New concept for the prevention and treatment of metastatic lymph nodes using chemotherapy administered via the lymphatic network

Abstract

Intravenous chemotherapy has poor access to metastatic lymph nodes (LNs) and is limited by short-lived drug concentrations. Here, we describe the administration of chemotherapy via the lymphatic network as a new concept for the prevention and treatment of metastatic LNs. A metastatic LN can be treated by the injection of drugs into an upstream LN, either the sentinel LN (SLN) or another upstream LN. In a mouse model, tumor cells were inoculated into the subiliac LN (SiLN) to induce metastasis to the proper axillary LN (PALN). Two routes were used for drug delivery to the PALN, namely from the SiLN and from the accessory axillary LN (AALN). We found that tumor masses were formed in lymphatic vessels between the SiLN and PALN. The flow of fluorescent solution injected into the SiLN towards the PALN decreased with tumor mass formation. Delivery from the AALN (free of metastatic tumor cells) to the PALN was identified as an alternative route. Intranodal injection can deliver high concentrations of drugs to secondary metastatic LNs. The study advocates a new concept for the prevention and treatment of metastatic lymph nodes whereby drugs injected into upstream lymph nodes can reach metastatic lymph nodes via the lymphatic network.

Figure 1. Establishment of a model for lymph node metastasis.

(A) Gross anatomy in MXH10/Mo/lpr mice. The efferent lymphatic vessels of the SiLN connect to the PALN, and the efferent lymphatic vessels of the PALN connect to the subclavian vein (SV). The efferent lymphatic vessels of the AALN connect to the PALN. Both the SiLN and AALN are located upstream of the PALN. The thoracoepigastric vein (TEV) connects to the SV and inferior vena cava (IVC) via the PALN and SiLN. LV: lymphatic vessel. Arrow: SiLN → PALN (metastatic route); AALN → PALN (lymphatic drug delivery route). Scale bar: 10 mm. (B) Induction of metastasis in the PALN. Tumor cells were inoculated into the SiLN to induce metastasis in the PALN. Metastasis to the PALN was detected on day 6 after inoculation. (C) Luciferase activity in the SiLN and PALN (n = 25). **P < 0.01, day 0 vs day 6 in the SiLN; *P < 0.05, day 0 vs day 6 in the PALN; one-way ANOVA. Mean ± SD.

Figure 2. Fluorescence intensity of ICG in the PALN.

(A) Representative images of mice after the injection of ICG solution into the SiLN. (B) Changes in the fluorescence intensity of ICG solution in the PALN over time. NS: not significant. Control (inoculation of vehicle without tumor cells), n = 4; day 6 (after inoculation of tumor cells), n = 7. (C,D) The PALN was homogenized and the fluorescence intensity of the supernatant was measured by IVIS. (C) Fluorescence images of individual wells of a 12-well plate. (D) Averaged values for the fluorescence intensity in the control group (inoculation of vehicle without tumor cells) and on day 6 and day 12 after inoculation of tumor cells (control, n = 4; day 6, n = 4; day 12, n = 4). The mean ± SD values are shown. *P < 0.05, control vs day 12 (Mann-Whitney U-test).

Figure 3. India ink flow from the SiLN to the PALN and histopathological analysis.

(A) Control group (n = 6). India ink was delivered to the PALN in all animals (delivery rate: 100%). Histopathology (H&E) revealed an artery and vein located between two lymphatic vessels stained with India ink. (B) Day 6 group (n = 6). (a) India ink was delivered from the SiLN to the PALN in 4/6 animals (delivery rate: 67%). (b) India ink was not delivered to the PALN in 2/6 animals (33%). The lymphatic vessels between the SiLN and PALN were harvested 30 min after the injection of India ink. Tissue sections stained with H&E demonstrated lymphatic vessels filled with tumor cells. Yellow arrow: PALN; LV: lymphatic vessel; T: tumor cells. The table indicates the rate of India ink delivery from the SiLN to the PALN.

Figure 4. Lymphatic delivery from the SiLN and AALN to the PALN.

(A) Visualization of two lymphatic routes. Green dye was driven to flow from the SiLN towards the PALN at a velocity of 50 μL/min using a syringe pump. At 120 sec after the initiation of green dye injection, yellow dye was driven from the AALN to the PALN at a velocity of 50 μL/min using a second syringe pump. Both administrations were terminated 180 sec after the initiation of green dye injection. The total volume of green dye injected was 150 μL, while that of yellow dye was 50 μL. (a) 45 sec after starting the injection of green dye. Approximately half of the SiLN was filled with green dye. (b) 105 sec after starting the injection of green dye. Green dye was visualized as running in the efferent lymphatic vessels of the SiLN towards the PALN. (c) 165 sec after starting the injection of green dye. Green and yellow dyes had converged in the PALN. (B) PALN showing the convergence of green and yellow dyes. (a) Anterior aspect. Two efferent lymphatic vessels extended from the SiLN to the PALN and two efferent lymphatic vessels extended from the AALN to the PALN. The area where the two dyes converged was dyed greenish-yellow. (b) Posterior aspect. Some of the green dye had flowed into the afferent lymphatic vessel of the PALN. (C) The marginal sinus of the PALN was filled with green and yellow dye (n = 3). (a) The dyes had spread into the cortex. (b) The square region in (a). H&E staining. Cor: cortex. Scale bar: (a) 100 μm, (b) 20 μm. (D) Metastatic tumor cells formed in the marginal sinus of the PALN on day 9 after inoculation of cells into the SiLN (n = 3). Tumor (T) was detected in the marginal sinus of the PALN. H&E staining. (a) India ink was delivered into the marginal sinus of the PALN. (b) The square region in (a). India ink was delivered below the tumor. Cap: capsule; Cor: cortex. Scale bar: (a) 100 μm, (b) 50 μm.