斑马鱼模型,黑色素瘤 z-bio 2021-02-21 371

　　Abstract: Dynamic cell heterogeneity is the cause of melanoma progression and treatment resistance. Advances in single-cell technology have revealed an increasing number of tumor and microenvironmental cell states in melanoma, but little is known about their function in the body. The zebrafish model is a powerful system for discovering, real-time imaging and studying the cell status during the progression and treatment of melanoma. By capturing the dynamic state of melanoma in living animals, zebrafish has the potential to solve the complexity of melanoma heterogeneity, which ranges from a single cell to the entire body of the disease process, revealing novel cancer biology and therapeutic targets . Melanoma is the highest of all cancers in terms of intratumoral heterogeneity and clonogenic potential, and is characterized by a poor prognosis. Heterogeneity stems from the combination of genetic mutations acquired in the course of disease development and the variation of non-genetic factors, such as gene transcription and metabolism. The transcription status of melanoma can reflect the developmental trajectory of neural crest, melanocytes or pigmented melanocytes, as well as status unrelated to lineage, such as stress or starvation. Another layer of heterogeneity stems from differences in cell cycle dynamics within tumors. Some populations are actively circulating, while others are slowly circulating or dormant. These populations can also exhibit stem cell characteristics. To make matters more complicated, the state of melanoma cells shows plasticity and can transition from one state to another. Understanding how transcriptional status affects the progression of melanoma in individual patients is critical to drug development and an important step in the development of personalized treatment options.

　　 The latest advances in single-cell RNA sequencing (scRNA-seq) technology provide an effective method to explore the degree of heterogeneity within tumors. Using scRNA-seq analysis, Tirosh and colleagues demonstrated that the transcriptome of melanoma tumor cells is arranged along a bipolar expression pattern. Untreated tumors mainly express MITF program genes at one end, and BRAF/MEK inhibitor-treated melanoma cells are The other end expresses abundant AXL program genes. Thus, the MITF gene expression program can directly differentiate (high MITF), proliferate (moderate MITF) and infiltrate (low MITF) cell states, as well as low MITF/AXL high cell states to show drug resistance. ScRNA-seq studies in short-term patient-derived cultures further validated these procedures and added diversity, including matrix and oxidative phosphorylation markers. It is becoming more and more obvious that in addition to extreme melanocyte and mesenchymal cell states, melanoma cell states can also be found in the dynamic spectrum along this trajectory, passing through one or more intermediate cell states, and even Transdifferentiation to the endothelial lineage. From our point of view, the zebrafish is a powerful lens through which we can resolve the complex intratumoral heterogeneity of melanoma. Similar to human skin, zebrafish melanocytes are found in the basal layer of the zebrafish epidermis. Humans and zebrafish share the developmental biology of many melanocytes. Transgenic and gene editing technologies are highly developed in zebrafish, tumor genomics, advanced imaging and functional manipulation of biological processes that can be compared at the single cell level. The zebrafish cancer avatar based on genetic engineering and transplantation is developing clinically relevant skin melanoma models, as well as rare melanoma subtypes, such as uveal and mucosal melanoma. Zebrafish has become a very successful phenotypic drug screening model, especially by screening the developmental lineage of melanocytes.

　　 scRNA-seq in zebrafish melanoma: The scRNA sequence of the zebrafish melanoma model revealed cancer cell states specific to the neural crest melanoma cell lineage, as well as those cancer cell states that extend beyond the neural crest melanoma cell lineage and are shared by other cancer types. Recently, a BRAF mutant and BRAF-independent melanoma model characterized by low MITF and high MITF transcription has been established. Low MITF zebrafish melanoma and MITF low melanoma patients have the same transcription status, which is a subtype of melanoma characterized by a poor prognosis. Through scRNA-seq analysis, we verified the transcriptome classification of melanoma, therefore, the status at the single cell level can reflect the characteristics of tumor samples. In addition, White, Yanai and colleagues recently reported that BRAFV600Ep53 mutant melanoma has three transcriptional states: melanocytes, neural crest, and stress-like, the latter manifested as pan-cancer cells. It is important to capture the fragility of melanoma as treatment changes and the disease subsides. Marine laboratories and our own laboratories use scRNA sequencing to solve the melanoma cell dynamics in the minimal residual disease (MRD) stage. There are persistent cells in the originally disease-free tissues, and they tend to or acquire the characteristics of tumor recurrence . These studies revealed the unexpected complexity of cells at MRD sites, indicating that multiple cell states coexist. It is worth noting that the persistent cells produced by BRAF/MEK targeted therapy in the PDX model share the aggressive cell state between the persistent cells produced by MITF lineage dependence in zebrafish, which indicates that in melanoma A common state of dedifferentiated cells appears during regression and collapse of large tumors. Our model further identified the G0-like cell population in the residual disease in the primary tumor, and traced back the G0-like cell population found in melanoma and other cancer types by others. Slow-cycle cell populations, some of which express epigenetic modifiers, including jarid1b, suggest that epigenetic control of transcription mechanisms is the basis for persistent cancer cell states. Functional analysis shows that these cells are necessary for continuous cell growth and appear after drug treatment, and even in this state, certain cells are still highly aggressive, but it has not been discovered how these cells promote melanoma recurrence. Together, these models provide strong evidence that the cell state in zebrafish is the same as that of human cancer, including G0-like, aggressive, and pigmented states.

