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. Conclusion: 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.
Highlights
Hypoxia is considered to be a hallmark of cancer which promotes angiogenesis, invasiveness, and metastasis, and has a major role in metabolic reprogramming of cancer cells which allows cells to survive in a hostile secondary metastatic environment [1]
For the first time, a hypoxia specific single-chain Fv (scFv) Ab binding to hepatocellular carcinoma (HCC) by phage library selection, and revealed that its antigen M2 splice isoform of pyruvate kinase (PKM2) is a potential biomarker for hypoxia in liver cancer tissues
Patients received different treatments, including hepatic artery ligation (HAL), trans-catheter arterial embolization (TAE), and trans-catheter arterial chemoembolization (TACE), and were incorporated into the library in an attempt to enhance the diversity of the hypoxia-responding Ig gene
Summary
Hypoxia is considered to be a hallmark of cancer which promotes angiogenesis, invasiveness, and metastasis, and has a major role in metabolic reprogramming of cancer cells which allows cells to survive in a hostile secondary metastatic environment [1]. Because of its central role in tumor progression and resistance to therapy, cancer cell adaptation to hypoxia has been used to develop tumor markers and target therapies [3]. HCC is generally developed through cirrhosis, which always causes fibrogenesis which demolishes the normal blood system, leads to a shortage of blood circulation and results in hypoxia. Conventional palliative therapies for HCC including hepatic artery ligation (HAL), trans-catheter arterial embolization (TAE), and trans-catheter arterial chemoembolization (TACE) can cause hypoxia. The principle of these palliative therapies is to restrict blood supply to suppress tumor growth, these therapies which induce hepatic ischemia always catastrophically result in hypoxia. Discovery of novel hypoxia-specific markers for HCC targeted therapies is urgently needed
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