Disrupted fibroblastic reticular cells and interleukin-7 expression in tumor draining lymph nodes

Abstract

The immune system of patients with cancer is usually in an inhibitory state. Lymph node (LN) draining of pathological sites provides a suitable microenvironment where adaptive immune responses mainly occur. However, the microenvironment in the tumor draining lymph nodes (TDLNs) of patients with cancer appears to be in favor of tolerance. The effects of tumor cells on TDLNs have not been elaborated clearly. The present results have indicated that tumor cells may directly affect TDLNs by decreasing the fibroblastic reticular cell population that led to less interleukin-7 secretion. As a result, the number of T cells in TDLNs declined with reduced survival signals. A decreased number of T cells in TDLNs means weakened ability of immune surveillance. Clinically, these results were also confirmed in LN biopsies from patients with colon cancer at different clinical stages. Results of the present study showed that tumor cells may directly inhibit the immunological function of TDLNs.

Keywords: cancer; fibroblastic reticular cells; interleukin-7; tumor draining lymph node.

Figure 1.

FRCs and IL-7 were significantly decreased in TDLNs. (A) Flow cytometric analysis showed that the number of FRCs (gp38⁺CD31⁻) in CD45⁻ cells dropped in TDLNs, representative of 5 mice for the control group and 5 mice for the tumor-bearing group. (B) Immunofluorescent staining showed lower expression of ER-TR7 in TDLNs. (C) Gp38 detected by western blot analysis normalized to changes in the β-actin intensity. (D) The estimated amounts of IL-7 mRNA were calculated with the reverse transcription-quantitative polymerase chain reaction. IL-7 expression in TDLNs significantly decreased compared with the control group. The mean values ± standard error of the mean were analyzed from 4 mice per group, in 3 independent experiments respectively. (E) Confocal images of LNs sections stained for ER-TR7 (green) and IL-7 (red). **P<0.01, ***P<0.001. IL-7, interleukin-7; LN, lymph node; TDLN, tumor draining lymph node; FRC, fibroblastic reticular cell.

Figure 2.

The number of T lymphocytes in TDLNs was reduced. (A) Flow cytometric analysis showed that the number of CD4⁺ and CD8⁺ T cells decreased significantly in TDLNs, representative of 4 mice for the control group and 8 mice for the tumor-bearing group. (B) LNs biopsies from mice were sectioned and stained for CD3. Staining was quantified by the number of cells with CD3 positivity. (C) LN biopsies from mice were sectioned and stained for CD8. Staining was quantified by the number of cells with CD8 positivity. The mean values ± standard error of mean were analyzed from 3 mice per group. *P<0.05, **P<0.01. LN, lymph node; TDLN, tumor draining lymph node; CD, cluster of differentiation; nTDLN, non tumor draining lymph node.

Figure 3.

Desmin, IL-7 and CD8⁺T cells decreased in human TDLNs. (A) LNs biopsies from patients with colon cancer were stained for desmin. QIA showed that the expression area of desmin shrunk by up to 60% accompanied with tumor progression. (B) LNs biopsies from patients with colon cancer were stained for IL-7. QIA showed that IL-7 is significantly reduced in Dukes D stage. (C) Representative sections stained for CD8 were shown from patients at different Dukes stages. QIA demonstrated that CD8⁺T cells reduced with tumor progression. *P<0.05, **P<0.01; ***P<0.001. IL-7, interleukin 7; LN, lymph node; TDLN, tumor draining lymph node; QIA, quantitative image analysis.

Figure 4.

The Spearman correlation test was conducted to analyze the existing correlations. (A) Correlative analysis between desmin and IL-7 expression area were performed for all subjects. (B) Correlative analysis between IL-7 staining and the number of CD8⁺ T cells were performed for all subjects. IL-7, interleukin-7.