Team:BM-AMU/Human Practices

Team:BM-AMU- 2019.igem.org

Human Practices



Background

Epithelial–mesenchymal transition (EMT) plays a crucial role in embryonic development, wound healing, and cancer metastasis. It is a progressive process with many heterogeneous metastable states in the middle, not only the epithelial state and mesenchymal state. These hybrid phenotypes are endowed with combined epithelial (e.g., cell–cell adhesion) and mesenchymal (e.g., motility) capabilities. It is clearly of interest to explore heterogeneous metastable states in EMT for understanding cell-fate determinations during embryonic development and tumorigenesis.

After identifying this valuable field, we are not trapped in the lab, but reach out to and learn from diverse stakeholders (Histology and Embryology Laboratory; Department of Pathology; Biotechnology company). Turning ideas into reality requires us to consider influence to communities. Therefore, we need to collect multiple opinions to ensure that our project is beneficial and responsible to the world. And furthermore, the feedback can somehow optimize our project, while we are trying to give out accessible solutions to what has troubled local communities through our designed two-way dialogues.

Inspiration
  • Histology and Embryology Laboratory +

      Inspiration：Focus on the intermediate states in the EMT process

      In order to understand the research status of epithelial-mesenchymal transition (EMT) , we met with several professors specializing in Embryonic Development.

      “Epithelial-mesenchymal transition (EMT) plays a crucial role in embryonic development and cancer metastasis.” - Prof. Rui Jian

      We met with Prof. Rui Jian, a developmental biologist in Histology and Embryology Laboratory, AMU. He is very excited that we are interested in the process of EMT and enthusiastically introduced us to the current researches related to EMT. The most important feedback we got is that EMT plays a crucial role in embryonic development and cancer metastasis. Through EMT, the phenotypes of cells undergo many fluctuations, such as losing cell–cell adhesion and cell polarity, and acquiring migratory and invasive properties.

      HP

      Finally, he suggested us to get in touch with the pathologists in hospitals due to the importance of EMT in realistic clinical conditions.


      “EMT is a multi-step transition, and various degrees of the transition may have distinct physiological outcomes” - Prof. Junlei Zhang

      Prof. Junlei Zhang is interested in stem cell biology. And we have also learned from her about some frontiers of EMT research. The most important feedback we got is that she suggested us to focus on the intermediate states in the EMT process. She explained that cells that have undergone EMT sometimes revert back to the epithelial state. This mesenchymal-epithelial transition (MET) enables the migratory cells to settle and proliferate at a distant organ, suggesting that EMT is a reversible process. EMT does not give rise to terminal mesenchymal phenotype in some biological scenarios. These observations and hypothesis strongly suggest that EMT is a multi-step transition, and various degrees of the transition may have distinct physiological outcomes. And at the end of our talks, she summarized that few studies have tried to identify these complex transitory states now, mainly due to the tremendous difficulty in accurately capturing cells in specific intermediate EMT states.

      HP

      Feedback: We are suggested to focus on the intermediate states in the EMT process.


  • Department of Pathology +

      Inspiration：Cellular heterogeneity based on EMT hybridization

      Another important realistic pathological process associated with EMT is the tumor process, so we followed Prof. Rui Jian’s suggestion and visited three hospitals in Chongqing: Southwestern Hospital, Daping Hospital and Xinqiao Hospital, then got in touch with several pathologists.

      “The functions of these multiple intermediate states and the complex transitions among all the phenotypic states are unclear” - Dr. Shicang Yu

      We met with Dr. Shicang Yu, a pathologist in the Southwestern Hospital, and talked about the effects of intermediate states of EMT in tumor process. He thought that the study of the intermediate states during EMT is valuable for the detection of tumor heterogeneity.

      HP

      When learn about intermediate states of EMT from Dr. Shicang Yu, we got more useful information from him: these intermediate states are associated with stemness and invasiveness during cancer progression, but the functions of these multiple intermediate states and the complex transitions among all the phenotypic states are unclear.


  • Biotechnology company +

      Inspiration：Obtain cellular accurate data during EMT Inspiration

      We had the opportunity to visit Vilang Biotech. In Chongqing, and Dr. Jian Wang, CEO of Vilang, led us to visit their company. Then, we had an in-depth conversation on techniques for researching the intermediate states during EMT.

      HP

      “The bottleneck of identifying these complex transitory states is the tremendous difficulty in accurately capturing cells in specific intermediate EMT states and, and what's more obtaining their cell data.” - Dr. Jian Wang

      Dr. Jian Wang told us that most studies now rely on immunofluorescence staining of cell fate markers to distinguish between different intermediate states during EMT. And the remaining cells would be selected under the same conditions for further exploration, such as RNA sequencing. However, due to cellular heterogeneity, the cells used for sequencing are different from the stained ones. And just like Prof. Junlei Zhang's idea, he thought that the bottleneck of identifying these complex transitory states is the tremendous difficulty in accurately capturing cells in specific intermediate EMT states and obtaining their cell data.

    • Output of inspirations:

      After the first input from diverse stakeholders (Histology and Embryology Laboratory; Department of Pathology; Biotechnology company), we came up with our 1.0-vision: Develop a method for accurately capturing cells in the intermediate states of EMT, so captured cells are not destroyed and can be used for further exploration. After the team brainstorming, we designed the project: EMT markers labeled with fluorescence as a precise monitor to measure the degree of transdifferentiation from E to M. Thus, we can accurately derive cells in different intermediate EMT states based on distinguished fluorescence expression.

    • Execution

    A solution to defects of traditional inducer.

    TGF-β is a classical controller for inducing EMT transformation, but we found that the process of EMT induced by TGF-β is unstable and discontinuous. In order to control the EMT process more accurately, we consulted Prof. Rui Jian again and got some insights.

    “In addition to inducing EMT progression, TGF-β has many other physiological effects, such as inflammation, tissue repair, and immune regulation. It acts like a master switch that controls many secondary switches below. ”- Prof. Rui Jian

    Feedback: we can find the end switch that manipulates the EMT in one direction and construct an orthogonalized system. After looking through the literature, we intend to use fundamental transcription factors as more precise controllers.

    Adjustment: We chose Snail1 and Twist1 transcription factors as controllers. Additionally, the Tet-on system and the Tamoxifen-Ert2 system were selected to control the expression level of transcription factors.

    A two-way dialogue

    unlocking the secrets of transcriptional landscape from cellular phenotype

    We have tried to design a software. When we input a fluorescent image with EMT markers labeled, it can help output the most probable sketch map of transcriptome. In order to popularize our software tool and get some advice for further promotion, we have organized an interview towards our potential users.

    Prof. Jiaxiang Xiong

    In order to evaluate the application value of this software, we met Prof. Jiaxiang Xiong, Teaching & Experiment Centre of Basic Medicine.

    Input: we’ve explained the mechanisms of image recognition and its applications in medical sciences, and convinced him that our software tool was practicable.

    Output: he was doubted about the users’ accessibility due to the difficulty of getting a fluorescent protein knock-in model.

    Adjustment: we are developing an update version to make immunofluorescent samples accessible.

    Dr. Ying Wan

    In order to evaluate the feasibility of software design, we communicate with Dr. Ying Wan, CTO of Vilang.

    Input: He agreed with us about that idea of predicting after our introductions.

    Output: Dr. Wan believed that our software is feasible, but he proposed that the credibility of the experimental results is not strong enough. Therefore, he suggested us to promote the algorithm to adapt high throughput samples in the future.

    Adjustment: Convolutional neural networks (CNN) models were developed here to expand the Supporting Vector Machine (SVM) models’ capability of handling high throughput data.