　　 Live cell subpopulation imaging: Dynamic visualization of cell subpopulations and their relationship with the surrounding microenvironment is essential for understanding the evolution of tumors and the phenotypic transition from one cell state to another. The zebrafish embryo is transparent, capable of real-time imaging of development, carcinogenic transformation, and interaction with neighboring cells at single-cell resolution over an extended period of time. The immune system of zebrafish is conserved to humans, and studies of zebrafish melanoma have begun to incorporate components of the immune response. For example, Feng et al. were the first to use real-time imaging of zebrafish embryos to find that the immune system can recognize transformed RAS mutant melanocytes at the earliest stage of melanoma development.

　　 The parallel study of zebrafish developmental lineage and melanoma model has given us a deeper understanding of the lineage disorders of melanoma. Real-time imaging has always been the core of these studies. White, Zon and his colleagues revealed the reappearance of neural crest expression characteristics during the development of melanoma, which led to the first clinical trial of melanoma cell transcription prolongation. Through real-time imaging, Kaufman et al. further used neural crest-specific lineage transgenes to prove that in the cancerous area, only cells that regain the characteristics of the early neural crest will evolve into a malignant population. White and colleagues used transparent adult zebrafish to capture some of the earliest metastatic melanoma cell populations. Real-time imaging of adult fish is also critical for capturing subpopulations of melanoma cells at the site of residual disease in situ. It provides the first image of persistent cells within the endogenous MRD site. These studies illustrate a special advantage of the zebrafish system: real-time imaging can be used to link the scRNA-seq cell status information captured from a fixed time point with the dynamic changes of melanoma cells in their own microenvironment.

　　 Functional analysis of melanoma subpopulations and microenvironment: Functional verification and testing of the role of new cell populations and cell states on the prognosis of melanoma is needed. The status of individual cells, as well as the components of the tumor microenvironment, can be experimentally changed and tracked through the use of fluorescent marker genes. The zebrafish-based platform can achieve tissue-specific and rapid gene overexpression, editing or electroporation, so that gene expression can be controlled in space and time. When combined with genetics, drug therapy and real-time imaging, zebrafish can provide a workable model for xenotransplantation during embryonic and adult stages. Similar to the mouse PDX model, human or zebrafish melanoma cells can be transplanted into zebrafish embryos with functional innate immunity, or into transparent adult zebrafish with weakened immune function, and tracked over time. Hurlstone and colleagues used xenotransplantation to discover the cooperation between cells of different phenotypes to promote melanoma metastasis, a phenomenon called cooperative invasion. In addition, White et al. by transplanting melanoma cells into early zebrafish embryos demonstrated the direct connection between the transcriptional characteristics of the cell state obtained from scRNA-seq analysis and its biological function. Studies have shown that subgroups of melanoma are the most powerful in generating new tumors and providing resistance to BRAF/MEK inhibitors. In the microenvironment, cancer-related cell populations have a profound impact on the growth of melanoma, which can be used for functional verification and mechanism exploration in the zebrafish model. For example, using exogenous endothelin 3 (EDN3) peptides and edn3b mutants, Kim and colleagues found that the EDN3 microenvironment of melanoma controls the transition of the phenotype to proliferation and differentiation. Zhang and his colleagues demonstrated that cancer-related fat cells directly transfer lipids to zebrafish melanoma to promote growth. Ferreira et al. constructed a tert mutant chimeric zebrafish to prove that non-cancer cells with short telomeres can promote aging and inflammation, thereby promoting melanoma in a non-cell-autonomous manner. This type of functional testing combined with reporter gene systems used in imaging and gene editing platforms provides tools for revealing new biological characteristics of cancer-related cell populations in the complex microenvironment of living animals.

　　 Conclusion: Drugs targeting melanoma cell status and subpopulations have appeared. Zebrafish is a powerful model system for rebuilding melanoma tumors in vivo, and a unique platform for phenotypic drug screening that targets the state of melanoma cells and tracks the fate of these cells during treatment. With the development of single-cell omics technologies (such as RNA, DNA, methylated DNA, or open chromatin location sequencing), zebrafish are being used to classify mutations and transcription dynamics in the progression and treatment of melanoma in the whole organism. The first example of combining single-cell RNA-seq with spatial transcriptomics has identified drug-resistant cell populations in the context of tumor morphology. Combined with in vivo imaging, these technologies will be a powerful method to capture how tumor cell populations interact and interact with the microenvironment within the tumor structure over time and subsequent treatment. Although the zebrafish system has the potential to study cell heterogeneity, there are still some challenges. In vivo imaging of adult zebrafish is still limited by resolution and feasibility. The pedigree tracking and site-specific gene editing techniques of adult zebrafish are unconventional. However, looking forward, we will be able to combine scRNA-seq with DNA mutation-based lineage tracking to track clone evolution and integrate proteomics and metabolomics. Gene editing and tissue-specific cell ablation technologies will further help study the function of cell states and types, and validate drug targets. The ultimate goal is to better predict the patient's condition and discover the new treatment that best meets the needs of the patient.