Contributions Of Circulating Tumor Cells With Epithelial-mesenchymal Transition In Proliferation, Drug Resistance And Bone Metastasis Of Lung Cancer

Background and objectivesLung cancer is the leading cause of cancer related death globally, accounting for almost 20% of cancer related fatalities. According to the pathological type and clinical feature, lung cancer are classified into two major types: non-small cell lung cancer(NSCLC) and small cell lung cancer(SCLC). The NSCLC represents about 85% of all lung cancers. Lung cancer is commonly associated with multiorgan metastasis. More than 65% of patients present local or disseminated metastatic disease at diagnosis. Bone metastasis represents one of the most deleterious clinical consequences of lung cancer. These skeletal lesions involve significant morbidity, metabolic syndromes, pathologic fractures, and spinal cord compression, which entail an overt reduction in quality of life and require costly treatments with limited impact on overall survival. As many as 50% patients with NSCLC develop bone metastases with a median survival rate in these patients of about 6 months, the lowest for all solid tumors metastasizing to bone. Five-year survival for these patients with current therapies is less than 5%. The main treatments for bone metastases are surgical resection and antiosteolytic therapy. However, there is still an urgent need for new agents targeting lung cancer derived bone metastases.It is well recognized that metastasis consists of distinct steps in tumor cells: 1. detach and migrate away from the primary tumor site, 2. invade neighboring tissue and penetrate through basement membrane, 3. enter the blood or lymphatic vessels, 4. survive the condition of anoikis while they are detached from the tumor mass and in circulation, then exit the blood or lymphatic vessels at a distant organ, 5. form micrometastatic nodule, adapt and reprogram the surrounding stroma, and form macrometastases. Investigators seeking to understand the cellular and molecular bases of tumor metastasis inevitably are challenged by the fact that metastasis is a complex, multistep biological process that most likely is controlled by distinct genes and signaling pathways during each step. Changes in cell phenotype from the epithelial to mesenchymal state is defined as the epithelial–mesenchymal transition(EMT) Epithelial carcinomas acquire fibroblastlike properties and exhibit reduced cell-cell adhesion and increased motility via EMT, which facilitates the escape of tumor cells from primary tumors.Unlimited growth is one of classic features of cancer cells. However, people find that not every tumor cell possess the ability to infinite proliferation. The term cancer stem cells(CSCs) refers to a minute subset of exclusively tumorigenic cells with the ability to self renew and to generate the diverse cell types present within a given tumor. CSCs less than 1% of the primary cancer cell number are sufficient initiate tumors in mice. Induction of EMT in differentiated cells by either overexpression of Snail or Twist caused the cells to acquire stem cell profile.Circulating tumor cells CTCs were defined as tumor cells originating from either a primary tumor or as metastatic cells that circulate freely in the peripheral blood of carcinoma patients. CTCs can be detected in almost all patients with solid tumors and associated with prognosis. CTCs are composed of several subpopulation and the mesenchyma types account for only a small percentage. Basically, each type of tumor cell can be ‘circulating’. It is likely, however, that metastases develop from a restricted population of tumor cells that have undergone EMT and that have acquired the ability to migrate to distant sites via the blood circulation, to colonize other tissues, and to initiate de novo tumor growth. Hence isolating and analyzing CTCs that have undergone EMT will propell a deeper understanding of metastatic progression and unveil novel therapeutic targets for drug development.Research contents and methodsWe collected typical tumor specimens from patients with NSCLC who were surgical treated at the Changzheng Hospital Affiliated to the Second Military Medical University.The specimens include primary lung tumor, lung cancer bone metastasis and para-tumor lung tissue. The expression of CDH1, EPCAM and VIM were detected with immunohistochemical assay. Then we injected the high levels of bone seeking PC-9 cells(PC-9H) with luciferase expression into the lung of the nude mice that established in our previous research.We analyzed and isolated the EMT cells of the mouse model by in vitro bioluminescence and flow cytometry and named PC-9EMT.In the following, western blotting, cell proliferation assays, immunofluorescence test, transwell migration assay, colony formation assays, chemotherapy resistance test, sphere forming test, cell cycle assay, apoptosis assay and osteoclast differentiation assay were used to detect the properties PC-9EMT.Finally, we used RNA-sequences array of transcriptome to identify the differentially expressed genes of PC-9EMT and confirmed gene of interest by using real-time RT-PCR. To further elucidate the important role of those selected genes, we analyzed the relationship between these genes m RNA expression level and the first progression time of NSCLC patients from 2437 lung tumor samples using publicly available datasets(2015 version).Results and conclusionIn this study, we found the following results:1.We successfully established the human lung cancer bone metastasis mouse models for in vivo imaging with luciferase expression.2. We can isolate EMT cells from these models for studying the mechanisms of lung cancer progression and development of anticancer drug3.We found the PC-9EMT cells generate with properties of both mesenchymal cell and stem cells。4.Comparative transcriptomic analysis and Real-time PCR analysis showed that expression of signaling molecules regulating several tumor properties including migration(MMP9), EMT(SNAI1、TWIST1), adhesion(FN1、COL1A1、COL6A1、JUP、OCLN、L1CAM) and drug resistance(KRT17、S100A4、KPTN、DLC1 和 EMP3) were altered in PC-9EMT cells.Our fndings should be helpful for a better understanding of the molecular aspects of the metastatic process in different organs, and could lead to molecular target based anticancer drugs and prevention of metastasis, especially bone metastasis. These findings also illustrate a direct link between the EMT and the gain of epithelial stem cell properties.