Cellular microenvironment in Burkitt’s lymphoma : gene expression profiling of tumour-associated macrophages in situ
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Date
30/06/2012Author
Petrova, Sofia
Metadata
Abstract
Tumour-associated macrophages (TAM) are a major component of the
inflammatory infiltrate that typifies most malignancies. Among them, Burkitt’s
lymphoma (BL), a high-grade non Hodgkin lymphoma (NHL) of B-cell origin,
represents a characteristic example. Studies from our group have shown that TAM
in BL exert pivotal roles that are mainly supportive of tumourigenesis such as
maintaining an immunosuppressive microenvironment. In order to unravel the
molecular mechanisms underlying TAM functions in BL, solid tumours from a mouse
xenograft model of BL have been used to obtain TAM and assess their activation
status in vivo. Laser-capture microdissection has been successfully used to procure
intact macrophage sections from the tumour site, allowing the production of a pure,
in situ gene expression signature of TAM in BL. Tingible-body Mφ from lymph node
germinal-centres and resident tissue Mφ from resting lymph nodes of non-tumour
bearing mice were chosen for direct comparison with TAM. Whole-genome
microarray technology has revealed a distinct TAM gene expression profile, with
454 genes being significantly up-regulated (fc ≥ 2, p<0.05) and 1293 genes being
significantly down-regulated (fc ≤ -2, p<0.05) between TAM and either of the two
normal Mφ populations. Further bioinformatics analysis of gene functions has
highlighted matrix remodeling, phagocytosis, and immune response among the
processes most highly enriched in TAM. Importantly, mRNA and tissue expression of
selected differentially expressed genes relevant to these processes was validated by
real-time qPCR and immunofluorescence labeling respectively. Following the
generation of the TAM profile in situ, in vitro experimental approaches were
undertaken in order to investigate how specific elements of the BL
microenvironment drive the observed TAM signatures. Specifically, the direct role
of apoptotic tumour cells, a key component of the BL microenvironment, versus
that of viable tumour cells in driving TAM matrix remodelling gene expression was
assessed in short-term mouse and human Mφ-NHL cell co-cultures. From the
aforementioned cluster, emphasis was given to MMP12 and MMP2 transcripts:
mRNA and protein expression of these MMPs was found to be up-regulated in Mφ
following viable tumour cell co-cultures and this effect was further enhanced
following apoptotic tumour cell co-cultures, implying that apoptotic NHL cells could
directly shape TAM matrix remodeling phenotype in BL in vivo. Whereas the mRNA
of both MMPs was solely Mφ-derived in this system, MMP12 and MMP2 protein
was surprisingly found also to be increased in NHL cells in the apparent absence of
increased mRNA. Detailed examination of MMP12 production by NHL cells revealed
that it is most likely an apoptosis-dependent process, since apoptotic NHL cells
generated through different apoptosis stimuli, as well as apoptotic cell-derived
microparticles, showed markedly increased MMP12 protein levels. In conclusion,
the data presented in this thesis, provide the first insight into the in vivo activation
status of TAM in high-grade NHL, through generation of the TAM gene signature in
situ. Upon further in vitro studies, apoptotic NHL cells were shown to directly
modulate the matrix remodelling component of the TAM signature as well as to
actively produce matrix remodelling mediators themselves, suggesting distinct roles
for tumour cell apoptosis within the NHL microenvironment that can profoundly
influence the disease outcome