POSTER 48

							                                     POSTER 48


Exposure to Necrotic Debris in Tumours Induces an Aggressive, Tumour-Promoting
Phenotype in Macrophages: Role of Toll-Like Receptor 4.

Russell Hughes1, Esha Kothari1, Craig Murdoch2 & Claire Lewis1
1
Academic Unit of Pathology, University of Sheffield Medical School. 2Dept. Oral and
Maxillofacial Surgery, University of Sheffield School of Clinical Dentistry, Sheffield.

The presence of areas of hypoxia (extremely low oxygen tension) and the consequent death of
oxygen-starved tumour cells by necrosis is a hallmark feature of murine and human tumours. In
previous studies we showed that inflammatory cells called macrophages accumulate in and
around such hypoxic/necrotic areas in tumours. Here we show that exposure not only to hypoxia
but also to necrotic debris (ND) in these areas has a profound effect on the phenotype of
macrophages. We generated ND by the repeated freeze-thawing of various human tumour cell
lines and show that primary human macrophages respond to ND with the expression of high
levels of various pro-angiogenic and growth-promoting enzymes/cytokines in vitro (VEGF, EGF,
IL-6, COX-2 and IL-1β – assessed by real time RT-PCR, arrays and ELISAs). As TLRs 2 and 4
have been implicated in the way that macrophages ‘sense’ such dead or dying cells, we
transiently transfected primary human monocytes/macrophages with siRNA to knockdown these
two TLRs and then exposed the cells to ND. In parallel experiments we also used neutralising
antibodies to functionally block these TLRs. Both approaches revealed that the pro-tumour
responses of macrophages to ND in vitro is mediated, in part, by TLR4 on the surface of
macrophages. We also show that TLR4 activation by ND initiates a signalling pathway involving
NFkB, p38MAPK and JNK. We are currently attempting to identify the factor(s) present in ND
that bind to TLR4 on macrophages.