Shared extracellular vesicle miRNA profiles of matched ductal pancreatic adenocarcinoma organoids and blood plasma samples show the power of organoid technology

Abstract

Extracellular vesicles (EV) are considered as a promising diagnostic tool for pancreatic ductal adenocarcinoma (PDAC), a disease with a poor 5-year survival that has not improved in the past years. PDAC patient-derived 3D organoids maintain the intratumoral cellular heterogeneity, characteristic for the tumor in vivo.Thus, they represent an ideal in vitro model system to study human cancers. Here we show that the miRNA cargo of EVs from PDAC organoids largely differs among patients. However, we detected a common set of EV miRNAs that were present in matched organoids and blood plasma samples of individual patients. Importantly, the levels of EV miR-21 and miR-195 were higher in PDAC blood EV preparations than in healthy controls, albeit we found no difference compared to chronic pancreatitis (CP) samples. In addition, here we report that the accumulation of collagen I, a characteristic change in the extracellular matrix (ECM) in both CP and PDAC, largely increases EV release from pancreatic ductal organoids. This provides a possible explanation why both CP and PDAC patient-derived plasma samples have an elevated amount of CD63 + EVs. Collectively, we show that PDAC patient-derived organoids represent a highly relevant model to analyze the cargo of tumor cell-derived EVs. Furthermore, we provide evidence that not only driver mutations, but also changes in the ECM may critically modify EV release from pancreatic ductal cells.

Keywords: Collagen; Exosome; Extracellular matrix; Organoid; PDAC; miR-195; miR-21.

Fig. 1

Experimental strategy to characterize EVs in PDAC. a–b Flow chart for determining PDAC-specific EV miRNAs (a) and to compare EV release (b)

Fig. 2

PDAC cell lines produce more EVs than control pancreas ductal cells both in 2D and 3D cultures. a The morphology of different cell lines in 2D and their colonies in 3D. Note that HPDEC is an immortalized pancreatic ductal control cell line. b CD63 + /CD81 + extracellular vesicle release of the indicated cell lines, measured by antibody-coated beads and flow cytometry (normalized to 10⁶ cells). The numbers after ‘D’ mean 2D or 3D cultures. c–d The distribution of particle size from 3D cultures of PDAC cell lines, determined by NTA. Note that cells, cell debris and large EVs were removed by serial centrifugation at 300 g and 2000 g. e Quantification of the particle concentration from 2 and 3D cultures (n = 4, data are normalized to 10⁶ cells). f miRNA levels in EV preparates isolated from Panc10.05 cells with serial centrifugation and ultracentrifugation (UC) or by a mixture of anti-CD81 and anti-CD63-coated beads (beads), measured by miRNA assays. Note that the empty, serum-free medium was used as control (Cells-). Scale bars: 50 µm (a, upper panels) or 10 µm (a, lower panels)

Fig. 3

Effect of 3D culturing on the miRNA cargo of PDAC cell-derived EVs is dependent on the matrix. a–b The morphology of the colonies in different matrices (left panels) and correlations between the miRNA levels of EV samples from 2D and 3D cultures in the indicated matrices for Panc08.13 (a) and Panc10.05 (b) cells (low-density miRNA cards). Ct values were normalized to the level of miR-19b. c The number and percentage of the overlapping and non-overlapping miRNAs of Panc08.13 (blue color) and Panc10.05 (yellow circles) cell-derived EVs cultured under the indicated conditions. Scale bars: 50 µm (a, b)

Fig. 4

Human PDAC patient-derived organoids release EVs. a The RNA levels of ductal, acinar and endocrine markers in patient-derived organoids (n = 5, RT-qPCR). b Percentage of CD63 + beads from the supernatants of PDAC organoids, detected by flow cytometry. Note that all cultures released CD63 + EVs. c Detecting particles from organoid cultures with TRPS after centrifugation with 300 g and 2000 g. Membrane with pore size of 400 nm was applied (measuring range 185–1100 nm) and the calibration beads were used in the same medium. d–e Representative image of an NTA measurement (d) and transmission electron microscope (TEM) (e). Scale bar: 500 nm (e)

Fig. 5

Screen of EV-miRNAs derived from five human PDAC organoid lines shows a common miRNA set. a Venn-diagram of the number of miRNAs detected in PDAC organoid lines. Note that a Ct value < 42 was regarded as positive hit. Supernatants were isolated from cultures with identical cell numbers. b–c Comparison of PDAC organoid-derived EV-miR profiles with plasma EVs of three individual patients. Data for the individual patients are shown in (b). Note that the majority of organoid-derived EV miRNAs are present in the plasma EVs as well (c). Numbers in parenthesis indicate the percentage of miRNAs detected in organoid and/or plasma EVs

Fig. 6

miRNAs present at an elevated level in PDAC blood-derived EVs are not specific for this disease. a The percentage of CD63 + beads after incubating them in the plasma samples from the indicated patient groups, detected by flow cytometry (n = 8–11). CP chronic pancreatitis, Ctr healthy control subjects. Note that identical volume of blood was applied in the experiments. b Comparison of the normalized levels of miRNAs present in all PDAC plasma EV samples (PDAC) to miRNA levels of CP and control subjects. Grey squares depict miRNAs that were absent in the samples. miRNAs that were present in the POCS set are marked with a red color. Note that since data were normalized to miR-19b, this miRNA is not shown on the graph. c Normalized miR-21 and miR-195 levels of the EV preparates. ANOVA and Tukey’s post hoc test (a) or Kruskal–Wallis and Dunn test (c) were used. **p < 0.01, ***p < 0.005

Fig. 7

Collagen I induces EV release from normal mouse pancreatic ductal organoids. a The morphology of organoids grown in Matrigel, Matrigel and collagen I (50–50%) or in collagen I 3 and 6 days after splitting. Scale bars: 100 µm. b The relative percentage of CD81 + beads after the indicated treatments (n = 4–5). Cultures were treated for 3 days before collecting EVs for 2 days. c CD81 + beads from organoids cultured in Matrigel with no collagen (0%) or 50% collagen I (n = 5). Positive beads were detected by either anti-CD81 or anti-CD63. d–e NTA representative images (d) and relative particle concentrations (e), measured from organoid supernatants at different collagen contents (n = 7). Data were normalized to cell number and then the Matrigel control was always taken as 100%. f The relative percentage of the KI67 + proliferating and active caspase-3 + apoptotic organoid cells in Matrigel without or with 50% collagen I (n = 3). g The relative percentage of CD81 + beads (detected by anti-CD81) from organoids cultured in Matrigel or 50% Matrigel/50% collagen (n = 5). Paired t test (b, c, f and g) or one-way ANOVA and Tukey post hoc test (e) were used. *p < 0.05