Detection of pancreatic ductal adenocarcinoma with galectin-9 serum levels

Abstract

Pancreatic ductal adenocarcinoma (PDAC) responds poorly to checkpoint blockade, such as anti-CTLA-4 and anti-PD-1. Galectin-9, a β-galactoside-binding lectin, promotes immune suppression through T-cell inhibition, and programming of tolerogenic macrophages. Of all cancers tested, PDAC showed the highest expression of LGALS9 (galectin-9) mRNA. We analyzed formalin-fixed and paraffin-embedded specimens from 83 patients with PDAC stained for galectin-9. Using flow cytometry, we determined galectin-9 expression on immune cells from tumor and matched blood samples from 12 patients with resectable PDAC. Furthermore, we analyzed galectin-9 serum levels by enzyme-linked immunosorbent assay using serum samples from 70 patients with PDAC, from 36 individuals with benign pancreatic disease, and from 28 healthy controls. Galectin-9 was highly expressed in human PDAC compared with normal pancreas and present on both tumor and immune cells. Tumor-infiltrating immune cells, especially CD3⁺ T cells, showed upregulation of galectin-9 compared with immune cells from matched blood. Blood γδ T cells from PDAC patients had higher galectin-9 expression than γδ T cells from healthy individuals. Galectin-9 polarized macrophages toward a protumoral M2 phenotype leading to suppressed T-cell cytokine secretion. Furthermore, serum concentration of galectin-9 was able to discriminate PDAC from benign pancreatic disease and healthy individuals, and was prognostic for stage IV patients. Galectin-9 is a new biomarker for the detection of PDAC.

Fig. 1. Human PDAC has high LGALS9 mRNA levels.

a Box plots of LGALS9 (galectin-9) mRNA expression measured in various human solid tumors (sample size in parentheses) assessed by RNA-seq. Tumors are sorted in order of decreasing median expression of LGALS9 mRNA. Of the pancreatic cancer samples from the TCGA database (n = 179), we analyzed only PDAC (n = 146). All expression values are log2-transformed. b Expression of CD274 (PD-L1) and LGALS9 mRNA was tested in human PDAC tissues using the TCGA database. c Correlation between high and low tertiles of LGALS9 mRNA expression and CD4, CD8A, and TRGC2 expression, d CD163, CD206, and TNF (TNFA) and e FUT4 (CD15) expression. Each point represents data from one patient. Data, median, unpaired t-test. *p < 0.05, ****p < 0.0001.

Fig. 2. Galectin-9 is expressed on tumor cells in human PDAC.

a Paraffin-embedded human PDAC specimens were tested for galectin-9 and CK19 co-expression by immunofluorescence microscopy. Representative images are shown. Nuclei counterstained with 4′,6-diamidino-2-phenylindole (DAPI). b Representative images from paraffin-embedded sections of human PDAC (n = 83) and normal pancreas tissue (n = 5) were tested for expression of galectin-9. c Quantification of galectin-9⁺ cells per high-power field (HPF). d Number of galectin-9⁺ cells per HPF among pathological T (left), N (middle), and UICC stages (right). Each dot represents a separate specimen. e Representative images from untreated human PDAC (left) and after neoadjuvant chemotherapy (right). Each point represents data from one patient. f Quantification of galectin-9⁺ cells per HPF. Bar graph, mean ± SEM, unpaired t-test. Dot plots, median, one-way ANOVA. **p < 0.01.

Fig. 3. PDAC-infiltrating immune cells express higher levels of galectin-9 compared with matched blood immune cells.

a Percentages of galectin-9 expression of indicated immune cells in matched blood and tumor specimens from PDAC patients. b Histogram and quantification of galectin-9 expression on all CD3⁺ T cells, c CD4⁺ T cells (CD3⁺CD4⁺CD8⁻, top), CD8⁺ T cells (CD3⁺CD4⁻CD8⁺, middle), γδ T cells (CD3⁺CD4⁻CD8⁻γδTCR⁺, bottom), d Monocytes/tumor-associated macrophages (TAMs, Lin⁻CD11b⁺CD14⁺, top) and monocytic myeloid-derived suppressor cells (M-MDSCs, Lin⁻CD11b⁺HLA-DR⁻CD14⁺, bottom). Each point represents data from one patient. Data, paired t-test. *p < 0.05, **p < 0.01.

Fig. 4. PDAC patients have increased galectin-9 expression on blood γδ T-cell subsets.

a Percentages of galectin-9 expression of indicated T-cell subset in blood from healthy individuals and patients with PDAC. b Percentages of galectin-9 expression of indicated T-cell subset in blood from patients with PDAC. c Percentages of galectin-9 expression on CD4⁺ T cells (left), CD8⁺ T cells (middle), γδ T cells (right) in blood from patients with PDAC among T, d N, and e UICC stages. Each point represents data from one patient. Data, median. one-way ANOVA or unpaired t-test. *p < 0.05, **p < 0.01, ****p < 0.0001.

Fig. 5. Galectin-9 polarizes macrophages toward a M2 phenotype.

a Macrophages were cocultured (1:4 ratio) with AsPC-1 cells with recombinant human galectin-9 or control for 48 h and stained for HLA-DR (top), CD86 (bottom) and b CD206. c After coculture for 48 h, macrophages were separated from AsPC-1 cells incubated with galectin-9 or control and then added to CD3⁺ T cells (1:2 ratio) for 48 h. T cells were stained for CD4, CD8, TNF-α (top) and IFN-γ (bottom). Bar graph, mean ± SEM, unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6. Galectin-9 serum levels discriminate PDAC patients from benign pancreatic disease and healthy individuals.

a Dot plots of galectin-9 concentrations in serum samples from health individuals (Healthy, n = 28), patients with chronic pancreatitis (CP, n = 18), intraductal papillary mucinous neoplasm (IPMN, n = 18), and PDAC (n = 70). b Dot plots of galectin-9 concentrations in serum samples from PDAC patients at indicated UICC stages. c Dot plots of galectin-9 concentrations in serum samples from stage IV PDAC patients displaying short- (<12 months) or long-term (>12 months) survival. d ROC curves for galectin-9 concentrations in serum samples from patients with CP (left), IPMN (middle), and PDAC (right) versus healthy controls (Healthy). e ROC curves for galectin-9, CA19-9, and CEA concentrations in serum samples from PDAC versus CP (left) and IPMN (right). Each point represents data from one patient. Data, median. one-way ANOVA or unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001.