Evolution of the immune landscape during progression of pancreatic intraductal papillary mucinous neoplasms to invasive cancer

Abstract

Background: Intraductal papillary mucinous neoplasms (IPMNs) are precursor lesions of pancreatic cancer, which is characterized by an immunosuppressive microenvironment. Yet, the spatial distribution of the immune infiltrate and how it changes during IPMN progression is just beginning to be understood.

Methods: We obtained tissue samples from patients who underwent pancreatic surgery for IPMN, and performed comprehensive immunohistochemical analyses to investigate the clinical significance, composition and spatial organization of the immune microenvironment during progression of IPMNs. Survival analysis of pancreatic cancer patients was stratified by tumour infiltrating immune cell subtypes.

Findings: The immune microenvironment evolves from a diverse T cell mixture, comprising CD8⁺ T cells, Th/c1 and Th/c2 as major players combined with Th9, Th/c17, Th22, and Treg cells in low-grade IPMN, to a Treg dominated immunosuppressive state in invasive pancreatic cancer. Organized lymphoid clusters formed in IPMN surrounding stroma and accumulated immunosuppressive cell types during tumour progression. Survival of pancreatic cancer patients correlated with Th2 signatures in the tumour microenvironment.

Interpretation: The major change with regards to T cell composition during IPMN progression occurs at the step of tissue invasion, indicating that malignant transformation only occurs when tumour immune surveillance is overcome. This suggests that novel immunotherapies that would boost spontaneous antitumor immunity at premalignant states could prevent pancreatic cancer development.

Funding: The present work was supported by German Cancer Aid grants (70,112,720 and 70,113,167) to S. R., and the Olympia Morata Programme of the Medical Faculty of Heidelberg University to S. R.

Keywords: Intraductal papillary mucinous neoplasm; Pancreatic cancer; Premalignant lesion; Tumour immunology; Tumour microenvironment.

Fig. 1

Tumour microenvironment compartments. Representative image of a human low-grade IPMN tissue section stained with H&E. Immune cell populations were quantified in distinct areas, juxtatumoral (red) and peritumoral (yellow) stroma, tertiary lymphoid structures (green), and normal adjacent pancreatic tissue (purple). Scale bar represents 100 μm². The H&E image was acquired at 20x magnification. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Immune cells accumulate in human IPMN already at an early stage. (a and b) CD45⁺ immune cell numbers per 100 μm² in normal adjacent (N) pancreatic tissue, within the juxtatumoral stroma (a), or in the peritumoral stroma (b) of low-grade IPMN (L), high-grade IPMN (H), and IPMN-associated invasive pancreatic cancer (IC) as determined by immunohistochemistry (IHC). All data are presented as box and whisker plots. ***p < 0.001, Kruskal-Wallis test with Dunn‘s posthoc analysis. (c), Representative images of CD45 IHC on indicated tissue areas. Scale bar represents 100 μm². Images were acquired at 20x magnification.

Fig. 3

Immune cell composition changes from low-grade IPMN to invasive pancreatic cancer. (a) Box and whisker plots comparing CD3⁺ T cell numbers per 100 μm² in normal adjacent (N) pancreatic tissue, within the juxtatumoral stroma (left), or in the peritumoral stroma (right) of low-grade IPMN (L), high-grade IPMN (H), and IPMN-associated invasive pancreatic cancer (IC) as detected by IHC. Representative image of CD3 IHC on an IPMN tissue section. (b) Stacked barplots showing leucocyte subtypes as percentage of CD45⁺ total leucocyte counts in juxtatumoral (left) and peritumoral stroma (right) of IPMN lesions as determined by IHC for CD1a, CD4, CD8, CD20, CD68 and CD208. (c and d) CD8⁺ T cell (c) and CD68⁺ macrophage (d) numbers per 100 μm² in juxtatumoral, or peritumoral stroma of low-grade IPMN (L), high-grade IPMN (H), and IPMN-associated invasive pancreatic cancer (IC) based on IHC analyses. Representative images of CD8 (c) and CD68 (d) IHC on IPMN sections. e, Box and whisker plots comparing CD4⁺ T cell numbers per 100 μm² in normal adjacent (N) pancreatic tissue, within the juxtatumoral stroma (left), or in the peritumoral stroma (right) of low-grade IPMN (L), high-grade IPMN (H), and IPMN-associated invasive pancreatic cancer (IC) as detected by IHC. Representative image of CD4 IHC on an IPMN tissue section. Scale bars represent 100 μm². Images were acquired at 20x magnification. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Kruskal-Wallis test with Dunn‘s posthoc analysis; ns, not significant.

