Lymph node effective vascular permeability and chemotherapy uptake

Abstract

Objective: Lymph node metastases are a poor prognostic factor. Additionally, responses of lymph node metastasis to therapy can be different from the primary tumor. Investigating the physiologic lymph node blood vasculature might give insight into the ability of systemic drugs to penetrate the lymph node, and thus into the differential effect of therapy between lymph node metastasis and primary tumors. Here, we measured effective vascular permeability of lymph node blood vessels and attempted to increase chemotherapy penetration by increasing effective vascular permeability.

Methods: We developed a novel three-dimensional method to measure effective vascular permeability in murine lymph nodes in vivo. VEGF-A was systemically administered to increase effective vascular permeability. Validated high-performance liquid chromatography protocols were used to measure chemotherapeutic drug concentrations in untreated and VEGF-A-treated lymph nodes, liver, spleen, brain, and blood.

Results: VEGF-A-treated lymph node blood vessel effective vascular permeability (mean 3.83 × 10^-7 cm/s) was significantly higher than untreated lymph nodes (mean 9.87 × 10^-8 cm/s). No difference was found in lymph node drug accumulation in untreated versus VEGF-A-treated mice.

Conclusions: Lymph node effective vascular permeability can be increased (~fourfold) by VEGF-A. However, no significant increase in chemotherapy uptake was measured by pretreatment with VEGF-A.

Keywords: chemotherapy; drug penetration; intravital microscopy; lymph node; vascular permeability.

FIGURE 1

Blood vessel effective vascular permeability. (A) Intravital multiphoton microscopy demonstrates the presence of FITC-BSA in lymph node blood vasculature of a BALB/c mouse ~ 30 seconds after intravenous injection. (B) Image of the same location as A, ~14 minutes later shows FITC-BSA extravasation is most apparent in left side. C–D) Images at 30 s (C) and 14 min (D) after injection show extravasation of FITC-BSA in the interstitial space after VEGF-A treatment in different BALB/c mouse with extravasation most apparent in the top right corner. (E) EVP measurements performed in normal, histamine-treated and VEGF-A-treated lymph nodes, as well as subcutaneous blood vessels. Mean and SEM are reported. ** = P<.01 using Dunn’s multiple comparisons test (ANOVA)

FIGURE 2

Chemotherapeutic drug concentrations in normal and VEGF-A treated lymph nodes. This figure shows data for all measured chemotherapeutic drugs (5-FU, paclitaxel, cisplatin, and doxorubicin) at 2 hours and 4 h after drug injection, with and without VEGF-A treatment. N=4 per group, mean, and SEM are reported. * = P<.05 using an unpaired nonparametric Mann-Whitney t test or Dunn’s multiple comparisons test (ANOVA)