The Study On Targeted Detection Of Lymph Node Metastasis In NSCLC With Water-soluble CdSe/ZnS-CKpan-Abfluorescent Quantum Dots Probes

BackgroundLung cancer has beenthe most popularcancerin our country, and most of them isnon-small celllung cancer(NSCLC).According tothe NCCN guidelines, non-small celllungcancerin early or mid stage shall be preferred surgery as quickly as they can, andlymphnode dissectionis recommendedto reduceearlyrecurrence rate. Butthesystemlymphadenectomysurgical means more harm with increased inthe probabilityofpostoperative adverse events, and even some patients with early stagelung cancerdue tothepassivenon-essential lymphadenectomy for a vague lymph node status.Therefore, ifthepreoperativeor intraoperativereal-time detectionof lymph nodestatus can be offered,extent of surgical resectioncan beaccurately, thereby reducing trauma. This is alsoin linewiththe development trend ofminimally invasivesurgeryanddamage controlconcept.ObjectivesLooking for specific tumor lymphatic metastasis markers; their coupled with the lowtoxicity of water-soluble dyes, CdSe/ZnS quantum dots.Tthe novel fluorescent tracerTshallbe special to identify non-small cell lung cancer cells andmetastasislymph nodes. Andverify its applicabilityin the cell level.Methods1Randomly select40non-small cell lung cancer patientsto receive surgical treatment(20cases of adenocarcinoma,20cases of squamous cell carcinoma).With the way ofimmunohistochemistry (Elivison two-step) to detect the expression of the CKpan, CK8/18,CK19, CEA, NSE, MUC1, EMA among the metastasislymph nodes, non-metastatic lymphnodes and normal lymph nodes. Screen out the right target probe with analysis theexpression of biomarker which relating to lymphatic metastasis.2. Use EDC method to coupleCdSe/ZnS and CKpan-Ab, and optimize the couplingsystemby adjusting the PH value and the ionic concentration buffer system. Measuringoptical properties and physical and chemical characteristicsof CdSe/ZnS-CKpan-Ab, toverify whether it meets the clinical needs.3. Use CdSe/ZnS-CKpan-Ab on cultured tumor cells to identifyAD and SSD cell line,which normal human lung epithelial cell line as a control, while also identifying highmetastatic lung adenocarcinoma cell line95-D for the coming animal testing. AssayCdSe/ZnS-CKpan-Ab fluorescent probes in vitro cytotoxicity, biological safety testing byMTT, and preliminaryexplore its applicable range of concentrations in the body.Results1, Imaging of lymph node metastasis in vivo has the following clinical value:identifying lymphatic metastasis, accurately determine regional lymph node dissection,precise surgical resection, improve the detection rate of lymph node metastasis.2, Among the cytokeratin (CK) family, CEA, NSE, MUC1and EMA, NSE has alowest detection rate(a significant difference, P <0.05) compared with other markers.MUC1and NSE have a vacation positive, Both EMA and CK19also exist gland, scaledifferences.Sofinally choose CKpan as the destination target.3, Use EDC to successfully synthesize CdSe/ZnS-CKpan-Ab.4, With changes PH value and ion concentration of the buffer system,CdSe/ZnS-CKpan-Ab spectral characteristics remain stable (P>0.05).5, Compare the coupled CdSe/ZnS-CKpan-Ab and unconjugated CdSe/ZnS, there isno significant difference with the ultraviolet absorption spectrum curve, whileCdSe/ZnS-CKpan-Ab slightly below the CdSe/ZnS. But both the two identical peaksabsorption peaks are at about560nm, which shows CdSe/ZnS with antibodies effectivecoupled,and after the coupling it remains good dispersibility and good spectral propertiesand no agglomeration occurred.6, With UV excitation,with the concentration of3.125nmol/L (diluted512-fold),CdSe/ZnS-CKpan-Abcan still be visually identified confirmed the excellentproperties of quantum dots high fluorescence intensity.7, Two weeks after contained in the dark,4℃,the fluorescence emission spectra ofCdSe/ZnS-CKpan-Abdid not change significantly.(P>0.05)8, We have used CdSe/ZnS-CKpan-Ab to lable for cell line, human squamouscarcinoma cell line (NCI-H226), human adenocarcinoma cell line (NCI-H1299), highlymetastatic human lung cancer cell line (95-D) and normal human lung epithelial cells(BEAS-2B), and we find that all cell were identified correctly by the new fluorescent tracerbesides BEAS-2B cells.9. We have used CdSe/ZnS-CKpan-Ab to stain the tissue slice of lung cancer, lung cancer metastasis-positive lymph node, normal lung tissue of their own, andmetastasis-negative lynph node. We observed correctly idetification of cancer cell in lungcancer tissue, and lung cancer metastasis-positive lymph node,while only a few basalepithelial cells are occasionally marked in normal lung tissue and metastasis-negativelynph node.10. Comparing CdSe/ZnS and CdSe/ZnS-CKpan-Ab found that no conjugatedCdSe/ZnS quantum dots can not imag, and after incubation with the monoclonal antibodyto block CKpan antigen, we also found that CdSe/ZnS-CKpan-Ab lost the ablility ofidentifing cancer cells. It show that CdSe/ZnS-CKpan-Ab end located within the cellcytoplasm, the possible mechanisms through receptor-mediated endocytosis by blockingreceptors can prevent the dye and the developer failed to enter the cell.11, MTT test confirmed that CdSe/ZnS-CKpan-Ab, CdSe/ZnS and CKpan-Ab havegrowth inhibitory monoclonal antibody for95-D cells.ConclusionWe have synthesized a new fluorescent tracer, which is conjugated of CKpan (aspecific tumor lymph node metastasis marker) antibody and low toxicity of water-solubletype Ⅰfluorescent dye (CdSe/ZnS quantum dot), to targeted identify cancer cells andmetastasizing lymph nodes of non-small cell lung. Further experiments verified that thenew fluorescent tracer has better optical properties and stability of the physical andchemical characteristics compared with ordinary organic dyes, and it could target cancercells effectively in votro cell, thus success in imaging cancer cell in vivo. All the rsults mayprovide useful theoretical and technical support for celective mediastinal lymph nodedissection of lung cancer.