Zebrafish modelling of apoptosis and inflammation in aggressive B-cell lymphoma
Apoptosis is a well-orchestrated programmed cell death. In cancer biology the evasion of apoptosis has been considered as one of the key events for tumour development and paradoxically, studies also show that apoptosis has detrimental effects that may even promote cancer. High rates of apoptosis have been observed in many cancers and aggressive B cell lymphoma (prototypically Burkitt’s lymphoma (BL)) present characteristic ‘starry sky’ appearance due to extensive apoptotic tumour cells engulfed by infiltrating tumour-associated macrophages (TAM). Previous work using a murine BL cell model has shown that constitutive apoptosis promotes both angiogenesis and the accumulation of pro-tumour TAMs. However, the detailed cellular and molecular mechanisms underlying how apoptosis fosters this pro-tumour growth microenvironment are still not fully understood, especially the role of apoptosis during early steps of tumour initiation. The aim of this project is to establish an in vivo model system to dissect the mechanisms as to how apoptotic B lymphoma cells enhance angiogenesis and how apoptotic B lymphoma cells interact with cells within the host microenvironment to promote tumour progression. Zebrafish (Danio Rerio), a small tropical fresh water fish, has become increasingly popular as a biomedical research model organism. Not only is it amenable for genetic manipulation, but also due to its transparency in the larval stages, is one of the most important models for in vivo live imaging studies. Therefore efforts were made to establish a novel transgenic B lymphoma model in zebrafish. Complementary to the transgenic model, I also established a Xenograft model using a B lymphoma cell line BL2 and its apoptosis resistant derivative BL2-bcl2 cell lines. Using a Tol2 based transgenesis system and zebrafish promoter for IgM heavy chain, I have generated transgenic zebrafish with either constitutive B cells expressing oncogenic cmyc, Tg (IgM1::cmyc-eGFP), or a tamoxifen inducible version, Tg(IgM1::CreERT2/IgM::lox-H2BmCherry-lox-cmyc-eGFP) which allows induction of oncogenic cmyc expression in B-cells with precise temporal control. Unfortunately, neither of these models developed B cell lymphoma, and fish appear to be generally healthy. Although flow cytometic analysis showed normal expression of the transgene in Tg(IgM::lox- H2BmCherry-lox-cmyc-eGFP), further analysis of the constitutive model failed to detect any CMYC expressing eGFP positive B-cells in the head-kidney. Therefore, unexpectedly IgM driven cmyc expression in zebrafish might drive B-cell death instead of B-cell malignancy. This is in contrast to the mouse B lymphoma model. More work is needed in choosing a suitable promoter and/or oncogene combination to generate a transgenic zebrafish B lymphoma model. Xenograft models using zebrafish larvae provided an additional opportunity to study interaction between tumour cell and cells within the tumour microenvironment. Available transgenic reporter zebrafish strains labelling various cell lineages facilitate in vivo imaging of host cellular responses to B lymphoma cells. In order to identify putative roles that apoptotic tumour cells might play in the tumour microenvironment, I have established a reliable and consistent xenograft protocol to graft tumour cells into yolk sack of 2 days post fertilization (dpf) zebrafish larvae. Consistent with previous observations in mice, BL2 (an apoptotic prone lymphoma cell line) cells survive better than their apoptotic resistant derivative BL2-bcl2 (over-expressing bcl2 in BL2 cells). However, the overall longest survival time is no more than 4 days post grafting even with BL2 cells. A possible explanation for this could be lack of some key B cell survival factors in zebrafish larvae, as normally mature B cells do not develop until two weeks post fertilization. The mouse models of BL indicate that TAMs have been attracted by apoptotic BL cells and accumulate at the BL microenvironment. To evaluate whether macrophages modulated by apoptotic cells promote BL survival in the zebrafish model, human monocyte-derived macrophages were activated by either apoptotic BL2 cells or IFN-γ / lipopolysaccharide (LPS) and co-injected with BL2 cells into zebrafish. Results showed that macrophages activated by apoptotic BL2 cells, but not IFN-γ /LPS, enhanced the survival of BL2 cells. In the next part I further investigated how apoptotic BL2 cells might modulate macrophages and the tumour microenvironment. Extracellular vesicles (EVs) are small membrane-bounded vesicles whose molecular profile is regulated by their cellular origin and the types of stimuli. EVs have been shown to be critical messengers in tumor progression and metastasis. The study of apoptosis-induced EVs (Apo-EVs) is sparse. I hypothesised that EVs released by apoptotic cells might mediate their pro-tumourigenesis properties. I used a recently developed novel protocol in my laboratory to isolate Apo-EVs from BL2 cells. EVs from apoptosis resistant BL2-bcl2 cells (non-Apo-EVs) were used as a control. I show here for the first time that Apo- EVs are pro-angiogenic in vivo. Further analysis of the secretome from apoptotic BL2 cells as well as their Apo-EVs indicates that soluble protein component(s) mediate the pro-angiogenic function. Combining macrophage reporter fish Tg(mpeg1::mCherry) with TNFα reporter Tg(tnfα::eGFP), I show that Apo-EVs promote macrophage activation but not TNFα in vivo. In conclusion, this project suggests that apoptotic tumour cells execute their pro-oncogenic functions by modulating macrophage activation, enhancing tumour angiogenesis, possibly through releasing Apo-EVs. Apo-EVs are recognized as a key player in fostering a pro-tumour growth microenvironment. Thus a further understanding of how apoptotic cells exert their tumour promoting roles will help us to optimise cancer therapy by maximizing tumour cell death while minimizing unwanted pro-tumorigenic effects. The models established during this project may be used to identify factors that are key to the survival and growth of B lymphoma.