Extended Data Fig. 7: (related to Sub-heading, CAN-3110 increases T cells in tumours). Quantitative IHC.
From: Clinical trial links oncolytic immunoactivation to survival in glioblastoma
IHC was performed by computer aided quantification of IHC stains (e.g., CD4, CD8, CD20) using slides scanned at 40X magnification using the Hamamatsu Nanozoomer S210. Using the Halo Image Analysis Sofware (PerkinElmer), 3 square regions of interest (approximately 160,000 mm2 each) are averaged for each case in in areas of tumour and in uninvolved/reactive brain tissue if present, and quantities are normalized by tissue area (mm2). (a-c) Pathological assessments of CD8+, CD4+, and CD20+ TILs are not systematically confounded by collection timepoint. Post-treatment IHC based counts of CD8+ (a), CD4+ (b), and CD20+ (c) TILs plotted versus the time of post-treatment tissue collection for the same IDHwt rGBM patients plotted in Fig. 2c (note that patient 045 was excluded due to early non-GBM mortality). Pearson’s correlation coefficient r and p values (2-sided, based on t-distribution) are provided above each plot calculated either using all patients or using only patients which were HSV1 seropositive before or after treatment. When counts were available for multiple post-treatment timepoints for a patient, the timepoint with the highest number of CD4+ or CD8+ or CD20+ cells were chosen. (d,e) Quantitative IHC for CD8+, CD4+ T cells and CD20+ B cells for each patient as a function of time. For each subject, the number of CD8+, CD4+ T and CD20+ B cells/mm2 are plotted as a function of time after CAN-3110 (i.e., when tumours underwent re-resection(s) and/or postmortem analyses after CAN-3110). Note that perinecrotic counts are not included here as they were only available for a few patients. n patients = 41. Kruskal-Wallis p = 0.33 (all patients), p = 0.16 (HSV1 seronegative patients), p = 0.45 (HSV1 seropositive/seroconverted patients) for CD8+. In (e), the same data is shown as in panel d but restricted to patients that have >1 post-treatment sample available (i.e., underwent more than 1 resection or had a resection and then also a postmortem analysis). n patients = 8. Kruskal-Wallis p = 0.39 (all patients), p = 0.30 (HSV1 seronegative patients), p = 0.44 (HSV1 seropositive/seroconverted patients) for CD8+. (f-i) Multiplex fluorescent imaging (mIF) for myeloid cell populations in pre- and post-CAN-3110 rGBM IDHwt. Each 20x region of interest (ROI) is plotted as a black dot. The overlaying bar graph is the mean of the ROIs and the error bars represent the standard deviation. For panels g and i, the values represent the percentage of the macrophage populations (CD68+ for panel g and CD68+ CD163+ for panel i) that are positive for PD-L1 expression. For panel h, the values are the cell density, or number of positive cells per mm2 of CD68+ CD163+ double positive cells. (f) Representative mIF images of post-treatment sample with quantified ROIs for comparison of solid tumour area and perinecrotic viral antigen positive tumour areas. Two subjects with pre-post-treatment pairs were examined (subjects 044 and 028). Scale bar = 50 μm. (g) Quantification of PD-L1 expression on total macrophage/microglial population in tumour near necrotic positive CAN-3110 region. (044: n = 3 ROIs (pre), 3 ROIs (Tumour), 6 ROIs (Necrotic); 028: n = 6 ROIs (pre), 3 ROIs (Tumour), 5 ROIs (Necrotic)) (h) post-treatment samples with CD163+ myeloid populations in both tumour and tumour-necrotic interface regions. Pre-treatment values are also shown. (044: n = 3 ROIs (pre), 3 ROIs (Tumour), 5 ROIs (Necrotic); 028: n = 6 ROIs (pre), 3 ROIs (Tumour), 5 ROIs (Necrotic)) (i) PD-L1 expression in CD163+ populations in perinecrotic interface regions. Pre-treatment values are also shown. DAPI blue nuclei enumeration, SOX2 white tumour nuclei enumeration, CD68 yellow pan-macrophage/microglia, CD163 orange macrophage/microglia. (044: n = 3 ROIs (pre), 3 ROIs (Tumour), 5 ROIs (Necrotic); 028: n = 6 ROIs (pre), 3 ROIs (Tumour), 5 ROIs (Necrotic)).
