Identification of CLEC10A as a human lectin for pancreatic ductal adenocarcinoma

Abstract

The mechanism by which glycans in pancreatic ductal adenocarcinoma (PDAC) interact with human endogenous lectins in the tumor microenvironment remains largely unknown. This study aimed to identify endogenous lectins that recognize and bind to pancreatic ductal adenocarcinomas. The reactivity of 43 human endogenous lectins belonging to the Galectin, Siglec, and C-type lectin families in PDAC cell lines and clinical PDAC samples was analyzed using flow cytometry and immunostaining of tissues. C-type lectin domain family 10 member A (CLEC10A), a C-type lectin, was a candidate endogenous lectin with high reactivity in some pancreatic cancer cells. CLEC10A lectin bound in approximately 60% of 113 clinical pancreatic cancer tissue sections. Immunohistochemistry with anti-CLEC10A antibody showed that CLEC10A was mainly expressed in CD163-positive monocytic cells. Of the 57 patients (out of 113) who achieved R0 surgical resection at stage II/III, those with high CLEC10A expression on macrophages and CLEC10A ligand expression on PDAC cells had significantly shorter overall survival. CLEC10A is a human lectin receptor for pancreatic ductal adenocarcinoma. The coexistence of CLEC10A-expressing cells in pancreatic cancer tissues and CLEC10A ligands on pancreatic cancer cells indicates poor prognosis.

Keywords: CLEC10A protein; Endogenous lectin; Pancreatic cancer; Tumor microenvironment.

Fig. 1

Three lectins, Galectin-4 C, Galectin-8 C, and CLEC10A-Fc, were extracted as candidate human endogenous lectins that recognize pancreatic cancer cells. (a) Among the 43 lectins, pancreatic cancer-reactive lectins were screened using flow cytometry. The difference (ΔMFI) between the median fluorescence intensity (MFI) of each lectin and the MFI of the control is shown in the heat map. Red arrows indicate lectins that met the criteria of MFI greater than 10⁴ for the pancreatic cancer cell lines, Capan-1 or AsPC-1, and less than 10⁴ for the pancreatic ductal cell line, hTERT-HPNE. (b) Histograms of Galectin-4 C, Galectin-8 C, and CLEC10A-Fc. The gray-filled histogram indicates control cells stained with PE-labeled BSA (for galectins) or PE-labeled anti-Fc antibody (for C-type lectins) used as negative controls, and the cyan line is the histogram of cells incubated with PE-labeled galectins or C-type lectins followed by PE-labeled anti-Fc antibody.

Fig. 2

CLEC10A is a human endogenous lectin with high affinity for pancreatic cancer cells. (a) Representative images of pancreatic cancerous and noncancerous tissues stained with three lectins (Galectin-4 C, Galectin-8 C, and CLEC10A-Fc). Scale bars, 10 μm. The arrowhead (▼) indicates the positive staining of the cell membrane. (b) Percentage of cases positive for lectin staining in pancreatic cancer cells (C) and pancreatic duct cells (N) in noncancerous tissue compared using Fisher’s exact test (NS: not significant, *P < 0.05).

Fig. 3

CLEC10A ligand is expressed in 60% of clinical pancreatic cancer cells. (a) Representative images of histochemically processed sections of clinical pancreatic cancer tissue analyzed for CLEC10A-Fc staining. Scale bars (Large window) 200 μm, (Small window) 10 μm. Staining intensity was scored according to the percentage of positive cells regardless of staining intensity as follows: 0, 0%; 1+, < 1%; 2+, 1–24; 3+, 25–49%; 4+, 50–74%; 5+, 75–100%. (b) A pie chart showing the percentage of each staining score in 113 processed sections of clinical pancreatic cancer tissue stained with CLEC10A-Fc.

Fig. 4

CLEC10A-expressing monocytic cells in pancreatic cancer stroma. (a) Left: Hematoxylin and eosin staining of representative pancreatic cancer tissue. Scale bars, 50 μm. Right: Immunostaining with anti-CLEC10A antibody in representative pancreatic cancer tissue. Scale bars, 50 μm. Cancer duct structures are surrounded by dotted lines (---); CLEC10A-expressing cells are indicated by arrowheads (▼). (b) Immunofluorescence staining of CLEC10A (red), CD163 (green), and nuclei (blue) in pancreatic cancer tissue; merge: yellow. Scale bars, 50 μm.

Fig. 5

Appearance of CLEC10A-expressing monocytic cells and coexistence of CLEC10A ligand on pancreatic cancer cells are poor prognostic factors in stage II and III cases. (a) Representative images of formalin-fixed paraffin-embedded (FFPE) sections of clinical pancreatic cancer tissues stained with anti-CLEC10A antibody. Scale bars, 100 μm. Staining intensity was scored according to the number of positive cells in the 20× field of view as follows: 0 (0 fields); 1+ (1–2 fields); 2+ (> 3 fields). (b) Classification based on the combination of CLEC10A and CLEC10A ligand expression intensity. (c) Kaplan–Meier curve comparing category Low (n = 23) with category High (n = 5). Log-rank test (*P < 0.05) was used.