| IntroductionLung cancer is one of the leading causes for cancer-related mortality. The incidenceand death rate increased significantly, as the aggravation of environment pollution andincreasing number of smokers. Despite of the advancement in diagnosis and chemotherapy,radiotherapy, immune therapy and target therapy of lung cancer, the overall survival rateremains extremely low (around15%), especially for the patients with lung adenocarcinoma,which was resistant to both chemotherapy and radiotherapy, and could lead to early distantmetastasis. Lacking of efficient methods for early diagnosis might be an important reasonfor poor clinical outcomes in patients with lung adenocarcinoma. Besides, the residualtumor cells which were resistant to chemo-or radiotherapy could cause recurrence, relapseand metastasis, thus largely decrease patient survival. Therefore, deeper understanding ofthe occurrence of lung adenocarcinoma and establishing the efficient ways for earlydiagnosis and treatment have been the concern for clinical physicians.Recently, there was increasing evidence indicating that the maintenance and spreadingof a variety of tumors was sustained by a small subset of cancer cells-cancer stem cells(CSCs), which have been successfully isolated and identified in many tumors, such asleukemia, melanoma, glioma, and breast, prostate, pancreatic and colon cancer. These cellspossess ability to self-renew, unlimited proliferation potential, and capacity to generatedifferentiated cells which constitute the major tumor population. CSCs may be alsoresponsible for the functional heterogeneity that is commonly observed in solid tumors, andthey are more resistant to conventional chemotherapy and radiotherapy. Thus, earlydetection of CSCs and CSCs targeted therapy may offer new diagnostic and therapeuticstrategies against malignant tumors, and further improve the patient survival. Screening for the appropriate molecular targets and preparing for the antibody are thekeys for targeted therapy. The process of traditional hybridoma technology is cumbersomeand time consuming. Besides, the antibody generated by hybridoma technology has certaindistinct defects, such as large molecular weight, low penetration ability, and low serumclearance ratio. The appearance of phage antibody library leads gene engineering antibodytechniques into a new stage, which could produce antibody in vitro. The generatedsingle-chain antibody possesses several advantages, such as low molecular weight, highimmune activity, and improved penetration ability.Therefore, in this study, we try to prepare the mouse single–chain variable fragment(scFv) antibody by phage display technology with the target of Lewis lung cancer stemcells. Our study might provide novel strategies for early diagnosis and targeted therapy forlung adenocarcinoma.Objectives: To prepare the Lewis lung carcinoma mouse single–chain antibodiesthough phage display technology, and further explore the immune activity of the antibody.This study lays the foundation for future establishment of the early detection and targetedtherapy for lung cancer stem-like cells.Methods and ResultsPart1. Isolation and characterization of Lewis lung carcinoma stem-like sidepopulation (SP) cellsIsolation of Lewis lung carcinoma SP cells: Lewis lung cancer cells (LLCs) inlogarithmic growth phase were obtained, and we isolated the SP cells throughfluorescence-activated cell sorting (Hoechst33342and propidium iodide double staining).SP cells were further cultured in Dulbecco's modified Eagle's medium-high glucose, with10%fetal bovine serum,100U/ml streptomycin, and100U/ml penicillin, in humidifiedatmosphere of95%air and5%CO2at37℃, according to the supplier's instruction.Characterization of LLC stem-like SP cells: The ability of self-renewal, differentiatedprogeny, chemosensitivity, and tumorigenic properties in SP and non-SP cells wereinvestigated through in vitro culture and in vivo serial transplantation. The differentialexpression profiles of stem cell markers were examined by RT-PCR. We found that the SP cell fraction comprised1.1%of the total LLC population. SP cells were available to growin serum-free medium, and had significantly enhanced ability of cell proliferation andcolony formation. SP cells were more resistant to Cisplatin in comparison to non-SP cells,and displayed increased tumorigenic ability. Moreover, SP cells showed higher mRNAexpressions of Oct-4, ABCG2, and CD44.Part2. Construction of phage antibody library1. Construction of the lung adenocarcinoma model with conditional knock-out mouse:After amplifying and purifying AdCre, the Lox-stop-lox K-ras G12D mice were nasallyinfected with AdCre following intraperitoneal injection of nembuta. The mice wereexecuted after30days, and the lungs were fixed, embedded, sectioned, and furtherexamined by hematoxylin and eosin staining.2. Generation of mouse scFv antibody gene repertoires: spleen tissues fromLox-stop-lox K-ras G12D mice with lung adenocarcinoma was used as the B cells source,and we extracted the total RNA of the mouse spleen. The primers were designed accordingto literature, and we amplified cDNA gene library of VH and VL fragments using RT-PCR.The VH-linker and VL-linker were connected by SOE-PCR, and the Not I and Sfi Irestriction site were inlet in it. The scFv gene is the products purified from SOE-PCR. Ourresults demonstrated that the total RNA of the spleen tissue had two clear bands in agar gelelectrophoresis (28S and18S). In total5VHfragment were successfully amplified and wereabout350bp, and the4VL fragment were about350bp. Finally, in total of20scFv genewere20,which were about750bp.3. Ligation of scFv fragments into phage vector: After Sfi I/Not I digestion, the scFvgene repertoires were further purified from gel. They were then cloned into phage displayvector pCANTAB-5E, which was also digested by the Sfi I and Not I restriction enzyme.The mixes of scFv and pCANTAB-5E were used to be transformed into electro competenceEscherichia coli TG, and1.7×108clones were obtained. Random digestion reaction wasperformed, and the positive insert rate was86.36%(19/22).4. Display of phage antibody: the positive plasmids were obtained by2-YT culturemedium. After overnight induction in non-glucose medium, the scFv fragments were obtained using M13KO7help phage, with the final titer of1012cfu/ml.Part3Screening for phage antibody library1. Screening on Lewis lung CSCs: before selection, conventional Lewis lung cancercells were used to deplete the nonspecific binders from the phage library. Thereafter, wescreened the phage library on Lewis lung CSCs. After4rounds of panning, the number ofeluted phages significantly increased. The first phage yield is1.37×10-9, the fifth phageyield is2.47×10-7, and the fifth phage yield is as180times as the first phage yield.2．Immune activity analysis by competitive ELISA: ELISA was used to identify theactivity of soluble scFv. The results showed that5phage antibody could bind to LLCstem-like cells with high efficacy and s specificity. The P/N value is more than2.3. Immunochemistry analysis: Immunochemistry analysis was used to further detectthe immune efficacy and specificity of selected phage antibody. The results showed thatantibody from positive clone could bind to LLC stem-like SP cells, and the DAB stainingshowed deep-brown color.Conclusion:In the present study, we successfully construct LLC stem-like SP cell single-chainantibody using phage-display technology. Our results showed that this method is veryefficient and time-saving, as compared to traditional hybridoma procedure. The ELISA andimmunochemistry analysis confirmed that the selected phage antibody possess highimmune acitvity. Our study might provide useful information for novel strategies for earlydiagnosis and targeted therapy for lung adenocarcinoma.
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