Aberrant Glycosylation in Pancreatic Ductal Adenocarcinoma 3D Organoids Is Mediated by KRAS Mutations

Abstract

Aberrant glycosylation in tumor cells is a hallmark during carcinogenesis. KRAS gene mutations are the most well-known oncogenic abnormalities but their association with glycan alterations in pancreatic ductal adenocarcinoma (PDAC) is largely unknown. We employed patient-derived 3D organoids to culture pure live PDAC cells, excluding contamination by fibroblasts and immune cells, to gasp the comprehensive cancer cell surface glycan expression profile using lectin microarray and transcriptomic analyses. Surgical specimens from 24 PDAC patients were digested and embedded into a 3D culture system. Surface-bound glycans of 3D organoids were analyzed by high-density, 96-lectin microarrays. KRAS mutation status and expression of various glycosyltransferases were analyzed by RNA-seq. We successfully established 16 3D organoids: 14 PDAC, 1 intraductal papillary mucinous neoplasm (IPMN), and 1 normal pancreatic duct. KRAS was mutated in 13 (7 G12V, 5 G12D, 1 Q61L) and wild in 3 organoids (1 normal duct, 1 IPMN, 1 PDAC). Lectin reactivity of AAL (Aleuria aurantia) and AOL (Aspergillus oryzae) with binding activity to α1-3 fucose was higher in organoids with KRAS mutants than those with KRAS wild-type. FUT6 (α1-3fucosyltransferase 6) and FUT3 (α1-3/4 fucosyltransferase 3) expression was also higher in KRAS mutants than wild-type. Meanwhile, mannose-binding lectin (rRSL [Ralstonia solanacearum] and rBC2LA [Burkholderia cenocepacia]) signals were higher while those of galactose-binding lectins (rGal3C and rCGL2) were lower in the KRAS mutants. We demonstrated here that PDAC 3D-cultured organoids with KRAS mutations were dominantly covered in increased fucosylated glycans, pointing towards novel treatment targets and/or tumor markers.

Figure 1

(a) Experimental scheme. Diagram of steps for the glycosyltransferase and glycan analysis of patient-derived organoids. (b) Flow diagram of sample collection. Samples were collected from 24 patients. Collected and cultured tissues were IPMN (N = 4), PDAC (N = 24), and normal pancreatic ducts N = 4. Organoids successfully established were IPMN (N = 1), PDAC (N = 19), and normal pancreatic duct (N = 1). The cases that could be further analyzed were IPMN (N = 1), PDAC (N = 14), and normal pancreatic duct (N = 1).

Figure 2

KRAS mutation profile of the organoids. (a) Red areas in the left chart indicate KRAS -positive while blue indicates KRAS wild -type. (b) Brightfield images, H&E staining and CK19 immunostaining of 2 organoids cultures are shown (left: KRAS wild -type organoid, right: KRAS mutant organoid). Scale bar: 50 μm.

Figure 3

Comparison of glycan expression based on lectin microarray results between KRAS mutant and KRAS wild-type organoids. (a) List with t values of lectins significantly changed between KRAS mutation positive organoids and KRAS wild organoids. Labels and bars at the top of the figure indicate the type of glycan structure to which each lectin binds. Fucose (Fuc); red bar, Mannose (Man); green bar, Galactose (Gal); orange bar, sialic acid; purple bar, O-glycan; gray bar. Statistically significant differences are calculated by unpaired student's t test and the lectins with p < 0.05 are selected. Lectins are categorized based on their binding specificities. (b) The volcano plot of differential intensity of lectins in KRAS mutation organoids and KRAS wild organoids represents fold change and p values of the selected lectins on x- and y-axis, respectively. (Lectins listed in Figure 3(a)) (c) Lists of representative lectins that are listed by type in order of p value.

Figure 4

Comparison of glycosylation-related genes expression based on the result of RNA-seq between KRAS mutant and KRAS wild-type organoids. (a) The principal component analysis of gene expression data (glycosyltransferase n = 212) for the 16 organoids. Red and blue dots and circles are indicated to KRAS mutation positive organoids and KRAS wild organoids, respectively. (b) The volcano plot of mRNA differential expressions in KRAS mutation organoids and KRAS wild organoids represents fold change and p values on x- and y-axis, respectively. The vertical red and blue lines represent a fold-change cutoff of ≥2.0. (c) List with t values of fucosyltransferase between KRAS mutation positive organoids and KRAS wild organoids. Statistically significant differences are calculated by unpaired student's t test and p < 0.05 are selected. (d) The FUT6 mRNA expression from RNA seq, and the list of details of results. ^∗p < 0.001.

Figure 5

Analysis of glycosyltransferase and glycan expression in organoids established from the same patient. Case1 (a–c), Case2 (d–f). (a, d) Schema indicating the pancreatic lesion of Case1 used for the organoid and mutation site of each organoid. (b, e) Statistically significant differences were calculated by unpaired student's t test and p < 0.01 were selected. Lectins were categorized based on their binding specificities. Data are shown with t values. (c, f) Volcano plot with fold change and p values on x- and y-axes, respectively. The vertical line represents a fold-change cutoff of ≥2.0.