Evaluation of metastatic niches in distant organs after surgical removal of tumor-bearing lymph nodes

Abstract

Background: Surgical removal of primary tumors can promote the incidence of tumor metastasis. However, molecular mechanisms underlying this process remain unclear.

Methods: We inoculated tumor cells expressing luciferase gene into subiliac lymph node (SiLN) of the MXH10/Mo-lpr/lpr mice. The tumor-bearing SiLNs were surgically removed at a certain period of time after inoculation.

Results: In vivo bioluminescence imaging system and histological staining revealed metastasis in lung, proper axillary lymph node (PALN) and liver. The lung metastasis rate in SiLN removal groups was significantly higher than in the control group using Fisher exact test. Mann-Whitney U-test indicated that the luciferase-positive tumor cells in the lung and liver were significantly higher than in the control groups. The lung samples in SiLN removal groups had strong expression of lysine oxidase (LOX). Moreover, the number of CD11b⁺ cells in the lung and liver in the SiLN removal groups was significantly increased, which was positively correlated with LOX expression level. In addition, the condition of LOX and CD11b in liver was similar to lung. In the SiLN surgical removal groups, the matrix metalloproteinase (MMP)-2 and VEGFA expression in the lung tissues was significantly higher than in the control groups; the collagen fibers per area around the pulmonary vessels was quite significantly lower and negatively correlated with the expression of MMP-2 by Spearman's analysis. Our data indicated that the reticular fibers were deposited and disordered in the tumor tissues of the lungs in the removal groups, and the reticular fibers per area was higher than in the control groups. The tumor cells in the PALN of control groups were significantly higher than in the SiLN removal groups, and CD169⁺ and CD11c⁺ cells were also higher than in the SiLN removal groups.

Conclusions: Altogether, surgical removal of the tumor-bearing lymph node promoted tumor metastasis through changing the niche in lung and liver. Treatment targeting the metastatic niche might be an effective strategy to prevent tumor metastasis, thereby possibly increasing the survival and reducing the incidence of metastasis in cancer patients.

Keywords: Metastasis; Metastatic niche; Surgical removal; Tumor-bearing lymph node.

Fig. 1

In vivo and ex vivo detection of metastases using in vivo bioluminescence imaging system. a KM-Luc/GFP cells were injected into the SiLN (n = 22) (KM group). The SiLN was removed 3 days (G-D3 group, n = 9), or 6 days (G-D6 group, n = 8) after tumor cell inoculation. SiLNs that were injected with KM-Luc/GFP cells but not removed were used as control (G-C group, n = 5). b FM3A-Luc cells were injected into the SiLN (n = 15) (FM3A group). The SiLN was removed 3 days (G-D3 group, n = 4), or 7 days (G-D7 group, n = 6) after tumor cell inoculation. SiLNs that were injected with FM3A-Luc cells but not removed were used as control (G-C group, n = 5). c-d The lung, PALN and liver metastasis rate in the control and SiLN removal groups. e-f The ex vivo luciferase activity of the lung in the control and SiLN removal group

Fig. 2

The expression of luciferase in PALN, Lung and liver. The Immunohistochemical (IHC) staining of luciferase in PLAN (left panel), lung (middle panel) and liver (right panel) of the KM (a) and FM3A (b) groups. Bar: 100 μm

Fig. 3

The changes of metastatic niche in lung samples. The expression of LOX, CD11b, MMP-2 and VEGFA were determined using IHC staining in the KM (a) and FM3A (b) groups. Bar: 50 μm

Fig. 4

Location and expression of collagen fibers and reticular fibers. Location and expression of collagen fibers (left panel) and reticular fibers (right panel) were analyzed by Elastic-Masson (EM) staining and silver impregnation method in the KM (a) and FM3A (b) groups. Bar: 100 μm

Fig. 5

The changes of metastatic niche in liver samples. The expression of LOX and CD11b were observed using IHC staining in the KM (a) and FM3A (b) groups. Bar: 20 μm

Fig. 6

Histological changes in tumor-draining PALN. a-b HE staining of PALN in mice in KM (a) and FM3A (b) control groups. As time went by, tumor cells were found in lymphatic sinuses and tumor nests formed. c-d The expression of F4/80, CD169 and CD11c in PALN were analyzed by IHC staining. Bar: (a-c) 100 μm, (d) 50 μm