Topographical Distribution and Spatial Interactions of Innate and Semi-Innate Immune Cells in Pancreatic and Other Periampullary Adenocarcinoma

Abstract

Background: The clinical management of pancreatic and other periampullary neoplasms remains challenging. In contrast to other cancer types, immunotherapies are largely ineffective, and the reason for the deprived immune response and the immune inhibiting cellular composition is only fragmentarily understood. The aim of this study was to comprehensively map the abundance, topographic distribution and spatial interaction of innate and innate-like immune cells in the tumor microenvironment of periampullary adenocarcinoma.

Methods: Multiplexed immunofluorescent imaging was performed on tissue microarrays with tumors from a consecutive cohort of 175 patients with resected periampullary adenocarcinoma. To obtain a detailed spatial analysis of immune cell infiltration, two multiplex immune panels including antibodies against CD3, NKp46, CD56, CD68, CD163 and CD1a, CD208, CD123, CD15, CD68 and pan-cytokeratin were applied.

Results: The infiltration of natural killer (NK) and NK-like T (NKT) cells was lower in malignant compared to benign tissue. NKT cells were more abundant in intestinal type compared to pancreatobiliary type tumors, and were associated with more favorable clinicopathological features and a prolonged survival. The interaction of NKp46⁺ NKT cells with macrophages was also associated with a prolonged survival.

Conclusions: This study provides a comprehensive map of the innate immune landscape in periampullary adenocarcinoma. NK cells, and even more so NKT cells, are revealed to be central players in the local immune response in a clinically relevant context.

Keywords: dendritic cells; innate immunity; natural killer T-cells; natural killer cells; tumor microenvironment.

FIGURE 1

Representative immunofluorescence images. (A) Representative image of a TMA core with heterogeneous infiltration of leukocytes in panel 1; red denoting CD68, yellow denoting CD56, green denoting CD3, pink denoting NKp46, cyan denoting CD163 and magenta denoting cytokeratin. (B) Representative image of a TMA core with heterogeneous infiltration of leukocytes in panel 2; green denoting CD1a, red denoting CD208, yellow denoting CD15, pink denoting CD123, cyan denoting CD68 and magenta denoting cytokeratin.

FIGURE 2

Associations of immune cell subsets with tumor morphology. Radar plots of leukocyte infiltration levels in PB-type tumours (purple) and I-type tumors (blue). Data shown separately for total tissue (left), the tumor compartment (middle) and the stromal compartment (right).

FIGURE 3

Associations of immune cell subsets with clinicopathological factors. Heatmap illustrating differences in densities of leukocyte subclass infiltration according to anatomical origin.

FIGURE 4

Prognostic impact of immune cell subsets in the entire cohort. (A) Forest plots depicting hazard ratios of death within 5 years according to the density of leukocytes characterized in panel 1. (B) Forest plots depicting hazard ratios of death within 5 years according to the density of leukocytes characterized in panel 2.

FIGURE 5

Identification of immune profiles in the entire cohort. (A) Heatmap illustrating unsupervised hierarchical clustering of patients by normalized infiltration densities of leukocytes in the tumor compartment. Blue color indicates low levels of infiltrating immune cells and red color indicates high levels of infiltrating immune cells. (B) Heatmap illustrating unsupervised hierarchical clustering of patients by normalized infiltration densities of leukocytes in the stromal compartment. Blue color indicates low levels of infiltrating immune cells and red color indicates high levels of infiltrating immune cells.

FIGURE 6

Cellular interactions and their impact on patient survival in the entire cohort. (A) Representative microphotograph of immunofluorescence staining illustrating NKp46⁺ NKT cells (green) within the interaction zone of a CD68⁺ macrophage (red). Cancer cells are visible as CK⁺ cells (purple). (B) Forest plots depicting hazard ratios of death within 5 years according to the interaction of NKp46⁺ NKT cells with other immune cells and cancer cells. (C) Forest plots depicting hazard ratios of death within 5 years according to the interaction of CD68⁺ macrophages with other immune cells and cancer cells.