Proteomics Study On Identification Of Human Clara-like Lung Cancer Cells And Alveolar Type Ⅱ Epithelial-like Cancer Cells

Background and objective: According to Chinese cancer registrationreport2013, lung cancer incidences in China have been very severe, bothmorbidity and mortality have ranked the first among the malignanttumors.Although large research investments, therapies of lung cancerhave not been satisfactory. With the clinical benefits gained from themolecular targeted drugs in recent years, people have graduallyrecognized the importance of individual treatment of lung cancer. Thepremise of individual treatment of lung cancer is to elaborate the lungcancer classification. In the past, lung cancers were simply divided intosquamous cell carcinoma, adenocarcinoma, small cell carcinoma andlarge cell carcinoma. In2011, new international multi-disciplinaryclassification of lung adenocarcinoma was issued, and lungadenocarcinoma (operation samples) can be divided into pre-invasivelesion, micro-invasive carcinoma, invasive adenocarcinoma and variantinvasive adenocarcinoma. In fact, the histological classification of lungcancer is far from enough, the facts that different therapeutic reaction oftyrosine kinase inhibitors in EGFR (epidermal growth factor receptor)wild type or mutant type patients have inspired many doctors and researchers, that lung cancer classification just from histology aspect istoo rough, and cytologic, even molecular, gene classification should becarried on. From cytologic classification aspect, on the basis ofultrastructure under electron microscope, primary lung adenocarcinomacan be divided into: airway epithelial-like cell type, goblet-like cell type,bronchial gland-like cell type, clara-like cell type, alveolar type IIepithelial-like cell type and mixed type. Especially clara-like lung cancercells and alveolar type II epithelial-like lung cancer cells areindistinguishable，because cell morphology are extremely similar underlight microscope. The iconic structure of clara-like lung cancer cell issimilar to clara cell secretory granules--clara particles, and iconicstructure of alveolar type II epithelial-like lung cancer cells is similar toplate lamellar bodies in alveolar type II epithelial cells, these two kinds ofcells can only be identified by morphological observation under electronmicroscope. The electron microscope detection are expensive, tissuesample cutting and preparation are complicated, and the work cycle islong, so electron microscope detection is difficult to be widely used inclinic.so it is difficult to carry out electron microscopy detection intoclinic, therefore clinical lung cancer cell typing is difficult, while clinicalresearches based on cell typing have been stalled. This study intended tosearch for the tumor markers of clara-like lung cancer cells and alveolartype II epithelial-like cancer cells by proteomics methods, facilitating the clinicians and researchers to identify the two cell types with easier andcheaper methods such as immunohistochemistry.Methods: Human clara-like lung cancer cell line NCI-H358and alveolartype II epithelial-like lung cancer cell line A549were cultured undersame condition, cells of4different generations were chosen, and dividedinto NCI-H358group and A549group. When the cells grew into thelogarithmic growth phase, total proteins were extracted, and proteinswere separated with2D-DIGE (two-dimensional fluorescence differencegel electro-phoresis), protein expression were compared and analysedwith analysis software DeCyder. Protein dots differentially expressed5times more than or close to5times between two groups were selected,digged from preparation gel, identified by mass spectrometry, andverified by western blot. Proteins of significantly different expressionbetween two groups were chosen for immunohistochemistry and electronmicroscope detection.32cases of surgical tissue specimens werecollected, every tissue sample was divided into2blocks,1block wasfixed in10%neutral buffered formalin, and levels ofERO1L(endoplasmic reticulum oxidoreductin1like protein alpha) weredetected by immunohistochemical method, the other block was fixed in2.5%glutaraldehyde solution and stored in dark place at4℃. Accordingto immunohistochemical results, tissues of average positive cell rateexceeding50%or total negative were selected for cell typing under transmission electron microscope.Results: After the proteomics research, a total of38different proteinspots were found between the two groups, including8protein differencesmore than5times or close to5times.5proteins were identified by massspectrometry, including UCHL1(ubiquitin carboxyl-terminal hydrolaseisozyme l1, high expressed in A549cells), ERO1L, CK19, CK8andPRDX2(cytoskeletal19, cytoskeletal8, peroxiredoxin2, high expressedin NCI-H358cells). The expressions of ERO1L in NCI-H358cells weresignificantly higher than that in A549cells through western blotverification(p=0.02), but differences of UCHL1level in A549cells andNCI-H358cells were not significant. Immunohistochemical stainingshowed that ERO1L was mainly expressed in the cytoplasm, andoccasionally in nucleus, it was mainly focally expressed, the positive rateof ERO1L in lung cancer was72.4%, the positive rate in lung squamouscell carcinoma was83.3%, the positive rate in lung adenocarcinomacarcinoma was66.7%, and there was no statistical difference between thepositive rate in lung squamous cell carcinoma and lung adenocarcinomacarcinoma(P=0.41). ERO1L rarely expressed in diffuse type, theconstituent ratio of ERO1L average positive cell rate more than76%wasonly17.3%, while the constituent ratio of ERO1L average positive cellrate between0-25%was62.1%. The expression of ERO1L in lung cancerwas very clear, negative, weak positive, moderate positive or strong positive expression could be found. According to the results ofimmunohistochemistry,5ERO1L positive lung adenocarcinoma tissues,5ERO1L total negative lung adenocarcinoma tissues,2ERO1L positivesquamous cell carcinoma tissues,1ERO1L total negative squamous cellcarcinoma tissue,1large cell carcinoma(positive) and1small cellcarcinoma(negative) were chosen for electron microscope observation，itwas found that4of5ERO1L-positive cases of lung adenocarcinomashowed pulmonary clara-like cancer cells, and none of ERO1L-negativetissues showed clara-like lung cancer cells under electron microscope.Conclusion: ERO1L is expected to become the tumor marker foridentification of human clara-like lung cancer cell subtype in lungadenocarcinoma, ideal markers for identification of alveolar type IIepithelial-like lung cancer cells have not been found.