Figure 1.
Expression of glial cell markers and NK cell ligands on U87MG tumour cells.
(A) Mean Fluorescence Intensity (MFI) histograms and % cells expressing glial markers (GFAP,Nestin, Vimentin, and A2B5), (B) MFI histograms and % cells expressing class I HLA ligands (HLA-A,-B,-C), non-classical HLA-G and HLA-E, as well as HLA-DR,DP,DQ. (C) MFI histograms and % cells expressing ULBP 1, 3, 2/5/6, MICA and MICB activating ligands. Dark histograms represent negative control, light histograms represent stained cells.
Table 1.
U87MG tumour cells' expression of glial markers and ligands for NK cell receptors.
Figure 2.
Longitudinal T1-weighted images and tumour growth.
(A) Longitudinal axial post-contrast T1-weighted images of nude rats bearing U87MG tumours treated with combination NK+mAb9.2.27, NK cell monotherapy, mAb9.2.27 monotherapy, and vehicle controls, showing the same animal after 7 days, 17 days and 3 months post NK+mAb9.2.27 treatment. (B) Tumour volumes (#voxels) quantified on post-contrast T1-weighted images, before and after 7 and 17 days treatment. Data represents mean ±SEM of all animals treated.
Figure 3.
Longitudinal T2-weighted images.
Longitudinal corresponding axial T2-weighted images of same animals as in Figure 2. Nude rats bearing U87MG tumours treated with combination NK+mAb9.2.27, NK cell monotherapy, mAb9.2.27 monotherapy, and vehicle controls, showing the same animal after 7 days, 17 days and 3 months post NK+mAb9.2.27 treatment.
Figure 4.
Immunohistochemical staining and cell proliferation.
(A, top panel) haematoxylin and eosin staining showing leucocyte packed necrosis in U87MG tumours treated with NK+mAb9.2.27 and mAb9.2.27 monotherapy, (arrows), Magnification 200X; Scale bar 100 µm. Cellular dense tumours treated with NK cell monotherapy and haemorrhaging control, untreated tumours. (A, middle panel) Ki67 staining of proliferating tumour cells (A, bottom panel). Magnification 200X; scale bar 100 µm. Tunel stained apoptotic cells, Magnification 200X; Scale bar 100 µm. (B) Quantified Ki67 labelling index, data represents mean ±SEM of all animals treated. (C) Quantified Tunel labelling index, data represents mean ±SEM of all animals treated,*p<0.05; **p<0.001, ***p<0.0001. D Ratio of proliferation: apoptosis index.
Figure 5.
Perfusion parameters and maps.
(A) Elevated extravascular extracellular volume fraction ve, in NK+mAb9.2.27 compared to monotherapy animals from cohort 1 (received 1 million NK cells), *p<0.05, **p<0.001 ***p<0.0001. (A left panels) Parametric maps visualising intratumoral heterogeneity in ve in representative control, monotherapy and NK+mAb9.2.27 treated animals at 7 days. Intensity scale shows minimum (blue voxels) and maximum (red voxels) intensity levels. (B) Increased interstitial extracellular volume fraction, ve, elevated in NK+mAb9.2.27 compared to NK cells and mAb9.2.27 monotherapy animals, and decreased ve in NK cell monotherapy compared to untreated control from cohort 2 (treated with 2 million NK cells), *p<0.05, **p<0.001, ***p<0.0001. (B, left panels) Parametric maps visualising intratumoural heterogeneity in ve in control, monotherapy and NK+mAb9.2.27 treated animals at 17 days. (C) Significant association between ADC and ve in the NK+mAb9.2.27 (R2 = 0.798, p = 0.041) and NK cell monotherapy animals (R2 = 0.993, p = 0.004). Graphs in A and B represent estimated marginal mean±95% confidence intervals.