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
