Direct Delivery of a Cytotoxic Anticancer Agent into the Metastatic Lymph Node Using Nano/Microbubbles and Ultrasound

<div><p>Direct injection of an anticancer agent into a metastatic lymph node (LN) has not been used as a standard treatment because evidence concerning the efficacy of local administration of a drug into a metastatic LN has not been established. Here we show that the combination of intralymphatic drug delivery with nano/microbubbles (NMBs) and ultrasound has the potential to improve the chemotherapeutic effect. We delivered <i>cis</i>-diamminedichloroplatinum (II) (CDDP) into breast carcinoma cells <i>in vitro</i> and found that apoptotic processes were involved in the antitumor action. Next, we investigated the antitumor effect of intralymphatic chemotherapy with NMBs and ultrasound in an experimental model of LN metastasis using MXH10/Mo-<i>lpr</i>/<i>lpr</i> mice exhibiting lymphadenopathy. The combination of intralymphatic chemotherapy with NMBs and ultrasound has the potential to improve the delivery of CDDP into target LNs without damage to the surrounding normal tissues. The present study indicates that intralymphatic drug delivery with NMBs and ultrasound will potentially be of great benefit in the clinical setting.</p></div

Histopathology analysis.

<p>(A) Representative sections stained with HE are shown for (Aa) control, (Ab) US + ALs, (Ac) CDDP + ALs, and (Ad) CDDP + ALs + US groups. Panels (Ae) and (Af) show higher magnification views of the regions highlighted in panels (Aa) (white dotted square) and (Ad) (black dotted square), respectively. CD31-immunostained sections are shown for the control (Ag) and CDDP + ALs + US (Ah) groups. Black dotted lines indicate carcinomatous lesions and white arrows indicate necrotic lesions. Black triangles indicate the border between normal tissue and necrotic areas. In the control and US + ALs groups, widespread cancerous lesions can be seen (Aa, b), while smaller lesions were evident in sections from the CDDP + ALs group (Ac). In sections from the CDDP + ALs + US group, carcinomatous lesions could not be detected, but necrotic lesions were observed (Ad, f). In the control group, CD31-positive vasculature was distributed over the entire lesion (Ag), whereas in the CDDP + ALs + US group CD31-positive vasculature was rarely observed in necrotic lesions (Ah). (B) Quantification of macrovessel and microvessel densities of tumor-bearing SiLNs. No significant differences in macrovessel and microvessel densities were detected between any of the experimental groups.</p

Blood vessel extraction images of tumor-bearing SiLNs.

<p>(A) Representative vascular extraction images for each group, taken from the widest cross-section of the tumor-bearing SiLN. The white circles indicate the boundaries of each SiLN. In the control, US + ALs and CDDP + ALs groups, no obvious changes in vessel structures were observed between days 0 and 9. In the CDDP + ALs + US group, the proportion of the tumor-bearing SiLN colored green in the vascular extraction image was lower on day 9 than on day 0. (B) Normalized blood vessel densities of tumor-bearing SiLNs (<i>n</i> = 4 for each group). Values at different time points were normalized to those on day 0. There was a trend for normalized blood vessel density on day 9 to be lower in the CDDP + ALs + US group than in the other 3 groups, although there were no significant differences between any of the groups. Mean ± SEM values are shown. Scale bars represent 2 mm.</p

Parameters related to the incidence of metastasis.

<p>A. Effects of inoculation conditions on the incidence of lymph node metastasis. Metastatic incidence increased with injection of a larger number cells (a), did not vary significantly with injection duration (b), but decreased with a larger SiLN volume (c). In the values presented above the bars, the denominator represents the number of inoculated mice while the numerator represents the number of mice with metastases in the proper-ALN. NS indicates P>0.05; *P<0.05 calculated by Fisher’s exact probability test. B. Assessment of the correlation between the above 3 parameters and metastasis incidence, using a new parameter, cells mm⁻³ min⁻¹. When metastasis incidence was set at 1, 100% metastasis was achieved when the cells mm⁻³ min⁻¹ value exceeded 4.72×10² (n = 39). Analyses were performed with the Mann-Whitney U test.</p

The expression levels of apoptosis-related genes.

<p>The mean fold-change in the expression level of each gene, relative to the control group, was measured for the US + ALs, CDDP + ALs and CDDP + US + ALs groups (<i>n</i> = 5 for each group). Expression levels of caspase-8 (a), caspase-9 (b), caspase-3 (c) and p53 (d) in FM3A cells. There was a trend towards higher expression of all 4 genes in the CDDP + ALs + US group compared with the other 3 groups. Mean ± SEM values are shown.</p

Establishment of the model of lymph node metastasis.

