Application Of SNFYMPL Fluorescence Probe In Diagnosis Of Gastric Cancer Using Confocal Laser Endomicroscopy

?Background? In recent years,incidence of gastric cancer has significantly decreased over the world,while it is still the fifth popular cancer and gives rise to the third most deaths.East Asia is the highest incidence area.Early diagnosis and early treatment are pivotal ways to raise the survival time of GC patients.Detection of early gastric cancer has been difficult for general white light endoscopy,and calls for the experience of endoscopists.Confocal laser endomicroscopy is a new-developed endoscopy technology emerging in the past decade.It has evident advantage in the diagnosis of tiny lesions on gastrointestinal mucosa.CLE examination with molecular probe,said m CLE,is able to get fluorescence images of special target molecule to detect molecular abnormal in the epithelial tissues.At present,probes in related studies include fluorescein labelled antibodies or peptides.Compared with antibodies,peptides have several advantages.Linear heptapeptide SNFYMPL was screened on Barrett's esophagus tissues with phage display technic and subtractive whole cell approach.This peptide could be labelled to synthesis a fluorescence probe,which has demonstrated its affinity with esophageal adenocarcinoma cell line OE33,and also with high grade dysplasia mucosa by detection on tissues ex vivo.Affinity of the probe with HGD was significantly higher than that with squamous epithelium and intestinal metaplasia,which meant that it had the ability of discovering esophageal adenocarcinoma and its premalignant lesions in topical administration on mucosa.Since the similar histologic origin and evolution process of gastric cancer and esophageal adenocarcinoma,SNFYMPL fluorescence probe is possibly used to the diagnosis of gastric cancer.In previous work,we have investigated the binding capacity of the peptide on gastric cancer cell lines.While the specificity of its binding on the gastric cancer cells and tissues needs to be further validated.And the application value of the peptide in vivo is still not clear.?Objective? 1.To synthesize the fluorescence probe SNF~*-FITC,and investigate the binding capacity of heptapeptide SNFYMPL to gastric cancer cells.2.To investigate the binding capacity of fluorescence probe SNF~*-FITC to gastric cancer tissues.?Methods? 1.To synthesize the fluorescence probe SNF~*-FITC,and investigate the binding capacity of heptapeptide SNFYMPL to gastric cancer cells.1)Synthesize the fluorescence probe from the beginning with the help of the agent company.Purify and examine its quality with HPLC and ESI-MS.2)Slides of two kinds of gastric cancer cell lines were stained with fluorescence probe SNF~*-FITC and the fluorescence intensity of which was observed with confocal laser microscope.Unrelated sequence peptide probe GGG*-FITC and immortalized gastric mucosa epithelial cell line GES were taken as the negative control agent and cell.3)Suspension of two kinds of gastric cancer cells were incubated with fluorescence probe SNF~*-FITC.The binding ratio and fluorescence intensity of which were examined by flow cytometry.GGG*-FITC and GES were taken as the negative control agent and cell.4)Immunocytochemistry of two kinds of gastric cancer cell lines was conducted with biotin labelled peptide SNFYMPL-biotin.Compare its binding capacity with unlabeled peptide SNFYMPL by competitive binding test.2.To investigate the binding capacity of fluorescence probe SNF~*-FITC to gastric cancer tissues.1)Fluorescence staining was performed on 48 pairs of cryosections of operation-removed tissues of gastric cancer patients.Fluorescence intensity binding on the slides were observed with confocal laser microscope,and positive ratio was calculated.2)Fluorescence staining was performed on 48 pairs of operation-removed tissue pieces of gastric cancer patients,and observed with experimental probe-based confocal laser endomicroscope.The positive ratio was also calculated.3)Hematoxylin and eosin staining of each sample was completed to be taken as the golden standard.3.To evaluate the capacity of the fluorescence probe SNF~*-FITC in detecting gastric cancer tissues in undergoing endoscopy,a nude mice subcutaneous xenograft model of gastric cancer was established.Fluorescence imaging was performed on xenograft-bearing mice in vivo with IVIS.?Results? 1.Fluorescence probe SNF~*-FITC was synthesized,and purity of the finished product was 95.99%.2.In fluorescence staining images of gastric cancer cell lines,probe SNF~*-FITC preferentially binds to plasma membrane and cytoplasm of tumor cells.And its fluorescence intensity was significantly higher than that of controlled probe GGG*-FITC.In contrast,fluorescence signal binding on GES cells were extremely weak.3.In flow cytometry,the intensity of fluorescence binding on two kinds of gastric cancer cell lines was significantly higher than that on GES(102 VS 100),and the binding rates were both higher than that of GES(95% VS 1%).In contrast,the controlled probe also has a much lower fluorescence intensity of smaller than 100 and binding rate of less than 1% on all kinds of cells.4.In immunocytochemistry test,the binding of SNFYMPL-biotin and cancer cells was strongly positive,and which of GES was weakly positive.In competitive binding assay,overdosed unlabeled peptide was add in,while the stained result was still moderately positive.It indicated that labelled peptide has similar binding capacity to tumor cells with bald peptide.5.In fluorescence staining of cryosections,the positive ratio of SNF~*-FITC stained gastric cancer tissues was 81.25 %(39 / 48),while the unrelated sequence peptide probe had a much lower positive ratio(14.58 %,7 / 48)(P < 0.001).The positive ratio of SNF~*-FITC stained adjacent non-tumor tissues was also much lower than that on cancer tissues(27.08 %,13 / 48)(P < 0.001).6.In fluorescence staining of tissue pieces,the positive ratio of cancer tissues was 83.33%(5 / 6).?Conclusion? Fluorescence probe SNF~*-FITC synthesized with heptapeptide SNFYMPL had high affinity and specificity with gastric cancer cell lines.The target of SNF~*-FITC on tumor cells was located on plasma membrane and cytoplasm.SNF~*-FITC has much higher binding capacity to gastric cancer cells and tissues than GGG*-FITC.SNF~*-FITC could be topically administrated on the mucosa to achieve molecular imaging of cancer tissues,thus it could be a potential targeting probe in the diagnosis of gastric cancer using confocal laser endomicroscopy.