Mast Cell Accumulation in Glioblastoma with a Potential Role for Stem Cell Factor and Chemokine CXCL12

Glioblastoma multiforme (GBM) is the most common and malignant form of glioma with high mortality and no cure. Many human cancers maintain a complex inflammatory program triggering rapid recruitment of inflammatory cells, including mast cells (MCs), to the tumor site. However, the potential contribution of MCs in glioma has not been addressed previously. Here we report for the first time that MCs infiltrate KRas+Akt-induced gliomas, using the RCAS/TV-a system, where KRas and Akt are transduced by RCAS into the brains of neonatal Gtv-a- or Ntv-a transgenic mice lacking Ink4a or Arf. The most abundant MC infiltration was observed in high-grade gliomas of Arf−/− mice. MC accumulation could be localized to the vicinity of glioma-associated vessels but also within the tumor mass. Importantly, proliferating MCs were detected, suggesting that the MC accumulation was caused by local expansion of the MC population. In line with these findings, strong expression of stem cell factor (SCF), i.e. the main MC growth factor, was detected, in particular around tumor blood vessels. Further, glioma cells expressed the MC chemotaxin CXCL12 and MCs expressed the corresponding receptor, i.e. CXCR4, suggesting that MCs could be attracted to the tumor through the CXCL12/CXCR4 axis. Supporting a role for MCs in glioma, strong MC infiltration was detected in human glioma, where GBMs contained significantly higher MC numbers than grade II tumors did. Moreover, human GBMs were positive for CXCL12 and the infiltrating MCs were positive for CXCR4. In conclusion, we provide the first evidence for a role for MCs in glioma

CXCL12 and CXCR4 expression in mouse RCAS-<i>KRas</i>+RCAS-<i>Akt</i> induced gliomas.

<p>Immunofluorescence staining for CXCL12 and CXCR4 was performed in both <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse gliomas. The quantification of intensity signal for CXCL12 (lower left panel) and CXCR4 (lower right panel) revealed statistically significant difference between tumor and nontumor areas of the objective fields in both <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse brain tumors (* p<0.05). Error bars show SD. Scale bar = 50 µM.</p

Summary of patient characteristics and treatment received.

<p>OII = oligodendroglioma grade II, AII = astrocytoma grade II, Primary GBM = primary glioblastoma, F = female, M = male.</p

Demonstration of MC migration toward glioma-conditioned medium.

<p>Co-expression of CXCL12 and CD31 in mouse RCAS-<i>KRas</i>+RCAS-<i>Akt</i> induced gliomas. (<b>A</b>) Trans-well assay using CXCL12-neutralizing antibodies revealed statistically significant decreased migration of BMMCs towards glioma-conditioned medium. (<b>B</b>) Trans-well assay using antibodies to block CXCR4 receptor expressed on the BMMC surface demonstrated statistically significant decrease in BMMC migration towards glioma-conditioned medium. Appropriate isotype controls were used (* p<0.05, ** p<0.01). (<b>C</b>) Immunofluorescence staining demonstrated co-localization of CXCL12 and CD31 in both <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse gliomas. Image analysis revealed an average of 16% and 14% of total CXCL12-positive cells in <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse gliomas respectively were co-localized with CD31-positive endothelial cells. However, almost 94% and 82% of total CD31-positive cells in <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse gliomas correspondingly were co-localized with CXCL12-positive staining. No statistical difference between <i>Ntv-a-</i> and <i>Gtv-a</i> lines was found. Scale bar = 50 µM. Error bars show SD.</p

MC distribution in the mouse RCAS-<i>KRas</i>+RCAS-<i>Akt</i> induced brain tumors.

