The effect of photoimmunotherapy followed by liposomal daunorubicin in a mixed tumor model: a demonstration of the super-enhanced permeability and retention effect after photoimmunotherapy

Abstract

In general, de novo solid tumors are composed of phenotypically and functionally heterogeneous malignant cells. This heterogeneity interferes with the effectiveness of targeted molecular cancer therapies. Even if most of the tumor is killed by a targeted treatment, recurrences are common and can be lethal. In this study, a mixed tumor model, which is predominantly a population of epidermal growth factor receptor (EGFR)-positive A431 cells combined with a smaller population of EGFR-negative Balb3T3/DsRed cells, was established. This mixed tumor was then treated with photoimmunotherapy, a newly developed target-cell-selective cancer therapy using a monoclonal antibody (mAb)-photosensitizer (IR700 fluorescence dye) conjugate and exposure of near-infrared light. Although photoimmunotherapy successfully treated EGFR-positive A431 cells in the mixed tumor, EGFR-negative Balb/DsRed cells were not responsive. However, photoimmunotherapy also induced a large increase in tumor permeability, known as the super-enhanced permeability and retention (SUPR) effect, which allowed a 5-fold increase in the accumulation of a liposomal chemotherapy (DaunoXome) and resulted in more effective therapy than either photoimmunotherapy or liposomal daunorubicin alone. The liposomal daunorubicin, administered 1 hour after EGFR-targeted photoimmunotherapy, was homogeneously distributed, allowing delivery to tiny surviving nests of EGFR-negative Balb3T3/DsRed cells, resulting in prolonged survival of mice.

Figure 1

The mixed tumor model composed of EGFR-positive (A431) and EGFR-negative (Balb/DsRed) tumor cells. Pan-IR700 (green) was intravenously injected 1 d before PIT. Scattered Balb/DsRed tumor foci (red) were observed in A431 cells (Green) before PIT. After PIT, severe damage was shown only in EGFR-positive A431 tumors, and the proportion of Balb/DsRed increased with time after PIT. Balb/DsRed cells eventually almost replaced the entire tumor at 7 days after PIT. Histological evaluation before and after PIT. EGFR-targeted PIT induced selective intense necrosis of EGFR-positve A431 cells as indicated by hematoxylin and eosin (H&E) staining. In contrast, spindle shaped Balb/DsRed cells remain intact even after PIT.

Figure 2

Enhanced delivery of liposomal daunorubicin based on PIT-induced SUPR effects. (A) In vivo fluorescence images of Pan-IR700 (green). PIT was performed only on the right-side tumor with the left-side tumor serving as a control. (B) Liposomal daunorubicin (yellow) preferentially accumulated in PIT-treated mixed tumors (red circles) at 30 min post injection of liposomal daunorubicin, but did not accumulate in no NIR light exposure control tumors (blue circles) growing in the same mice. (C) Dynamic fluorescence intensity curves show that daunorubicin accumulated and was retained by PIT-treated tumors selectively. ‘Back’ indicates the fluorescence in the back representing background signal. Data are means ± s.e.m. (n=3). (D) Intratumoral distribution of daunorubicin. Daunorubicin was broadly and homogeneously distributed post-PIT encircling the surviving tumor cells (Balb/DsRed and A431). DsRed: red, DX (daunorubicin): yellow, IR700: green. Scale bars: 100 μm.

Figure 3

In vivo therapeutic effect induced by the combination of PIT and liposomal daunorubicin. (A) Tumor growth inhibition by a combination therapy of Pan-IR700-mediated PIT and liposomal daunorubicin in A431 and Balb/DsRed tumors. Data are mean ± s.e.m. n ≥ 10 mice in each group, ***p< 0.01, *p< 0.05 for treatment compared to DX-only groups and PIT-only groups. (B) Analysis using a KaplanMeier survival curve of the combination therapy of PIT and liposomal daunorubicin in A431 and Balb/DsRed tumors. n ≥ 10 mice in each group, ***p< 0.05 for treatment compared to the other control groups using a log-rank test with a Bonferroni’s correction for multiple comparisons.