Analysis of Donor Pancreata Defines the Transcriptomic Signature and Microenvironment of Early Neoplastic Lesions

Abstract

The adult healthy human pancreas has been poorly studied given the lack of indication to obtain tissue from the pancreas in the absence of disease and rapid postmortem degradation. We obtained pancreata from brain dead donors, thus avoiding any warm ischemia time. The 30 donors were diverse in age and race and had no known pancreas disease. Histopathologic analysis of the samples revealed pancreatic intraepithelial neoplasia (PanIN) lesions in most individuals irrespective of age. Using a combination of multiplex IHC, single-cell RNA sequencing, and spatial transcriptomics, we provide the first-ever characterization of the unique microenvironment of the adult human pancreas and of sporadic PanIN lesions. We compared healthy pancreata to pancreatic cancer and peritumoral tissue and observed distinct transcriptomic signatures in fibroblasts and, to a lesser extent, macrophages. PanIN epithelial cells from healthy pancreata were remarkably transcriptionally similar to cancer cells, suggesting that neoplastic pathways are initiated early in tumorigenesis.

Significance: Precursor lesions to pancreatic cancer are poorly characterized. We analyzed donor pancreata and discovered that precursor lesions are detected at a much higher rate than the incidence of pancreatic cancer, setting the stage for efforts to elucidate the microenvironmental and cell-intrinsic factors that restrain or, conversely, promote malignant progression. See related commentary by Hoffman and Dougan, p. 1288. This article is highlighted in the In This Issue feature, p. 1275.

Figure 1.

The adult human pancreas harbors frequent PanIN lesions. A, Schematic of workflow to recover and process Gift of Life donor pancreas organs. B, Right, population distribution bar plot of the donor cohort. Tan bars represent donors that were found to have PanIN lesions on histologic examination. Blue bars represent donors in whom no neoplastic lesions were found. Left, pie chart of donor cohort by sex. Middle, pie chart of donor cohort by race. C, Hematoxylin and eosin (H&E) sections of representative PanIN lesions found in donor pancreata. Each H&E section represents a different donor.

Figure 2.

PanIN lesions in healthy pancreata are surrounded by a unique microenvironment. A, mfIHC composite images of FFPE donor tissue specimens, highlighting acinar, normal duct, ADM, and PanIN structures. Antibodies and colors of the immune panel used are in the legend below. PanCK, pancytokeratin. B, mfIHC composite images of FFPE donor tissue specimens, highlighting acinar, normal duct, ADM, and PanIN structures. Antibodies and colors of the fibroblast panel used are in the legend below. C, Quantification of percent positive CD163⁺ cells (top left), CD8⁺ T cells (top right), CD4⁺ T cells (bottom left), and Tregs (bottom right) surrounding acinar, ADM, duct, and PanIN populations, respectively. Asterisks denote a P value of <0.05, as determined by ANOVA. HPF, high-power field. D, Quantification of percent positive SMA⁺ cells (top left), vimentin⁺ cells (bottom left), FAP⁺ cells (top right), and PDGFR⁺ cells (bottom right) surrounding acinar, ADM, duct, and PanIN populations, respectively. Asterisks denote a P value of <0.05, as determined by ANOVA.

Figure 3.

Healthy pancreata contain several nonepithelial populations, including myeloid cells, lymphocytes, fibroblasts, and endothelial cells. A, UMAP of all cells captured from single-cell RNA sequencing of six donor pancreata. Populations are identified by color. B, Histogram of cell-type abundance of all captured cells by donor. C, UMAP of extracted myeloid cells from donor pancreata. Populations are identified by color. AltAct, alternatively activated macrophages. D, Histogram of cell-type abundance of specific myeloid cell populations by the donor. E, UMAP of extracted lymphocytes from donor pancreata. Populations are identified by color. F, Histogram of cell-type abundance of specific lymphocyte populations by donor. G, UMAP of extracted fibroblast, pericyte, and endothelial populations from donor pancreata. Populations are identified by color. H, Histogram of cell-type abundance of specific fibroblast, pericyte, and endothelial populations by donor.

Figure 4.

Comparison of the microenvironment in healthy pancreata and pancreatic tumors reveals distinct stromal features. A, UMAP of all cells captured from single-cell RNA sequencing of six donor pancreata merged with 15 PDAC samples and 3 adjacent normal samples. Populations are identified by color. B, Histogram of cell-type abundance of all cell populations by disease state [healthy, adjacent normal (AdjNormal), and tumor]. C, Neighborhood graph differential abundance plot of the merged tumor, healthy, and adjacent normal samples. Size of dots represents neighborhoods, whereas edges represent the number of cells shared between neighborhoods. Neighborhoods colored in red represent significantly increased abundance in healthy samples, whereas neighborhoods colored in blue represent significantly increased abundance in tumor samples. logFC, log-fold change; Nhood, neighborhood. D, Beeswarm plot of differential abundance by cell type. X-axis represents a log-fold change in abundance between tumor and healthy states. Each dot is a neighborhood; neighborhoods colored in red represent significantly increased abundance in healthy samples, whereas neighborhoods colored in blue represent significantly increased abundance in tumor samples. E, Correlation heat map of ­pseudobulk-aggregrated counts of 15 tumor samples, 3 adjacent normal samples, and 11 donor samples (given that single-cell sequencing was performed on the head and tail sections separately in 5 out of 6 donors). Each row/line represents one aggregated single-cell sequencing sample. F, PCA plots of pseudobulk-aggregated counts from all cells, myeloid cells, T cells, and fibroblasts. Each dot represents one aggregated single-cell sequencing sample. G, Circos plots of putative ligand–receptor interactions that are upregulated in PDAC epithelial cells (left), fibroblasts (middle), and macrophages (right) compared with healthy cells. The heat map within the circos plots is the scaled average expression of each gene within PDAC tissue cell populations. The interactions plotted are those in which the expression level of the ligand is increased in PDAC samples compared with healthy tissues.

