Immune cell infiltration as an indicator of the immune microenvironment of pancreatic cancer

Abstract

Background: The host immune reaction is represented by immune/inflammatory cell infiltrates. Here we systematically analysed tumour-infiltrating immune/inflammatory cells in pancreatic ductal carcinoma (PDC) and evaluated their clinicopathological impact.

Methods: Using immunohistochemistry, we examined tumour-infiltrating CD68(+) pan-macrophages, HLA-DR(+)CD68(+) M1 macrophages (M1), CD163(+) or CD204(+) M2 macrophages (M2), CD66b(+) neutrophils (Neu), CD4(+) T cells (CD4(+)T), CD8(+) T cells (CD8(+)T), and FOXP3(+)CD4(+) regulatory T cells (Treg) in 212 cases of PDC, and conducted correlation and survival analyses using the Kaplan-Meier method and Cox proportional hazards model.

Results: Higher levels of tumour-infiltrating pan-macrophages, M2, Neu, or the ratio of Tregs to CD4(+)T (%Treg) were significantly associated with shorter survival, whereas higher levels of tumour-infiltrating CD4(+)T, CD8(+)T, or the ratio of M1 to pan-macrophages (%M1) were significantly associated with longer survival. Survival analysis of pairs of these variables revealed that some of the resulting patient groups had exclusively longer survival. We then connected the apparently related factors, and two significant variables emerged: tumour-infiltrating CD4(+)T(high)/CD8(+)T(high)/%Treg(low) and tumour-infiltrating %M1(high)/M2(low). Multivariate survival analysis revealed that these variables were significantly correlated with longer survival and had a higher hazard ratio.

Conclusion: Tumour-infiltrating CD4(+)T(high)/CD8(+)T(high)/%Treg(low) and %M1(high)/M2(low) are independent prognosticators useful for evaluating the immune microenvironment of PDC.

Figure 1

Immunohistochemical features of tumour-infiltrating immune/inflammatory cells. The representative photos are shown: (A) CD8⁺ T cells, (B) CD4⁺ T cells, (C) FOXP3⁺ T cells, (D) CD68⁺ pan-macrophages, (E) CD163⁺ M2 macrophages, (F) CD204⁺ M2 macrophages, (G) CD66b⁺ neutrophils, and (H) CD68⁺HLA-DR⁺ M1 macrophages (CD68 in brown and HLA-DR in purple). Scale bars show 10 μm in length. A comparison of infiltrating immune/inflammatory cells into normal pancreas, chronic pancreatitis (non-cancerous pancreas, and PDC is shown in I. Columns and bars show mean (n=20) and standard error, respectively. Significance values were *P<0.05 and **P<0.01.

Figure 2

Kaplan–Meier survival curves showing comparison of overall survival between high (orange) and low (blue) infiltration (inf.) of immune/inflammatory cells. P-values obtained from log-rank test. The ‘x' and ‘+' represent censoring and failure, respectively. The proportions of 5-year survival with 95% confidence intervals (in parentheses) are shown. *Ratio of CD68⁺HLA-DR⁺ M1 macrophages to CD68⁺ pan-macrophages, **ratio of FOXP3⁺CD4⁺ Tregs to CD4⁺ T cells.

Figure 3

Kaplan–Meier survival curves showing comparison of overall survival among four groups of patients divided on the basis of combinations of two variables with high and low infiltration of immune/inflammatory cells. A and B are representative profiles. The four groups are 1st variable^high and 2nd variable^high in blue, 1st variable^high and 2nd variable^low in pink, 1st variable^low and 2nd variable^high in red, and 1st variable^low and 2nd variable^low in green. The ‘x' and ‘circle' represent censoring and failure, respectively.