Fig. 4

Distribution of T cell subtypes varies between distinct compartments of IPMN lesions and evolves during tumour progression. (a) Balloonplots showing relative abundance of indicated T cell subtypes in juxtatumoral stroma (left), or in the peritumoral stroma (right) of IPMN lesions. The size of the balloons correlates with cell densities as determined by IHC for FOXP3, CD3/T-bet, CD3/GATA3, CD3/PU.1, CD3/RORγt, and CD3/AHR. b, FOXP3⁺ cell numbers per 100 μm² in juxta-, or peritumoral stroma of low-grade IPMN (L), high-grade IPMN (H), and IPMN-associated invasive pancreatic cancer (IC) based on IHC analyses. Representative image of FOXP3 IHC in the peritumoral stroma. (c and d) Box and whisker plots comparing CD3⁺T-bet⁺ (c), and CD3⁺GATA3⁺ (d) cell numbers per 100 μm² in normal adjacent (N) pancreatic tissue, within the juxtatumoral stroma (left), or in the peritumoral stroma (right) of low-grade IPMN (L), high-grade IPMN (H), and IPMN-associated invasive pancreatic cancer (IC) as detected by IHC. Representative images of CD3/T-bet (c), and CD3/GATA3 (d) IHC on IPMN sections. (e–g) Box and whisker plots comparing numbers of CD3⁺PU.1⁺ (e), CD3⁺RORγt⁺ (f), and CD3⁺AHR⁺ (g) T cells per 100 μm² in normal adjacent (N) pancreatic tissue and in the juxtatumoral stroma of low-grade IPMN (L), high-grade IPMN (H), and IPMN-associated invasive pancreatic cancer (IC) as detected by IHC. Representative images of CD3/PU.1 (e), CD3/RORγt (f), and CD3/AHR (g) IHC on juxtatumoral stroma of IPMN sections. Examples of CD3⁺T-bet⁺, CD3⁺GATA3⁺, CD3⁺PU.1⁺, CD3⁺RORγt⁺, and CD3⁺AHR⁺ cells are denoted with arrows. b-g, Scale bars represent 50 μm². Images were acquired at 20x magnification. *p < 0.05, ***p < 0.001, ****p < 0.0001, Kruskal-Wallis test with Dunn‘s posthoc analysis; ns, not significant.

Fig. 5

Correlation of T cell subtypes in the tumour microenvironment with IPMN histological grade. (a) Correlation matrix comparing the densities of T cell subtypes as determined by IHC in juxtatumoral areas of all IPMN lesions. Scale refers to the rank-based Spearman correlation coefficient. (b) Principal component analysis (PCA) of all IPMN tissue samples based on immunohistochemical quantification of juxtatumoral macrophages and T cell subtypes. The first three principal components (PC1, PC2, PC3), which explain most of the data variation, are shown in 3D. Each data point represents one sample, colours correspond to the three different histological grades (blue, low-grade IPMN; yellow, high-grade IPMN; grey, IPMN-associated invasive pancreatic cancer). *p < 0.05, **p < 0.01, ***p < 0.001, p-values are approximated by using the t distribution based on Spearman correlations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Tertiary lymphoid structures (TLS) evolve from low-grade IPMN to invasive pancreatic cancer. (a) Representative images of human low-grade IPMN (IPMN-L) and IPMN-associated pancreatic cancer (IPMN-IC) specimen containing TLS stained with H&E, or by IHC for CD3, CD20, or CD208, showing B cell follicles and T cell zones in lymphoid aggregates. (b) Percentage of low-grade IPMN (L), high-grade IPMN (H), and IPMN-associated invasive pancreatic cancer (IC) specimen with at least one tertiary lymphoid structure present in the peritumoral area. (c), Box and whisker plots comparing numbers of CD8 (left) and CD4 (right) T cells per 100 μm² in normal adjacent (N) pancreatic tissue and in TLS of low-grade IPMN (L) and IPMN-associated invasive pancreatic cancer (IC) as detected by IHC. (d) Stacked barplots showing relative densities of indicated T cell subtypes. The height of the bars is proportionate to the cell densities after normalization of cell counts as determined by IHC for FOXP3, CD3/T-bet, CD3/GATA3, CD3/PU.1, CD3/RORγt, and CD3/AHR. (e-j) FOXP3⁺ (e), CD3⁺T-bet⁺ (f), CD3⁺GATA3⁺ (g), CD3⁺PU.1⁺ (h), CD3⁺RORγt⁺ (i), and CD3⁺AHR⁺ (j) T cell numbers per 100 μm² in normal adjacent (N) pancreatic tissue and tertiary lymphoid structures of low-grade IPMN (L) and IPMN-associated invasive pancreatic cancer (IC) based on IHC analyses. (k) Stacked barplots showing relative densities of immature (CD1a) and mature (CD208) dendritic cells in juxtatumoral stroma (left), peritumoral stroma (middle), and TLS (right) of low-grade IPMN (L) and IPMN-associated invasive pancreatic cancer (IC). The height of the bars correlates with cell densities after normalization of cell counts as determined by IHC for CD1a and CD208. Scale bars represent 100 μm². Images were acquired at 20x magnification. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Kruskal-Wallis test with Dunn‘s posthoc analysis; ns, not significant.

Fig. 7

Association of T cell subtypes with pancreatic cancer prognosis. (a) Illustration of included samples and design of the Kaplan-Meier survival analysis based on the immune signature scores of the “PanImmune Feature Matrix“ published by Thorsson et al. (b), Survival plots of pancreatic cancer patient groups stratified by Th2 signatures. Overall survival time is shown in days. TCGA, The Cancer Genome Atlas; CD8, CD8⁺ T cells; B, B cells; MP, macrophages; DC, dendritic cells; **p < 0.01, Log-Rank test; ns, not significant.