| ObjectivesAs the data showed by the Centers for Disease Control (CDC) in 2012, breast cancer has been become the second most diagnosed cancer. While in the females worldwide, it is the most frequently diagnosed cancer and the leading cause of cancer death. The most common cause related with mortality is the present of metastasis, including lung, bone and brain metastasis. Epithelial-mesenchymal transition (EMT) has been accepted as a critical process that allows stationary epithelial cells to gain the motile ability needed to migrate and invade during embryogenesis, organ development, tissue regeneration, and organ fibrosis. Extensive studies over the past decade have provided strong evidence for the activation of the EMT program in cancer progression and metastasis. Thus, more research about the relationship between EMT and cancer progression needs to be uncovered.However, most data from the EMT researches got from cell lines and ex vivo tumor slices,we are lack of direct in vivo data to prove the existence of EMT in tumors and we still do not know about the in vivo characteristics related with EMT in the primary tumors. In this study,we try to use an transgenic EMT-driven fluorescent protein switching mice breast cancer model, combined with intravital two-photon microscopic imaging, to get the direct in vivo imaging evidence of the EMT process in primary breast cancer. We also analyzed the in vivo characteristics related with EMT to uncover the relationship between EMT and tumor metastasis. The EMT changes after anti-metastasis drug treatment was also visualized in this study.MethodsBreast cancer cells acquired from the MMTV-PyMT, Rosa26-RFP-GFP, and Fsp1-Cre triple transgenic mouse model, which can used to monitor the conversion of RFP-positive epithelial cells to GFP-positive mesenchymal cells in mammary tumors under the control of the Fspl (ATL1) promoter, a gate keeper of EMT initiation. RFP-positive cells were isolated from the tumors, sorted, and transplanted into mammary fat pads of SCID mice to monitor EMT during breast tumor formation. A skin flap contains the tumor and kept the blood supplies was made to expose the tumor for the intravital two-photon microscopic imaging.Continuous time lap imaging was acquired in the sorted RFP tumor cells culture, which was used to capture the dynamic changes of the EMT process. Similar time lap imaging was also performed in the intravital microscopic imaging, to obtain the EMT process in vivo. The GFP cells in the primary tumors were imaged and their morphology characteristics were analyzed.Cabozantinib (XL-184) was chosen as the anti-metastasis drug based on the gene database analysis. The drug effects on EMT process was first confirmed in the cell culturing.TGF-? was used to induce EMT in the sorted RFP cells, after treatment of XL-184, the cell signal and morphology was analyzed. The drug was also used in the tumor mice, the tumor volumes were recorded, intravital two-photon microscopic imaging was performed after the drug treatment. The morphology and distribution of GFP cells in the tumor was analyzed.Results1. Based on the immunocytochemistry staining results, we found that when the RFP tumor cell turn to GFP positive, the expression of E-cadherin will lost, which indicated that the switching of cell color from RFP to GFP represents the EMT process in tumor cells. The intravital two-photon microscopic imaging of the primary breast tumor shows sporadic distribution of GFP cells, indicated the EMT processes in primary tumor.2. Continuous time-laps imaging of cultured RFP cells captured the gradual GFP signal enhancement and the elongation of cell morphology. Similar signal and morphology changes also captured in the intravital microscopic imaging of the primary breast cancer.3. Based on the GFP cells morphology results, we found that vast majority (96.55%) of cells after EMT acquired morphology characteristics related with migration and invasion,only a small number of cells became cancer associated fibroblast cells, which contributed to the regulation of tumor progression. More invasion characteristics was obtained in cells after EMT, including more fusiform cell shape, more cell membrane protrusions and preferentially localized close to blood vessels. When the cell located in the center of the tumor lobule, the elongation of membrane protrusions is very slow, while when the cell move adjacent to blood vessels, the elongation velocity accelerated dramatically. What's more, we also observed that small clusters of GFP cells move together toward the blood vessel or tumor stroma.4. In the in vitro cell cultures, XL-184 inhibited the EMT process induced by TGF-?. In vivo, XL-184 slow down the growth of primary breast cancer dramatically; what's more,during the intravital two-photon microscopic imaging of the drug treated tumor, we found clusters of GFP cells which have similar distribution and cell morphologies, it's totally different from what we imaged in control groups. These clustered GFP cells were demonstrated as epithelial phenotype by IHC staining.ConclusionsIntravital two-photon imaging combined with the EMT driven fluorescent protein switching mice breast cancer model, the direct imaging of EMT process in primary breast cancer was successfully captured. Based on the morphology changes after EMT, the imaging results demonstrated that tumor cells mainly gained more characteristics related with cell migration and invasion, which indicated that the EMT process in primary breast cancer mainly correlated with tumor invasion and metastasis. The in vitro drug treatment demonstrated that the drug can inhibit EMT process in cells. The intravital imaging of the drug treated breast cancer showed that the drug could reverse the EMT process at the very early stage. These results indicated that the drug inhibition of tumor growth and metastasis may through inhibition of EMT process in the tumor, which may provide a new target for the breast cancer treatment. |
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