Whole-body Fluorescence Imaging Of GFP-expressing Tumor Growth And Metastasis

How tumor cells spread remains an area of intense medical investigation. More and more cancer genes involed in metastasis have been cloned. However, traditional histopathological examination or immunohistochemistry can't test gene function in vivo and evaluate drug efficacy at molecular targets associated with disease. In recent years, advances in the molecualr imgaing 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. Whole-body opitical imaging method provides the visual representation, characterization, and quantification of physiological and pathological processes at the cellular and molecular levels within intact living organisms. Small advantages of optical imaging method compared with other imaging modalities include 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 equipment. In our study, we have imaged, in real time, GFP-expressing tumors growth and metastasis.Human lung carcinoma cells(SPC-A1)were transfected with pEGFP-C1 by FuGENE 6 reagent, and selected by culture with geneticin (G418). A stable high GFP-expressing clone, SPC-A1-EGFP was obtained by limiting dilution in 96-well flat-bottomed culture plates. A comparison of the growth curves of the two cell lines, SPC-A1 and SPC-A1-EGFP, showed that SPC-A1 and SPC-A1-EGFP cells were not clearly different in growth rates. GFP expressing stability was tested, and showed that SPC-A1-EGFP cell was stably expressing, so we could substitute SPC-A1-EGFP cells for SPC-A1 cells to study tumor in vitro and in vivo.SPC-A1-EGFP cells were treated with cisplatin or Triton X-100 to induce apoptosis or necrosis, respectively. Fluorescence microscopy was used to image sing cell fluorescence during cell death. Cells in a given population were analyzed by flow cytometry for the percentage of fluorescence-positive cells during cell death. The results showed that single-cell experiment was consistent with population-cell experiment Live GFP-transfected cells showed a strong fluorescence intensity, which was significantly diminished upon induction of apoptosis, whereas necrotic GFP-transfected cells almost completely lost their GFP-associated fluorescence. Cell death correlated strongly with the fluorescent signal from stable SPC-A1 cell lines expressing GFP at high levels, making it an ideal tool for cell-based high throughput screening antitumor agents. A subcutaneous tumor growth model was established by injecting SPC-A1-EGFP cells subcutaneously into nude mice. Two types of metastasis model were established: spontaneous metastases by subcutaneous injection and experimental metastases by tail vein injection of SPC-A1-EGFP cells. Tumor-bearing mice were viewed with whole-body fluorescent imaging system. The results showed that growth of subcutaneous tumor and metastasis of intraperitoneal tumor were visualized clearly with this system, while lung metastases were imaged via a chest-wall skin-flap window. This study provides a platform for monitoring metastasis by orthotopic implantation and evaluating in vivo efficacy of antitumor drugs. Our work showed that GFP-expressing tumor cells could be used to not only study relationship between cell death and fluorescence intensity, but aslo non-invasively monitor tumor growth, metastasis and angiogenesis in real time. GFP-based optical imaging has potential to screen antitumor drugs at cellular and in vivo levels.