Fig. 6: The presence of stromal cell types sensitizes DIPG cells to antibody-based immunotherapy. | Oncogene

Fig. 6: The presence of stromal cell types sensitizes DIPG cells to antibody-based immunotherapy.

From: Multicellular tumor-stromal interactions recapitulate aspects of therapeutic response and human oncogenic signaling in a 3D disease model for H3K27M-altered DIPG

Fig. 6

A Schematic demonstrating components of the innate immune system that can be exploited to target GD2-expressing tumors. Yellow highlighted complement system and microglia are investigated in the present 3D DIPG model. B Expression of the tumor specific disialoganglioside GD2 on the surface of patient-derived DIPG cell lines (green: SU-DIPG-4, SF8628, SU-DIPG-17, SU-DIPG-13, SU-DIPG-6), measured by flow cytometry with primary labeled GD2 APC (clone 14G2a) mAb. Stain Index (SI) for GD2 expression was normalized to isotype control (mouse IgG2a APC ab). Neuroblastoma cell lines (red: L-AN-6, CHLA-15 & 9464D) served as additional positive controls. Medulloblastoma (UW-228-2) and glioblastoma (D54) cell lines served as negative controls. C Representative images of DIPG-6 cells cultured alone as neurospheres (T-6) or in co-culture as TTA (TEM-6), shown after treatment with dinutuximab (14G2a) mAb in the presence of human plasma (10%) donated from healthy volunteers (n = 4) as well as heat-inactivated human plasma (HI-HP; control) at 0 and 9 h D Relative DIPG tumor cell viability (integrated GFP green fluorescence intensity per treated well normalized to average fluorescence intensity of untreated wells) at 3 h and 9 h, showing stromal influence on extent of antibody-based complement-dependent lysis of GD2 expressing DIPG cells plated in variable density [T-6, light green] and T-6(3), Dark green] and in combination with microglia and endothelial cells (TEM-6, Red-Green). Results were averaged across 4 well replicates per experiment. Statistical significance was determined by one-way ANOVA and Tukey post-hoc test. * p-values < 0.05 for dinutuximab-induced cytotoxicity in GFP-expressing DIPG cells were defined significant. was investigated in a coincubation of dinutuximab (14G2a) mAb (0.5 µg/ ml) and of human plasma (10%) donated from healthy volunteers (n = 4) in the tumor cell only (SU-DIPG-6) neurospheres and 3D TTA comprised of GFP-expressing DIPG cells with endothelial cells and tagRFP expressing microglial cells columns act as negative control. Heat-inactivated plasma (HI) from the same donors served as control in the experiment. E DIPG-6 tumor cell viability in 3D TTA comprised of microglia preincubated in 40% human serum or 10% FBS at 3 h post-treatment with dinutuximab (0.5 µg/ml).

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