Development of lung cancer diagnostic method using exosomal protein DDOST

Abstract

폐암은 세계적으로 높은 사망률을 가진 치명적인 질병 중 하나이다. 많은원인들이 있지만 그 중에서도 효과적인 조기 진단 및 치료의 부재가 큰 원인이다. 사람들이 폐암 진단을 받았을 때에는 이미 치료하기에 늦은 시기이다. 폐암은 모양과 크기에 따라 4종류로 나뉜다. 폐암을 진단하고 치료하려는 시도는 다양하게 이루어져 왔다. 새롭게 뜨고 있는 진단법으로는 proteomic tools를 사용한 방법이다. Proteomic tools인 바이오마커를 사용하면 정상 사람과 폐암 환자를 비침습적인 방법으로 구별해 낼 수 있다. 특히 세포 표면 멤브레인 단백질은 효과적인 바이오마커가 될 수 있다. 비소세포폐암을 타겟으로 한 단클론항체들이 여럿 있으나 불행하게도 모든 폐암 세포를 타겟으로 한 단클론항체는 없다. 그러므로 우리는 4종류의 폐암 세포 분석을 통해 4종류의 폐암 세포 표면 모두에서 발현되는 세포 표면 단백질을 찾아 새로운 타겟 단백질로 선정하였다. 이러한 기준에 따라 DDOST (Dolichyl-diphosphooligo-saccharide-protein glycosyltransferase 48 kDa subunit) 단백질이 폐암 조기 진단 및 치료를 위한 표적 단백질로 선정되었다. 이전 연구를 통해 DDOST 다클론항체를 사용하여 DDOST 단백질이 폐암 진단 및 치료에 도움이 된다는 결과를 얻어 내었고 우리는 좀 더 정확한 진단을 하기 위해 단클론항체를 제작하였다. 또한 폐암 세포에서 분비되는 엑소좀을 통하여 폐암 진단에 사용하고자 하였다. 먼저 생산한 단클론항체의 적정을 통하여 우수한 항체를 골라내어 실험에 사용하였다. 다음으로 DDOST 단백질이 정상 폐 세포가 아닌 폐암 세포에서만 발현되는 지를 확인하였다. 그 결과 DDOST 단백질이 정상 폐 세포가 아닌 폐암 세포에서만 발현되는 것을 확인하였다. 또한 실제로 폐암 세포에서 엑소좀이 분비되는 지를 확인하여 엑소좀이 진단에 사용될 수 있는지를 확인하였다. 다음으로 ELISA 방법을 통해 엑소좀의 표면 단백질인 DDOST를 이용한 폐암 진단이 가능한지를 확인하였다. 세포에서뿐만 아니라 폐암 이식 동물 모델에서 얻은 혈청과 실제 폐암 환자에게서 얻은 혈청으로도 동일하게 실험을 진행하여 DDOST 단백질이 실제로 폐암 진단이 가능한지를 확인하였다. 실험 결과들을 통해 엑소좀이 폐암 진단에 사용될 수 있으며DDOST 항체가 폐암 조기 진단에 효과적인 바이오마커로서 사용이 가능할 것이라는 것을 확인할 수 있었다.|Lung cancer is a one of the fatal cancer with the highest morality in the world. There are many reasons, because of absence of effective early diagnosis and treatment. When people was diagnosed with lung cancer, they were already late for the treatment. Lung cancer divided 4 types with size and shape, Adenocarcinoma, Small cell lung carcinoma, Non-small cell lung carcinoma, Squamous cell lung carcinoma. There have been many trials to diagnosis or treatment of lung cancer. One of the rising diagnosis method is proteomic tools. Using proteomic tools, biomarkers of lung cancer which are able to estimate the differences between normal and lung cancer with non-invasive method. Especially, cell surface membrane protein can be an efficient biomarker. There are several monoclonal antibodies approved clinically that target non-small cell lung cancer. Unfortunately, they are not suitable for all types of lung cancer. Therefore, we investigate a new target protein covering with all types of lung cancers by analyzing cell membrane proteins expressed in four kinds of lung cancer cell lines, A549, H460, H146, SK-MES1. DDOST (Dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48kDa subunit) was selected candidate target of lung cancer among cell membrane proteins expressed in only lung cancer cell lines. We suggest that DDOST protein is a potential target for antibody based early diagnostic and therapy of lung cancer with polyclonal antibodies in previous experiments. So, for a more accurate and specific diagnosis and therapy, we produced monoclonal antibody. We make hybridoma cell and get antibody with serum free media or xenograft mice model. We found that DDOST monoclonal antibody works well through flow cytometry and western blot. And we confirmed antibody’s working with lung cancer growth by check. Our result explains that using monoclonal antibody is more specific method of early diagnosis of lung cancers. And we use exosome from cells for diagnosis of lung cancer. Exosome is a vesicle secreted from the cell and is known to reflect the properties of that cell. And Exosome has same proteins of the cells. Exosome is also secreted from early stage of cancer and is known to be secreted more from cancer cells. Also, there is a lot of Exosome in blood, about 〖10〗^10or more per ml of blood. We use Exosome with its property for marker to diagnose lung cancer. We suggested that Exosome is efficient biomarker to diagnosis of lung cancer with non-invasive method.; Lung cancer is a one of the fatal cancer with the highest morality in the world. There are many reasons, because of absence of effective early diagnosis and treatment. When people was diagnosed with lung cancer, they were already late for the treatment. Lung cancer divided 4 types with size and shape, Adenocarcinoma, Small cell lung carcinoma, Non-small cell lung carcinoma, Squamous cell lung carcinoma. There have been many trials to diagnosis or treatment of lung cancer. One of the rising diagnosis method is proteomic tools. Using proteomic tools, biomarkers of lung cancer which are able to estimate the differences between normal and lung cancer with non-invasive method. Especially, cell surface membrane protein can be an efficient biomarker. There are several monoclonal antibodies approved clinically that target non-small cell lung cancer. Unfortunately, they are not suitable for all types of lung cancer. Therefore, we investigate a new target protein covering with all types of lung cancers by analyzing cell membrane proteins expressed in four kinds of lung cancer cell lines, A549, H460, H146, SK-MES1. DDOST (Dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48kDa subunit) was selected candidate target of lung cancer among cell membrane proteins expressed in only lung cancer cell lines. We suggest that DDOST protein is a potential target for antibody based early diagnostic and therapy of lung cancer with polyclonal antibodies in previous experiments. So, for a more accurate and specific diagnosis and therapy, we produced monoclonal antibody. We make hybridoma cell and get antibody with serum free media or xenograft mice model. We found that DDOST monoclonal antibody works well through flow cytometry and western blot. And we confirmed antibody’s working with lung cancer growth by check. Our result explains that using monoclonal antibody is more specific method of early diagnosis of lung cancers. And we use exosome from cells for diagnosis of lung cancer. Exosome is a vesicle secreted from the cell and is known to reflect the properties of that cell. And Exosome has same proteins of the cells. Exosome is also secreted from early stage of cancer and is known to be secreted more from cancer cells. Also, there is a lot of Exosome in blood, about 〖10〗^10or more per ml of blood. We use Exosome with its property for marker to diagnose lung cancer. We suggested that Exosome is efficient biomarker to diagnosis of lung cancer with non-invasive method.