title: Phage display library selection of a hypoxia-binding scFv antibody for liver cancer metabolic marker discovery.
abstract: Hypoxia, which is frequently observed in liver cancer and metastasis, influences tumor progression and resistance to therapy. Although hypoxia-associated biomarkers are of use in other cancers, none is recognized as a surrogate for hypoxia in liver cancer. In this study, we generated seven unique human single-chain Fv (scFv) antibodies (Abs) specific to hypoxic liver cancer cells, using normoxia-depleted vs hypoxia-selected phage library panning technology. By developing the scFv immunoprecipitation-based mass spectrometry method, the antigen that bound with one of the Abs (H103) was identified as the M2 splice isoform of pyruvate kinase (PKM2), an enzyme that is a key regulator of aerobic glycolysis in cancer cells. Increased expression of PKM2 was induced by hypoxia in liver cancer cell lines. Immunohistochemical (IHC) staining showed that PKM2 was highly expressed in moderately and well differentiated hepatocellular carcinoma (HCC) tissues with a hypovascular staining pattern. High expression of PKM2 was also localized in the perinecrotic area of intrahepatic cholangiocarcinoma (ICC) tissues. The percentage of the HCC or ICC tumor expressing PKM2 was significantly higher with more tumor necrosis, low microvessel density, and advanced stage. Moreover, the H103 scFv Ab was efficiently internalized into hypoxic liver cancer cells and could have potential for targeted drug delivery.
  
  
  CONCLUSIONS
  our study, for the first time, developed hypoxia-specific scFv Ab H103 to liver cancer cells, and revealed that PKM2 is a promising biomarker for hypoxia in HCC and ICC tissues. These allow further exploration of this valuable Ab and PKM2 antigen for hypoxia targeting in liver cancer.
authors.affiliation: Department of Medical Biochemistry and Cell Biology, School of Medicine, Nankai University, Tianjin, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Clinical Laboratory, Cancer Hospital of Tianjin Medical University, Tianjin, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Medical Biochemistry and Cell Biology, School of Medicine, Nankai University, Tianjin, China.
authors.forename: Hang
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Medical Biochemistry and Cell Biology, School of Medicine, Nankai University, Tianjin, China.
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Gao
authors.affiliation: Department of Medical Biochemistry and Cell Biology, School of Medicine, Nankai University, Tianjin, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Li
authors.affiliation: MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing, China.
authors.forename: Zhongyuan
authors.initials: Z
authors.lastname: Sun
authors.affiliation: Department of Medical Biochemistry and Cell Biology, School of Medicine, Nankai University, Tianjin, China.
authors.forename: Rong
authors.initials: R
authors.lastname: Xiang
authors.affiliation: Department of Medical Biochemistry and Cell Biology, School of Medicine, Nankai University, Tianjin, China.
authors.forename: Sihe
authors.initials: S
authors.lastname: Zhang
mesh_terms: D014408:Biomarkers, Tumor; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D015687:Cell Hypoxia / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D045744:Cell Line, Tumor; D061505:Cell Surface Display Techniques / Q000379:methods*; D005260:Female; D015723:Gene Library; D057809:HEK293 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D008297:Male; D008875:Middle Aged; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D057127:Single-Chain Antibodies
keywords: M2 splice isoform of pyruvate kinase; biomarker; hypoxia; liver cancer; phage antibody library