MOESM2 of High expression of B7-H3 on stromal cells defines tumor and stromal compartments in epithelial ovarian cancer and is associated with limited immune activation
Creators: name: MacGregor, Heather
name: Sayad, Azin
name: Elia, Andrew
name: Wang, Ben
name: Katz, Sarah
name: Shaw, Patricia
name: Clarke, Blaise
name: Crome, Sarah
name: Celine Robert-Tissot
name: Bernardini, Marcus
name: Nguyen, Linh
name: Ohashi, Pamela
Publisher: name: figshare
Year: 2020
Subjects: subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer

description: Additional file 2 Fig. S1: B7-H3 immunohistochemistry of noncancerous human placenta, tonsil, spleen, and liver tissues. FFPE sections from various tissues were stained for B7-H3 expression by IHC. Fig. S2: B7-H3 and B7-H4 show different expression patterns on tumor and stromal cells in EOC. A: Flow cytometric staining of B7-H3 and B7-H4 on CD45− cells can define tumor and stromal populations. B, C: Levels of B7-H3 (B) and B7-H4 (C) expression on tumor (red) and stromal (blue) cells. D: Levels of HLA-ABC expression on tumor (red) and stromal (blue) cells in relation to B7-H3 (top) and B7-H4 (bottom). Matched FMO control shown in grey (B,C,D). Examples shown are from three patients’ tumors. Fig. S3: Top 50 genes whose mRNA expression most significantly positively correlates with CD276 mRNA expression across 22 TCGA datasets. Red text denotes genes with possible roles in the synthesis or modification of the ECM. Fig. S4: Gating schema for CD45− population positive for epithelial or stromal markers. B7-H3 gMFI was calculated for viable, CD45− singlets positive for epithelial (EpCAM, E-Cadherin) or stromal (FAP, PDGFRβ, PDPN, CD10) markers. Fig. S5: Comparative levels of B7-H3 expression between different tumor and stromal populations. A,B: Comparisons between levels of B7-H3 expression on FAPhigh (A) or PDGFRβ+ (B) stromal cell populations and EpCAM+ tumor cells. C: Comparisons between proportions of FAPhigh and PDGFRβ+ CD45− cells. D: Proportions of total FAPhigh with PDGFRβ+FAPhigh cells in EOC samples. Points from the same patient are connected by a line. Significance was determined by paired T test. Fig. S6: Example of methods used to quantify tumor and stromal content of tumors. A: H&amp;E stained slides categorized into tumor (red), stroma (green), and excluded (yellow) areas using HALO software. B: Flow plot of B7-H3 staining used to gate tumor (B7-H3low) and stromal (B7-H3high) cells. Fig. S7: Recurrence-free and overall survival in association with low or high tumor-to-stroma ratio (T:S). Recurrence-free (p = 0.098) and overall (p = 0.26) survival curves in patients with tumors with low (T:S 1.5; orange) T:S. Significance was determined by Mantel-Cox text. Fig. S8: Differences in immune cell phenotype is not due to NACT treatment. A,B,C: Comparison between frequency of PD-1high CD4+ and CD8+ T cells (A), CD16 expression on monocytes (B), and activating (ICOSL, B7-H3, CD40, CD86, CLEC9a) and inhibitory (B7-H4, B7-H3, PD-L1, PD-L2) expression on APCs (C) from patients who did (circle) or did not (open square) receive NACT treatment within 6 months prior to surgery. Significance was determined by Mann Whitney U test. Fig. S9: T:S is not associated with differences in expression of exhaustion or activation markers by infiltrating T cells. A: Gating schema for determining coexpression of high levels of PD-1 with TIM3 and/or LAG3 on CD4+ and CD8+ T cells. B: Proportion of PD-1high CD4+ (top) or CD8+ (bottom) T cells coexpressing TIM3, LAG3, or both. C,D: Expression of ICOS, TIA-1, GzmB, 2B4, CD107a, and GITR staining on CD4+ and CD8+ T cells directly ex vivo. Whisker and box plots summarize marker expression from tumors with low ( 1.5; orange) T:S. Statistical significance was determined by Mann Whitney U test (B,D). Fig. S10: Expression of activating, inhibitory, and maturation markers on B cells. A: Example stains of CD19+ B cells coexpressing CD20+CD22+, IgD+, BTLA+, or CD27+. B: Column plots showing marker expression on B cells from tumors with low (blue) or high (orange) T:S. Statistical significance was determined by Mann Whitney U test. Fig. S11: Immune cell frequency, but not phenotype, is associated with tumor location. A-D: Frequency of T cells (A), B cells (B), monocytes (C), and CD4+ and CD8+ T cells (D) isolated from ovary or omental tumors. E: Frequency of high levels of PD-1 expression on CD4+ or CD8+ T cells isolated from ovary or omental tumors. F: Level of CD16 expression on monocytes isolated from ovary or omental tumors. G: Level of expression of activating (ICOSL, B7-H3, CD40, CD86, CLEC9a) or inhibitory (B7-H3, B7-H4, PD-L1, PD-L2) markers on APCs isolated from ovary or omental tumors. Data from ovary tumors is shown in black, data from omental tumors is shown in purple. Statistical significance was determined by Mann Whitney U test.