<p>(<b>A</b>) Immunofluorescence staining for endothelial cell marker CD31 and MC tryptase mMCP-6 in <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse brain tumors revealed perivascular localization of MCs. Lower panel: quantification of perivascular MCs in mouse brain tumors revealed about 50% in the corresponding objective fields with no difference between <i>Ntv-a Arf−/−</i> (n = 5) and <i>Gtv-a Arf−/−</i> (n = 5). Error bars show SD. Scale bar = 50 µM. (<b>B</b>) Immunohistochemical analysis for SCF expression revealing marked expression of SCF in the glioma vascular structures in both <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mice (indicated by blue arrows). Expression of SCF was also observed in MC granules (indicated by red arrows). Lower panel: quantification of total absolute intensity signal for SCF revealed statistically significant difference between tumor and nontumor areas of the objective fields in both <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse brain tumors (* p<0.05). Error bars show SD. Scale bar = 50 µM.</p

MC infiltration of human gliomas.

<p>(<b>A</b>) Immunohistochemical analysis of human MC tryptase (hTRS) in human low-grade gliomas (grade II, n = 8) and glioblastomas multiforme (GBM) (grade IV, n = 10). Right panel: quantification of MCs. Error bars show SD, *** p<0.001. Scale bar = 50 µM. (<b>B</b>) Immunofluorescence staining for CXCL12 and CXCR4 in human GBMs. Scale bar = 50 µM. (<b>C</b>) Immunofluorescence staining for CXCR4 and hTPS in human GBMs displayed co-expression of CXCR4 and hTPS. Scale bar = 25 µM. The inset represents a MC with co-localization of CXCR4 and hTPS at the single-cell level where maximum intensity projection of z-stack confocal images was applied.</p

Accumulation of MCs in RCAS-<i>KRas</i>+RCAS-<i>Akt</i> induced tumors from <i>Ntv-a Ink4a−/−</i>, <i>Ntv-a Arf−/−</i>, <i>Gtv-a Ink4a−/−</i> and <i>Gtv-a Arf−/−</i> mice.

<p>(<b>A</b>) Chloroacetate esterase (CE) and H&E-stained <i>Ntv-a Ink4a−/−</i> and <i>Ntv-a Arf−/−</i> tumors. Arrows in left panels indicate MCs. Scale bar = 100 µM. (<b>B</b>) Chloroacetate esterase (CE)- and H&E-stained <i>Gtv-a Ink4a−/−</i> and <i>Gtv-a Arf−/−</i> tumors. Arrows indicate MCs. (<b>C</b>) Quantification of MCs in both non-tumor MHb (MHb, − tumor), cancerous MHb (MHb, + tumor) and in the tumor area (tumor) of mouse glioma samples. <i>Ntv-a Ink4a−/−</i> (MHb − tumor, n = 6; MHb + tumor, n = 12; tumor, n = 12), <i>Ntv-a Arf−/−</i> (MHb − tumor, n = 6; MHb + tumor, n = 12; tumor, n = 12), <i>Gtv-a Ink4a−/−</i> (MHb − tumor, n = 6; MHb + tumor, n = 2; tumor, n = 2), <i>Gtv-a Arf−/−</i> (MHb − tumor, n = 6; MHb + tumor, n = 12; tumor, n = 12) revealing statistically significant difference between all compared groups for <i>Arf</i> deficient mice (** p<0.01, *** p<0.001). Error bars show SD. (<b>D</b>) MC carboxypeptidase A (MC-CPA)- and mMCP-6 positive MCs from the <i>Ntv-a Arf−/−</i> tumor.</p

Co-expression of CXCR4 and mMCP-6 by MCs in mouse RCAS-<i>KRas</i>+RCAS-<i>Akt</i> induced gliomas.

<p>(<b>A</b>) Immunofluorescence staining for CXCR4 and mMCP-6 was performed in both <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse gliomas, demonstrating co-expression of CXCR4 and mMCP-6. Scale bar = 50 µM. (<b>B</b>) Immunofluorescence staining, demonstrated co-localization of CXCR4 and mMCP-6 at the single-cell level in both <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse gliomas. Maximum intensity projection of z-stack confocal images was applied. Scale bar = 20 µM. (<b>C</b>) The quantification of MCs in mouse brain tumors revealed about 90% to be CXCR4-positive in the corresponding objective fields with no difference between <i>Ntv-a Arf−/−</i> (n = 5) and <i>Gtv-a Arf−/−</i> (n = 5). Error bars show SD.</p