Fig 2: Western blot and densitometry results for NRP1 (A,B), SDC2 (C,D), and GPC4 (E,F) from NE-targeted BMDM lysates. Three separate blood monocyte donations were obtained from healthy donors and cultured in RPMI with GM-CSF for 8–10 days to differentiate cells to blood monocyte derived macrophages. Cells from each donor were treated with control vehicle or NE (200 or 500 nM) for 1 or 2 h. Cell lysates were collected, protein quantified, and Western analyses were performed for NRP1 (A), SDC2 (C), and GPC4 (E). Left panels are representative Western blots for protein targets and as a control, β-actin. After densitometry of bands using ImageJ, relative expression corrected for β-actin was compared to control treated samples and summary data of relative expression for each protein shown in the panel on the right, NRP1 (B), SDC2 (D), and GPC4 (F). Significant differences in relative expression for N = 3 per protein was determined; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig 3: Human CAR and multifunctional CAR lymphocytes kill glioma cell lines and glioblastoma cells from patient samples with an intact microenvironment(A) Human LN-229 and ZH-161 glioma cells were co-cultured with human T cells or NK cells that were mock transfected or transfected with mRNA coding for CAR or CARΔ(CD3ζ) at different E:T ratios for 24 h. Glioma cell lysis was assessed using flow cytometry (mean ± SD of n = 3, one-way ANOVA with ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).(B) Same setup as in (A) but with human macrophages that were polarized using M-CSF or GM-CSF/IFNγ and co-cultured at an E:T ratio of 8:1. Total remaining glioma cells after co-culture are shown (mean + SD of n = 3, one-way ANOVA with ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).(C and D) Human immune cells transfected with ZsGreen mRNA and mRNA coding for CAR or cytokines or both were ex vivo co-cultured with surgically derived patient glioblastoma samples, and images of co-cultures were quantified using pharmacoscopy. (C) Remaining fractions of glioblastoma cells from n = 10 glioblastoma patient samples are represented as boxplot with median +/− quartiles and min to max. Significances were quantified by comparing co-cultures with mock-transfected immune cells and co-cultures with mRNA-transfected immune cells using paired nonparametric t test (∗p < 0.05; ∗∗p < 0.01). (D) Representative immunofluorescence images of co-cultures are shown with CD45+ immune cells in red, tumor cells in orange, and CAR immune cells in green. White arrows denote immune cell clusters. Scale bar, 40 μm.