A26
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
1Tumour Targeting Group, University of Sheffield Medical School, Sheffield, South Yorkshire, UK, 2Dept Oral and Maxillofacial Surgery, School of Clinical Dentistry, Sheffield, South Yorkshire, UK
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 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 the pro-tumour responses of macrophages to ND in vitro to be 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.
