Fig. 6
Increased macrophage recruitment and high local concentration of TNFα drives resistance and predicts failure of response to AATx a NOD/SCID mice with reconstituted RFP-Bone Marrow cells, used to generate intracranial gliomas with GFP-U87 cells (n = 20). Ten reconstituted mice were injected with GFP-U87 to generate intracranial gliomas. Ten mice from both tumor bearing and non-tumor group were treated with B20.4.1.1 treatments and vehicle controls one-week post intracranial injection (n = 5 for each). Tumors were harvested after two weeks of treatment, small portion from each tumor was saved for protein extraction and morphological studies, the rest of tumors were processed into single cell suspension and pooled prior to FACS. BM-derived macrophages (RFP+/F4/80+ cells) were sorted and pooled for RNA and protein extraction. b TNFα ELISA was performed on both untreated and B20.4.1.1 treated U87 xenograft tumor tissues (n = 5, left panel). Percentage of BM-derived macrophages (GAMs, RFP+/F4/80+ cells) in tumor cells (GFP cells) were analysed by flow cytometry (middle panel, pooled sample). TNFα gene expression in GAMs was determined by quantitative PCR (right panel, pooled sample). c Heatmap of fourteen genes associated with TNFα signaling pathway in untreated or B20.4.1.1 treated GAMs. d Double immunofluorescence staining was performed with CD31 and VCAM1 on the tumor sections. Percentage of bone marrow derived cells (BMDC, red fluorescence) and activated microvascular density (aMVD, CD31 and VCAM1 double positive cells) were determined by digital analysis in normal brain (NB) and tumors, with and without treatment of B20.4.1.1. e Thirteen human GBM samples collected prior to Bevacizumab treatment were stained by dual immunohistochemistry with anti-TNFα and anti-CD68. The percentage of double positive cells in CD68(+) cells per mm2 of tumor section was determined by digital analysis and two groups were divided based on median value. f Kaplan–Meier survival curve of high TNFα (n = 7) and low TNFα (n = 6) groups, p = 0.0212. g Graphic summary: GBM cells secrete IL-8 and CCL2 which stimulate GAMs to produce TNFα. Subsequently, TNFα induces a distinct gene expression signature of activated ECs including VCAM-1, ICAM-1, CXCL5, and CXCL10. High expression of VCAM1 correlates with worse survival outcome in IDH-wt glioma patients. Inhibition of TNFα with antibodies or drugs inhibits GAM-induced EC activation, improves survival and prolongs durability of response to AATx. *p < 0.05