Figure 5.

Myeloid and fibroblast populations from tumor-bearing pancreata display distinct transcriptomic signatures compared with their nontumor counterparts. A, Left, UMAP of extracted myeloid cells from the single-cell dataset of healthy, adjacent normal, and tumor samples. Populations are identified by color. Right, UMAP overlay of disease states on extracted myeloid cells from the single-cell dataset of healthy, adjacent normal, and tumor samples. AdjNorm, adjacent normal; AltAct, alternatively activated macrophages; cDC, conventional dendritic cells; pDC, plasmacytoid dendritic cells; Mac, macrophage. B, PCA plots of pseudobulk-aggregated counts from specific myeloid cell populations. Each dot represents one aggregated single-cell sequencing sample. C, Top differentially expressed genes between alternatively activated macrophages (left) and resident macrophages (right) from healthy (blue) and tumor (orange) samples. D, Violin plots of normalized expression of select TAM markers comparing healthy to tumor samples. Adjusted P value for significantly differentially expressed markers: CXCL8 4.64E-59. E, Left, UMAP of extracted fibroblast and pericyte cells from the single-cell dataset of healthy, adjacent normal, and tumor samples. Populations are identified by color. Right, UMAP overlay of disease states on extracted fibroblast/pericyte cells from the single-cell dataset of healthy, adjacent normal, and tumor samples. F, Violin plots of normalized expression of select fibroblast markers mapped across fibroblast and pericyte populations. Adjusted P value for significantly differentially expressed markers: PDGFRA 1.58E-308, ACTA2 1.85E-261, TAGLN 1.18E-117, DPT 2.00E-67, and FAP 3.63E-308. G, PCA plots of pseudobulk-aggregated counts from fibroblast (top) and pericyte (bottom) populations. Each dot represents one aggregated single-cell sequencing sample. H, Top differentially expressed genes between fibroblasts from healthy (blue) and tumor (orange) samples. I, Violin plots of normalized expression of select fibroblast markers comparing healthy to tumor samples.

Figure 6.

Spatial transcriptomics reveals a unique epithelial gene signature of PanIN lesions that aligns closely with tumor epithelium. A, Left, GeoMX tissue section from a donor pancreas stained for panCK and CD45. panCK⁺ segments are pseudocolored in green, whereas panCK⁻CD45⁻ segments are pseudocolored in red. Right, GeoMX tissue section from surgically resected, treatment-naive PDAC stained for panCK and CD45. panCK⁺ segments are pseudocolored in purple. B, Sankey plot showing the distribution of ROIs in donor or PDAC samples. C, Heat map of cell type–specific markers derived from differential gene expression using the linear mixed model on spatial transcriptomic ROIs. D, UMAP overlay of disease states on extracted epithelial cells from the single-cell dataset of healthy, adjacent normal, and tumor samples. E, AUCell gene set scoring mapped to the epithelial single-cell dataset of healthy, adjacent normal, and tumor samples using signatures derived from acinar, normal duct, PanIN, and ADM spatial transcriptomic ROIs. The top row represents spatial transcriptomic signatures obtained from healthy tissue; the bottom row represents spatial transcriptomic signatures obtained from tumor tissue. F, AUCell gene set scoring mapped to the epithelial single-cell dataset of healthy, adjacent normal, and tumor samples using signatures derived from glandular tumor and poorly differentiated tumor ROIs.

Figure 7.

Claudin18, MUC5AC/B, and AQP1/3 distinguish normal ducts, ADM, and PanIN in a healthy pancreas. A, Donor tissue stained with antibodies against MUC5B (green) and E-Cadherin (red), along with DAPI. B, Donor tissue stained with antibodies against MUC5AC (green) and E-Cadherin (red), along with DAPI. C, Donor tissue stained with antibodies against Claudin18 (green) and E-Cadherin (red), along with DAPI. D, Donor tissue stained with antibodies against AQP3 (green) and E-Cadherin (red), along with DAPI. E, Donor tissue stained with antibodies against AQP1 (green) and E-Cadherin (red), along with DAPI. F, Donor tissue stained with RNAScope probe for TFF1 (red) and antibody against Claudin18 (green), along with DAPI. G, Donor tissue stained with antibodies against p-ERK (green), Claudin18 (orange), and E-Cadherin (red), along with DAPI. E-Cad, E-Cadherin.