The role of substance P in the progression and complications of secondary brain tumours.

Abstract

Secondary brain tumours occur when cancer cells enter the circulation from their primary site and colonise the brain, previously shown to occur across the blood-brain barrier (BBB). Substance P (SP), a neurogenic inflammatory mediator, acting predominantly through NK1 receptors plays a role in opening the BBB and in the formation of oedema following stroke and brain trauma. It is hypothesised that SP may also promote the extravasation of tumour cells through the BBB, formation of peritumoral oedema and progression of secondary brain tumours. Walker 256 rat breast carcinoma cells obtained from the Centre for Medical Research, Tohoku University had superior tumorigenic properties compared to cells from the American Type Culture Collection, and were therefore subsequently used in two albino Wistar rat models of tumorigenesis. Firstly, internal carotid artery tumour cell injection was used to establish the effect of tumour cell extravasation across the BBB on brain albumin, endothelial barrier antigen (EBA) and SP immunoreactivity. I then determined if NK1 receptor antagonists could prevent tumour cell extravasation, by evaluating tumour incidence and volume. Secondly, a stereotaxic direct inoculation model was used to investigate the effect of NK1 receptor antagonists on brain tumour growth and peritumoral oedema, compared with dexamethasone treatment. Evan’s blue extravasation and albumin immunoreactivity were used to assess BBB permeability, and brain water content to evaluate cerebral oedema. Tumour volume, Ki67 immunoreactivity, caspase-3 immunoreactivity and tumour cell density were used as measures of tumour growth. Furthermore, cell viability and cell death assays determined if NK1 antagonists or dexamethasone treatment cause alterations in tumour cell growth in vitro. In the carotid model, SP and albumin immunoreactivity increased in the brain during the extravasation of tumour cells, and in the peritumoral area of established tumours. The invaded blood vessels lacked EBA immunoreactivity, indicating loss of BBB properties. However, NK1 antagonists administered in the first three days following tumour cell injection failed to reduce tumour incidence or volume, suggesting that extravasation may be a multifactorial process, and that NK1 receptor antagonism alone is not sufficient to prevent tumour extravasation and growth. In the direct inoculation model, NK1 receptor antagonists did not reduce peritumoral oedema or decrease tumour growth when used to treat established brain metastases. In contrast, dexamethasone, the standard treatment for peritumoral oedema, caused a reduction in brain water content and decreased tumour volume, but not tumour growth. The decrease in tumour volume with dexamethasone reflects reduced fluid content, as there was increased tumour cell density with no change in immunoreactivity to Ki67 (marker for proliferation) or caspase-3 (marker for apoptosis). Furthermore, in vitro studies showed no effect for dexamethasone on tumour cell viability. These results suggest that peritumoral oedema is driven by classical inflammation rather than neurogenic inflammation in the direct inoculation model. In conclusion, in these models of secondary brain tumours, SP does not appear to play a role sufficient to promote NK1 receptor antagonism as an appropriate preventative treatment for brain metastasis, as an anticancer agent, or as an alternative to dexamethasone for the management of peritumoral oedema.