Optical Imaging Of Growth,Metastasis And Apoptosis Of Human Adenoid Cystic Carcinoma

How tumor cells spread remains an area of intense medical investigation. As more and more cancer genes involved in metastasis have been cloned, traditional methods of tumor detecting (immunohistochemistry or histopathological examination) can't test gene function in vivo or evaluate drug efficacy at molecular targets associated with disease. In recent years, advances in the molecular imaging have been accelerated. With small animal models widely used in the basic and pre-clinical sciences, non-invasive imaging of tumor growth and metastasis in vivo in real time becomes a reality. Optical imaging method provides the visual representation, characterization, and quantification of physiological and pathological processes at the cellular and molecular levels within intact living organisms. Obvious advantages of optical imaging method compared with other imaging modalities including the use of non-ionising low energy radiation, high sensitivity, capability of continuous data acquisition for real-time monitoring, no substrate, and the development of potentially cost-effective equipments. In this dissertation, we studied two aspects by optical imaging. On one hand, we monitored tumor growth, early stage tumor metastasis and tumor treatment by a fluorescent imaging system established in our laboratory, while at the same time monitoring microbe infection and therapy. On the other hand, by fluorescence resonance energy transfer (FRET) combined with fluorescent protein, we monitored apoptosis of TK-GFP-expressing ACC-M cells induced by ACV from cell level. The main contents and conclusions are as following: First human adenoid cystic carcinoma cell (ACC-M) was transfected with pEGFP-C1 by SuoHua reagent, and selected by culture with geneticin (G418). A stable high GFP-expressing clone, ACC-M-GFP was obtained. Five-week-old female nude mice were injected with ACC-M-GFP in the primary organ: submandibular gland. Metastases were only visualized by GFP expression in the lung by a whole-body fluorescent imaging system established for nude mice in our laboratory. However, metastatic lesions of ACC-M-GFP in the lung, muscle, bladder and bony were found by imaging of GFP expression in intact mice through tail vein injection of ACC-M-GFP cells. At the same time, we made metastasis tissues for pathological slices, which were proved tumor cells. The construction of highly fluorescent and stable GFP transfectants of ACC-M had revealed the multi-organ metastatic capability of ACC-M cells. In addition, the therapy model of subcutaneous tumor growth by subcutaneous injection of ACC-M-GFP was established. Tumor-bearing mice were viewed with whole-body fluorescent imaging system. The results showed that growth of tumor was visualized clearly with this system. And this simple, nonintrusive technique can show in great detail the temporal and spatial behavior of the infectious process of red fluorescent protein (DsRed2)-expressing bacteria from outside intact infected animals. This study provides a platform for monitoring tumor growth and evaluating in vivo efficacy of antitumor drugs. Based on the above study, HSV-tk/ACV induced and killed human adenoid cystic carcinoma cell (ACC-M) in vivo and in vitro, which were observed through optical imaging and green fluorescence protein (GFP) tagging technique. ACC-M was transfected with TK-GFP, and the single clone cell strain ACC-M-TK-GFP was selected by G418. With fluorescent microscope, whole-body fluorescent imaging system and fluorescent microscope, we could view ACV treated ACC-M-TK-GFP cells in cell level and in nude mice. The results showed ACV could induce apoptosis of ACC-M cells with TK gene, had no effect on ACC-M cells without TK gene. Tumor death inversed proportion with vessel density was also found by whole-body fluorescent imaging system. This study proved that optical imaging is very crucial for studying disease, monitoring tumor growth and evaluating in vivo efficacy of antitumor drugs with the development of fluorescent tagging technique. Inducing apoptosis of tumor cells is one of the important strategies in tumor therapy. To study the characteristics of suicide gene system of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells, the dynamic characteristic of apoptosis in this system and its effect on the human adenoid cystic carcinoma line ACC-M cell, CD3 (ECFP-DVED-DsRed)-and TK-GFP co-expressing ACC-M cells was used in our study. CD3 is a FRET-based indicator for activity of caspase3, which is composed of an enhanced cyan fluorescent protein, a caspase-sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. The cell apoptosis induced by acyclovir (ACV) was monitored using fluorescence resonance energy transfer (FRET) technique. FRET from ECFP to DsRed could be detected in normal CD3 and TK-GFP co-expressing ACC-M cell, while the FRET efficiency was remarkably decreased and then disappeared during cell apoptosis. It indicated that the apoptosis of TK-GFP-expressing ACC-M cells induced by ACV was via a caspase3 -dependentpathway. In this study, we monitored apoptosis of CD3 and TK-GFP co-expressing ACC-M cells,CD3-expressing ACC-M mixed TK-GFP-expressing ACC-M cells and CD3-expressing ACC-M cells induced by ACV were initiated caspase using FRET technique. The results showed ACV could induce apoptosis of CD3 and TK-GFP co-expressing ACC-M cells, but not CD3-expressing ACC-M cells. Bystander effects of HSV-tk/ACV were also found by FRET technique. It suggested that HSV-tk system combined with ACV was effective on inducing the apoptosis of cancer cell. The same as it was proved that FRET technique combined with CD3 probe, be promising rapid-screening potential drugs for cancer research.