence remained stable in all but2of the90samples with fluorescence level "2+" faded to "1+"(P=0.157); Twenty days after the experiment,7cases with fluorescence level "3+" faded to "2+", and9cases with fluorescence level "2+" faded to "1+"(P＜0.001); Thirty days after the experiment, the variation among each positive expression level was visible (P＜0.001),3cases with fluorescence level "1+" decayed and only11with fluorescence level "3+" remained, but nearly96.7%(87/90) of samples with positive expression could still be seen.4. Biological toxicity test of QDs on bladder cancer cell line EJ and prostate cancer cell line PC-3:Two kinds of live cells incubated with QDs for24hours, using cell counting kit-8(CCK-8) test, performed that the survival rate of cells was decreased with the increasing of QDs concentrations. However, in the common concentration (5nM, lOnM and20nM), no statistical difference in survival rate was observed between experimental and control groups (P>0.05), and the survival rates of bladder cancer cell line EJ and prostate cancer cell line PC-3could still remained92.6%and95.8%in the high concentration (80nM), respectively.5. The results of quantum-dot-based specific and nonspecific labeling used to live cells:Detecting CD44v6expression in the live bladder cancer cell line EJ by quantum-dot-based specific labeling, showed that CD44v6expressed on the cell membrane, and quantum-dot-based specific labeling exhibited good sensitivity. But in conventional culture environment, fluorescence of CD44v6expression began to fade after6to12hours owing to lack of enough stability. According to the result of Tat-conjugated quantum-dot-based nonspecific labeling on live prostate cancer cell line PC-3, it was found that the method was short of specificity, but QDs fluorescence maintained excellent constancy in the interior of the cell. Furthermore, the process of cell division was tracked dynamically under quantum-dot-based nonspecific labeling.6. Bi-color labeling in tissue sample and fixed cell:Detection of CD44v6and PCNA expression in bladder cancer tissue samples and fixed EJ cell by bi-color labeling, found that two markers were expressed in different location of the cell. CD44v6located in the cell member, and PCNA expressed in the nucleus.Conclusion Quantum-dot-based labeling technology not only exhibited excellent sensitivity and stability, but non-toxicity was found in biology application as well. In this study, according to the application of quantum-dot-based labeling on tissue sample and live cell, it showed broad application prospects in the field of genitourinary tract tumor research, while laid the foundation to improve the diagnosis and treatment for cancer. |
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