<p>A. Representative images captured by <i>in vivo</i> bioluminescence imaging of a mouse with tumor cells grafted into the SiLN to promote metastasis to the ALN. (<i>a</i>) <i>In vivo</i> and (<i>b</i>) <i>ex vivo</i> bioluminescence signals in the proper-ALN and SiLN on day 14 post-inoculation, indicating that the proper-ALN is the draining lymph node. B. Graph showing the high correlation between <i>in vivo</i> and <i>ex vivo</i> bioluminescence (<i>P</i> = 0.0023; Spearman’s rank correlation coefficient [rs]  = 0.9161; SiLN, <i>n</i> = 6; proper-ALN, <i>n</i> = 6). C. Results of histological verification. Tumor cells stained with H&E and luciferase-positive immunohistochemical signals in the proper-ALN and SiLN. MS: marginal sinus. T: tumor. D. Dissemination of KM-Luc/GFP cells (metastasis, <i>n = </i>4) or PBS alone (control, <i>n = </i>3) to each organ, assessed on day 14 post-injection of the SiLN. I, ipsilateral; C, contralateral; LN, lymph node. Error bars indicate the SEM values.</p

Metastatic flow and route. A.

<p>ICG flow from the SiLN to the proper-ALN, observed using an <i>in vivo</i> fluorescence imaging system (IVIS; <i>n</i> = 1). <b>B.</b> Representative PDE images, following an ICG injection speed of 0.5 mL/h. The speed of ICG flow was calculated by dividing the distance, <i>l</i>, by the duration of time that had elapsed post-injection. <b>C.</b> Graph of the relationship between ICG flow speed and intra-SiLN injection speed (low, 0.5; medium, 1.0; high, 3.0 mL/h; <i>n</i> = 4 per group), revealing a low level of variation between individual experiments in the low-speed group. D. HS-FVCS image of the afferent lymphatic vessels after intra-SiLN injection of FITC-BSA solution (<i>n</i> = 2). (<i>a</i>) Area near the SiLN and proper-ALN captured by a normal digital camera. Two regions of interest were selected. (<i>b</i>) Bright field images obtained by HS-FVCS, without use of a fluorescence filter. A thick superficial epigastric vein (→) was observed. (<i>c</i>) Fluorescence images obtained by HS-FVCS, with use of an appropriate fluorescence filter (bandwidth: 510±2 nm). A new flow channel filled with FITC-BSA solution (→) appeared at a distance of about 200 µm from the vein. (<i>d</i>) Results of hematoxylin and eosin (H&E) staining. The flow channel was identified as the afferent lymphatic vessels by injection of Indian ink. The vein was not stained (<i>n</i> = 1).</p

Monitoring of metastatic progression by CE-HFUS with Als.

<p>A. Imaging of the temporal changes in angiogenic vessel density in a cross-section of a metastatic proper-ALN, visualized by CE-HFUS with ALs. Blood vessel density increased with tumor progression. Red circles indicate the proper-ALN boundary, arrows and dotted lines indicate the AL-enhanced region, and green highlighting indicates the dense area of neovasculature in the proper-ALN. B. Results of 3D quantitative analysis of the temporal changes in blood vessel volume and density in metastatic proper-ALNs. The term “cells” indicates the metastasized group (1×10⁵ cells/min, <i>n</i> = 4) and “PBS” the negative control group (<i>n</i> = 3). Values for each group were normalized against the measurement on day 0. Error bars indicate the SEM values. * is for comparison of the temporal change within each group; # is for comparison between groups. * or #, <i>P</i><0.05; ** or ##, <i>P</i><0.01, calculated using two-way ANOVA followed by the Tukey-Kramer test.</p

Evaluation of the treatment efficacy of delivery of CDDP with ALs and US.

<p>(A) <i>In vivo</i> bioluminescence imaging on day 10. (B) Longitudinal analysis of normalized luciferase activity (<i>n</i> = 4 for each group). Values at different time points were normalized to those on day 3. Black arrows indicate the days on which treatment was administered (days 3, 4 and 7). On day 10, normalized luciferase activity in the CDDP + ALs + US group was significantly lower than that in the control group (<i>P</i> < 0.05) and showed a trend towards being lower than that in the US + ALs or CDDP + ALs group. Mean ± SEM values are shown. *<i>P</i> < 0.05.</p

<i>In vitro</i> cell viability.

<p>Normalized cell viability was obtained by dividing the value in each group by the mean value of the control samples (<i>n</i> = 6 for each group). (A) Normalized cell viability of FM3A cells. (B) Normalized cell viability of MH129F cells. (C) Normalized cell viability of FM3A cells when the level of CDDP was 0.5 μM. A significant difference in normalized cell viability between the CDDP + ALs and CDDP + ALs + US groups was demonstrated only at an US intensity of 1.0 W/cm². (D) Normalized cell viability of MH129F cells when the concentration of CDDP was 0.01 μM. There was a significant difference in normalized cell viability between the CDDP + ALs and CDDP + ALs + US groups at US intensities of 0.5 and 1.0 W/cm². Mean ± SEM values are shown. **<i>P</i> < 0.01.</p