Novel association between microglia and stem cells in human gliomas: A contributor to tumour proliferation?

Abstract

Brain tumour stem cells and microglia both promote the growth of astrocytomas, the commonest form of primary brain tumour, with recent emerging evidence that these cell types may interact in glioma models. It is unclear whether microglia and stem cells are associated in human gliomas. To investigate this question, we used the technique of tissue microarrays to perform a correlative study of a large number of tumour samples. We quantified immunostaining of human astrocytic tumour tissue microarrays (86 patients; World Health Organisation grade II-IV) for microglia Ionized calcium binding adaptor molecule 1 (Iba1) and CD68, and stem cell nestin, SOX2 and CD133. Ki67 was used to assess proliferation and GFAP for astrocytic differentiation. Immunoreactivity for both microglial markers and stem cell markers nestin and SOX2 significantly increased with increasing tumour grade. GFAP was higher in low grade astrocytomas. There was a positive correlation between: (i) both microglial markers and nestin and CD133, (ii) nestin and tumour cell proliferation Ki67 and (iii) both microglial markers and Ki67. SOX2 was not associated with microglia or tumour proliferation. To test the clinical relevance, we investigated the putative association of these markers with clinical outcomes. High expression for nestin and Iba1 correlated with significantly shorter survival times, and high expression for nestin, Iba1, CD68 and Ki67 was associated with faster tumour progression on univariate analysis. On multivariate analysis, nestin, CD133 and Ki67 remained significant predictors of poorer survival, after adjustment for other markers. These results confirm previous in vitro findings, demonstrating their functional relevance as a therapeutic target in humans. This is the first report of a novel correlation between microglia and stem cells that may drive human astrocytic tumour development.

Keywords: brain tumour; glioma; microglia; stem cells.

Figure 1

Quantification and illustration of the different markers in astrocytic tumours of grade II to IV. (A) GFAP is significantly decreased between tumours grade II and III (p = 0.034). (B) The proliferation marker Ki67 shows a significant increase in grade IV compared to grades II (p < 0.001) and III (p = 0.003). Microglial association with tumour growth is demonstrated by (C) the microglial marker Iba1 which is significantly increased between grades (II–III: p = 0.003; II–IV: p = 0.020) and (D) the increased microglial protein CD68 between grades II and IV (p < 0.001). For the stem cell markers, (E) nestin is significantly increased in tumour grade IV compared to grade II (p = 0.005) and grade III (p < 0.001), as also observed for (F) SOX2 (II–IV: p = 0.025; III–IV: p = 0.005). (G) CD133 shows a trend to increase in tumour between grades II to IV. Scale bar = 50μm.

Figure 2

Relationship between glial differentiation, proliferation, stem cells and microglia. (A) GFAP shows a positive correlation with cancer stem cell CD133 (p = 0.008). Ki67 is positively correlated with microglial markers (B) Iba1 (p = 0.006) and (C) CD68 (p = 0.002), and with (D) stem cell marker nestin (p < 0.001) indicating that both tumour stem cells and microglia correlate with proliferation. Statistical analysis shows (E) both microglial markers are associated (p < 0.001) as well as (F) the stem cell markers nestin and CD133 (p = 0.015); but also a positive correlation between microglia and stem cells with Iba1 correlated with (G) nestin (p < 0.001) and (H) CD133 (p = 0.006) and CD68 related to (I) nestin (p < 0.001) and (J) CD133 (p < 0.001).

Figure 3

Analysis with clinical information shows the probability of survival to be significantly associated with (A) the proliferation (high vs low Ki67; p = 0.033), (B) stem cell (high vs low nestin; p = 0.002) and (C) microglia (high vs low Iba1; p = 0.010). The probability of time to progression is also associated with the (D) proliferation (high vs low Ki67; p = 0.002), (E) stem cell (high vs low nestin; p = 0.007) and (F) microglia (high vs low Iba1; p = 0.005); but also with (G) cancer stem cell CD133 (high vs low p = 0.010) and phagocytic activity of microglia (high vs low CD68; p = 0.024).