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

Histidine-Rich Glycoprotein Can Prevent Development of Mouse Experimental Glioblastoma

Extensive angiogenesis, formation of new capillaries from pre-existing blood vessels, is an important feature of malignant glioma. Several antiangiogenic drugs targeting vascular endothelial growth factor (VEGF) or its receptors are currently in clinical trials as therapy for high-grade glioma and bevacizumab was recently approved by the FDA for treatment of recurrent glioblastoma. However, the modest efficacy of these drugs and emerging problems with anti-VEGF treatment resistance welcome the development of alternative antiangiogenic therapies. One potential candidate is histidine-rich glycoprotein (HRG), a plasma protein with antiangiogenic properties that can inhibit endothelial cell adhesion and migration. We have used the RCAS/TV-A mouse model for gliomas to investigate the effect of HRG on brain tumor development. Tumors were induced with platelet-derived growth factor-B (PDGF-B), in the presence or absence of HRG. We found that HRG had little effect on tumor incidence but could significantly inhibit the development of malignant glioma and completely prevent the occurrence of grade IV tumors (glioblastoma)

HRG had no effect on primary glial cell proliferation.

<p>(<b>A</b>) Primary glial cells were infected with RCAS-eGFP, RCAS-HRG or RCAS-PDGFB-HA and expression of the viral transduced proteins was analyzed with immunocytochemistry. The infection efficiency was similar in all conditions. (<b>B</b>) Proliferation assay on infected cells showed no effect of HRG compared to control cells on glial cell proliferation at day 7. Curves show the mean (±SEM) from three independent experiments for HRG and eGFP, and two independent experiments for PDGF-B.</p

Distribution of tumor malignancy grades.

<p>(<b>A</b>) Distribution of tumor grades (II–IV) in PDGF-B+X (P+X) and PDGF-B+HRG (P+H) injected <i>Ntv-a Arf-/-</i> mice. * p<0,05 (<b>B</b>) Distribution of low grade (II) versus malignant (III+IV) glioma. *p<0,05.</p

Presence and expression of viral transduced PDGF-B and HRG in tumors.

<p>(<b>A</b>) Insertion of the viral transduced human PDGF-B and HRG cDNA in genomic DNA prepared from PDGF-B+X (P+X) and PDGF-B+HRG (P+H) induced tumors. The tumor grade is given above each sample. Genomic DNA from U-706MG-a cells was used as positive control (+), and genomic DNA from an untreated mouse was used as negative control (−). (<b>B</b>) Expression of human PDGF-B and HRG mRNA in P+X and P+H tumors. RNA extracted from U-343MG and DF-1 RCAS-HRG cells were used as positive control for PDGF-B and HRG, respectively (+), and RNA from an untreated mouse brain was used as negative control (−).</p

RCAS-HRG infected DF-1 cells could produce functional HRG.

<p>(<b>A</b>) Viral produced HRG protein could be detected by western blot in conditioned media from DF-1 cells infected with RCAS-HRG but not from RCAS-PDGFB or RCAS-eGFP infected cells. Purified HRG was used as a positive control (+). (<b>B</b>) Viral produced HRG could significantly inhibit migration of HUVEC cells towards VEGF. t-test, ** p<0.01, *** p<0.001.</p

Tumor incidence in <i>Ntv-a Arf</i>-/- mice injected with PDGF-B+X (P+X) or PDGF-B+HRG (P+H) RCAS viruses.

<p>Tumor incidence in <i>Ntv-a Arf</i>-/- mice injected with PDGF-B+X (P+X) or PDGF-B+HRG (P+H) RCAS viruses.</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

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