The Expression Pattern and Clinical Significance of the Immune Checkpoint Regulator VISTA in Human Breast Cancer

Abstract

Background: Immunotherapies targeting CTLA-4 and PD-1 have elicited promising responses in a variety of cancers. However, the relatively low response rates warrant the identification of additional immunosuppressive pathways. V domain immunoglobulin suppressor of T cell activation (VISTA) plays a critical role in antitumor immunity and is a valuable target in cancer immunotherapy.

Methods: Here, we used single-cell RNA-seq to analyze the gene expression levels of 14897 cells from a breast cancer sample and its paired 7,320 normal cells. Then, we validated the protein expression of immune checkpoint molecules (VISTA, PD-1, PD-L1, TIGIT, TIM3, and LAG3) in 324 human breast cancer samples by immunohistochemistry and quantitative immunofluorescence (QIF) approaches.

Results: Single cell RNA-seq results show a higher level of immune checkpoint VISTA expression in breast cancer tissue compared to adjacent normal tissue. We also found that VISTA expressed highest in breast cancer tissue than other immune-checkpoints. Immunohistochemical results showed that VISTA was detected with a membranous/cytoplasmic staining pattern in intratumoral immune cells and breast cancer cells. Additionally, VISTA was positively correlated with pathological grade, lymph node status and the levels of PD-1 according to the chi-square test or Fisher's test. Furthermore, VISTA expression was higher in CD68+ tumor-associated macrophages (TAMs) than in CD4+ T cells, CD8+ cytotoxic T cells or CD20+ B cells.

Conclusions: These findings therefore support the immunoregulatory role of VISTA in breast cancer and indicate that targeting VISTA may benefit breast cancer immunotherapy.

Keywords: V domain immunoglobulin suppressor of T cell activation; breast cancer; quantitative immunofluorescence; single cell RNA-seq; tumor immune microenvironment.

Figure 1

Single-Cell RNA-Seq Experimental Initial Data Exploration (A) Heatmap of log counts of genes in signature. (B) t-SNE of complete cells isolated from the breast cancer tissue and matched adjacent normal breast tissue. (C) t-SNE of CD45+ cells isolated from the breast cancer tissue and matched normal breast tissue (Left). Pie charts of cell-type fractions for the patient’s tumor-infiltrating immune cells, colored by cell type (Right). (D) The violin plot shows the top 10 most variable genes among different cells in the breast cancer sample.

Figure 2

Immune Checkpoints expressions heterogeneity in immune cell populations in the breast cancer tissue. (A) t-SNE of normalized single-cell RNA-seq data for VISTA colored by markers. VISTA positive cells (orange), VISTA negative (blue). (B) Histogram of the expression of each immune checkpoint (PD-L1, PD-1, CTLA-4, TIGIT, LAG3, TIM3 and VISTA) in the microenvironment of breast cancer tissue. (C) Histogram of percentage of each immune checkpoint (PD-L1, PD-1, CTLA-4, TIGIT, LAG3, TIM3 and VISTA) in the primary immune cell subpopulation in the breast cancer microenvironment. (D) t-SNE of normalized single-cell RNA-seq data for ICs (immune checkpoints) colored by markers. ICs positive cells (orange), ICs negative (blue).

Figure 3

VISTA expression pattern in human breast cancer tissue. (A) Representative immunohistochemical staining for the VISTA protein in normal breast tissue (n=4), breast adenosis tissue (n=2), paracancerous tissue (n=13) and breast cancer tissue (n=324). Original magnification, 400 ×. (B) The expression of VISTA in breast cancer detected by immunohistochemistry. VISTA negative (n=186, 57.41%) vs. VISTA positive (n=138, 42.59%). (C) Representative positive VISTA staining in immune cells (110/324, 33.95%), tumor cells (47/324, 14.51%), and both types of cells (19/324, 5.86%). Original magnification, 200 ×.

Figure 4

Multiplex immunofluorescence for VISTA and selected tumor-infiltrating immune cell markers in human breast cancer tissue. Markers of tumor-infiltrating immune cells: CD68 (macrophages), CD4 (T cells), CD8 (cytotoxic T cells) and CD20 (B cells). (A) Representative images of biomarkers in human breast cancer tissue. CD4 staining is shown in green; CD8 staining is shown in red; CD20 staining is shown in yellow; CD68 staining is shown in cyan; VISTA staining is shown in magenta; and DAPI staining is shown in blue. 400×. (B) Co-localization of VISTA with selected tumor-infiltrating immune cell markers in breast cancer detected by immunofluorescence. CD4, CD8, CD20, and CD68 staining is shown in green; VISTA staining is shown in red; and DAPI staining is shown in blue. Areas of co-localization are indicated with yellow arrows. 400×. (C). Summary plot of the proportion of each subpopulation of cells among double-positive cells. Each dot represents data from an individual patient. P-values were obtained by an unpaired T-test. ^* p < 0.05; ^## p < 0.01; ^### p < 0.001. (D) Summary plot of the proportion of double-positive cells in each subpopulation of cells. Each dot represents data from an individual patient. P-values were obtained by an unpaired T-test. ^# p < 0.05; ^*** p < 0.001. NS, no significance.

Figure 5

(A) Representative immunohistochemical staining for immune checkpoint molecules (PD-L1, PD-1, TIGIT, TIM3 and LAG3) in breast cancer samples. (B) Representative multiplex immunofluorescence staining for immune checkpoint molecules (VISTA, TIGIT, TIM3 and LAG3) in breast cancer samples. VISTA staining is shown in green; TIGIT staining is shown in yellow; TIM3 staining is shown in red; LAG3 staining is shown in magenta; PANCK staining is shown in cyan; and DAPI staining is shown in blue. (C) Co-localization of PANCK with VISTA, TIGIT, TIM3 and LAG3 in breast cancer detected by immunofluorescence. VISTA, TIGIT, TIM3, LAG3 staining is shown in red; PANCK staining is shown in green; and DAPI staining is shown in blue. (D) Kaplan-Meier curves showing overall survival (OS) of breast cancer patients based on immune-checkpoints (VISTA, TIGIT, TIM3 and LAG3) status. **p < 0.01, ***p < 0.001. NS, no significance.