Early diagnosis of lymph node metastasis: Importance of intranodal pressures

Abstract

Regional lymph node status is an important prognostic indicator of tumor aggressiveness. However, early diagnosis of metastasis using intranodal pressure, at a stage when lymph node size has not changed significantly, has not been investigated. Here, we use an MXH10/Mo-lpr/lpr mouse model of lymph node metastasis to show that intranodal pressure increases in both the subiliac lymph node and proper axillary lymph node, which are connected by lymphatic vessels, when tumor cells are injected into the subiliac lymph node to induce metastasis to the proper axillary lymph node. We found that intranodal pressure in the subiliac lymph node increased at the stage when metastasis was detected by in vivo bioluminescence, but when proper axillary lymph node volume (measured by high-frequency ultrasound imaging) had not increased significantly. Intravenously injected liposomes, encapsulating indocyanine green, were detected in solid tumors by in vivo bioluminescence, but not in the proper axillary lymph node. Basic blood vessel and lymphatic channel structures were maintained in the proper axillary lymph node, although sinus histiocytosis was detected. These results show that intranodal pressure in the proper axillary lymph node increases at early stages when metastatic tumor cells have not fully proliferated. Intranodal pressure may be a useful parameter for facilitating early diagnosis of lymph node metastasis.

Keywords: EPR effect; Early diagnosis; intranodal pressure; lymph node metastasis; lymphatic network; mouse model of metastasis.

Figure 1

Tumor cell inoculation into the subiliac lymph node (SiLN) and cell characteristics. (A) Experimental schedule. The 72 mice included in the analysis were divided into three groups: control (n = 12), KM‐Luc/GFP (n = 24), and FM3A‐Luc (n = 36). The day of inoculation was defined as day 0. The KM‐Luc/GFP group was divided into two subgroups: day 3 and day 6 (n = 12, each subgroup). The FM3A‐Luc group was divided into three subgroups: day 6, day 10, and day 14 (n = 12, each subgroup). IVIS, in vivo luminescence imaging system; VEVO, high‐frequency ultrasound imaging system. (B) Intranodal pressure measurement. A hypodermic needle, connected to a pressure transducer, was inserted into the central region of the SiLN or PALN for 5 min. (C) Needle location in the SiLN before (a) and immediately after (b) inoculation. Cells were detected as a central shadow. Images were obtained with VEVO. (D) RNA isolation and PCR to detect the expression of the vascular endothelial growth factor (VEGF) family. Gel electrophoretic analysis of PCR for the VEGF family in KM‐Luc/GFP and FM3A‐Luc cells. Sterile distilled water was used for the negative control. B16F10 cells, which express VEGF‐C, were used as a positive control for VEGF‐C.

Figure 2

Induction of metastasis in a mouse model. (A–D) Changes in luciferase activity. (A,C) Luciferase activities induced by KM‐Luc/GFP cell injection into the subiliac lymph node (SiLN) (n = 24). Metastasis to the proper axillary lymph node (PALN) was detected on day 6. Luciferase activity increased in both the SiLN and PALN, but was higher in the SiLN. (B,D) Luciferase activities induced by FM3A‐Luc cell injection into the SiLN (n = 36). Metastasis to the PALN was detected on day 14. Tumor growth was slower than for KM‐Luc/GFP cells, but the trends in luciferase activity were similar. *P < 0.05, **P < 0.01 versus day 0 (one‐way anova and Tukey's test). Mean ± SD values are shown. (E–H) Changes in lymph node size. (E,G) Change in PALN volume, metastasized by KM‐Luc/GFP cells (n = 12). (F,H) Change in PALN volume, metastasized by FM3A‐Luc cells (n = 12). Lymph node volume was measured using high‐frequency ultrasound imaging. Mean ± SEM values are shown, normalized to those on day −1.

Figure 3

Intranodal pressure (INP) measurements in the subiliac lymph node (SiLN), and proper axillary lymph node (PALN). (A) KM‐Luc/GFP cells (SiLN and PALN, n = 36). *P < 0.05 for SiLN group versus control on day 3; **P < 0.01 for SiLN versus control on day 6; *P < 0.05 for PALN versus control on day 6 (two‐way anova and Tukey's test). (B) FM3A‐Luc cells (SiLN and PALN, n = 36). There were no significant changes in INP in the PALN. Mean ± SEM values are shown.

Figure 4

Leakage of indocyanine green (ICG) liposomes. Bioluminescence and fluorescence images of mice inoculated with KM‐Luc/GFP (A,C,E) or FM3A‐Luc (B,D,F) cells. Representative bioluminescence images: A(i), A(iii), B(i), and B(iii). Representative fluorescence images: A(ii), A(iv), B(ii), and B(iv). Solid tumor groups: A(i), A(ii), B(i), and B(ii). Metastasis groups: A(iii), A(iv), B(iii), and B(iv). Bioluminescence images were obtained 6 days after inoculation of KM‐Luc/GFP cells, and 14 days after inoculation of FM3A‐Luc cells. ICG liposomes were then injected i.v., and fluorescence images obtained 24 h later, that is, days 7 and 15 after inoculation of KM‐Luc/GFP and FM3A‐Luc cells, respectively. Quantification of luciferase activity (averaged values): KM‐Luc/GFP (C) and FM3A‐Luc (D). Luciferase activities were obtained 6 days after inoculation of KM‐Luc/GFP cells (**P < 0.01, solid tumor versus proper axillary lymph node [PALN]), and 14 days after inoculation of FM3A‐Luc cells (**P < 0.01, solid tumor versus PALN). E(i), E(ii), F(i), and F(ii): Following imaging, the subiliac lymph node (SiLN), PALN, and solid tumor were homogenized, and the fluorescence intensity of the supernatant measured by IVIS. E(i), F(i): Fluorescence images of individual wells of a 48‐well plate. E(ii), F(ii): Averaged values for the fluorescence/weight ratio. Mean ± SEM values are shown. **P < 0.01 versus solid tumor (one‐way anova and Tukey's test).

Figure 5

Representative histological images showing the internal structures of the subiliac lymph node (SiLN) and proper axillary lymph node (PALN) after initiation of metastasis. (A) Representative images of the SiLN and PALN stained with H&E, anti‐LYVE‐1 antibody, or anti‐CD31 antibody, under control conditions (n = 4) on day 3, or after inoculation with KM‐Luc/GFP cells (n = 4) on day 6 or FM3A‐Luc cells (n = 4) on day 14. In both the control and metastasis groups, the blood vessels remained discretely distributed. Cor, cortex; Paracor, paracortex; Meta, metastasis. (B) H&E staining of tumor‐containing PALN. Histiocytosis was evident in/around the lymphatic sinus (arrows). Bar = 50 μm.

Figure 6

Evaluation of subiliac lymph node (SiLN) and proper axillary lymph node (PALN) blood vessel density after initiation of metastasis. (A) Representative immunofluorescence images of the SiLN and PALN under control conditions (n = 4) on day 3 or after inoculation with KM‐Luc/GFP cells (n = 4) on day 6 or FM3A‐Luc cells (n = 4) on day 14, showing staining for CD31 as a marker of blood vessels. Bar = 200 μm. (B,C) Mean values for the percentage CD31‐positive area in the SiLN (B) and PALN (C). There were no significant differences in the entire lymph node blood vessel density between the control and metastasis groups (one‐way anova). Mean ± SEM values are shown. NS, not significant.