Pulsed high intensity focused ultrasound increases penetration and therapeutic efficacy of monoclonal antibodies in murine xenograft tumors

Abstract

The success of radioimmunotherapy for solid tumors remains elusive due to poor biodistribution and insufficient tumor accumulation, in part, due to the unique tumor microenvironment resulting in heterogeneous tumor antibody distribution. Pulsed high intensity focused ultrasound (pulsed-HIFU) has previously been shown to increase the accumulation of (111)In labeled B3 antibody (recognizes Lewis(y) antigen). The objective of this study was to investigate the tumor penetration and therapeutic efficacy of pulsed-HIFU exposures combined with (90)Y labeled B3 mAb in an A431 solid tumor model. The ability of pulsed-HIFU (1 M Hz, spatial averaged temporal peak intensity=2685 W cm(-2); pulse repetition frequency=1 Hz; duty cycle=5%) to improve the tumor penetration and therapeutic efficacy of (90)Y labeled B3 mAb ((90)Y-B3) was evaluated in Le(y)-positive A431 tumors. Antibody penetration from the tumor surface and blood vessel surface was evaluated with fluorescently labeled B3, epi-fluorescent microscopy, and custom image analysis. Tumor size was monitored to determine treatment efficacy, indicated by survival, following various treatments with pulsed-HIFU and/or (90)Y-B3. The pulsed-HIFU exposures did not affect the vascular parameters including microvascular density, vascular size, and vascular architecture; although 1.6-fold more antibody was delivered to the solid tumors when combined with pulsed-HIFU. The distribution and penetration of the antibodies were significantly improved (p-value<0.05) when combined with pulsed-HIFU, only in the tumor periphery. Pretreatment with pulsed-HIFU significantly improved (p-value<0.05) survival over control treatments.

Fig. 1

Tumor microvascular and antibody accumulation analysis of A431 tumors following treatment with HIFU and Alexa-647-B3. A) Tumor microvascular density (MVD) indicating overall spatial density of tumor microvasculature. B) Median distance from a tumor cell to the nearest vascular surface that describes overall microvascular architecture with smaller distances more efficient for transport. C) Blood vessel area to indicate blood vessel size, and D) Overall antibody intensity in the entire tumor section demonstrating the accumulation of antibody. Data are presented as individual data points and the bar represents the (n = 4–6).

Fig. 2

Fluorescence images of antibody distribution. Alexa-647-B3 antibody (green), blood vessels (red), and nuclei (blue) are shown for each group as a whole tumor (left column) and with higher magnification (right column). The white box corresponds to the region displayed in the right column. The Alexa-647-B3 antibody channel was acquired and displayed with consistent levels, while the blood vessels and nuclei are displayed to maximize contrast.

Fig. 3

Antibody penetration from the tumor edge (A) and blood vessel surface (B). Data are mean±SEM shown as a dashed line (A) or error bars (B), n=4–6. Control and HIFU are significantly different from the tumor edge up to 50 μm.

Fig. 4

Tumor growth (A) and survival (B) shown as a Kaplan–Meier plot. A) Tumor growth is delayed by treatment with ⁹⁰Y-B3 and ⁹⁰Y-B3+HIFU (mean±SD). B) Survival is extended by treatment with ⁹⁰Y-B3+HIFU versus all other groups (p-value < 0.05).