Real-time monitoring of in vivo acute necrotic cancer cell death induced by near infrared photoimmunotherapy using fluorescence lifetime imaging

Abstract

A new type of monoclonal antibody (mAb)-based, highly specific phototherapy (photoimmunotherapy; PIT) that uses a near infrared (NIR) phthalocyanine dye, IRDye700DX (IR700) conjugated with a mAb, has recently been described. NIR light exposure leads to immediate, target-selective necrotic cell death in vitro. Detecting immediate in vivo cell death is more difficult because it takes at least 3 days for the tumor to begin to shrink in size. In this study, fluorescence lifetime (FLT) was evaluated before and after PIT for monitoring the immediate cytotoxic effects of NIR mediated mAb-IR700 PIT. Anti-epidermal growth factor receptor (EGFR) panitumumab-IR700 was used for targeting EGFR-expressing A431 tumor cells. PIT with various doses of NIR light was conducted in cell pellets in vitro and in subcutaneously xenografted tumors in mice in vivo. FLT measurements were obtained before and 0, 6, 24, and 48 hours after PIT. In vitro, PIT at higher doses of NIR light immediately led to FLT shortening in A431 cells. In vivo PIT induced immediate shortening of FLT in treated tumors after a threshold NIR dose of 30 J/cm(2) or greater. In contrast, lower levels of NIR light (10 J/cm(2) or smaller) did not induce shortening of FLT. Prolongation of FLT in tissue surrounding the tumor site was noted 6 hours after PIT, likely reflecting phagocytosis by macrophages. In conclusion, FLT imaging can be used to monitor the acute cytotoxic effects of mAb-IR700-induced PIT even before morphological changes can be seen in the targeted tumors.

Figure 1

Samples of IR700-conjugated Panitumumab (Pan-IR700) at concentrations of 2.5, 5, 20, and 40 µg/mL were prepared by diluting with PBS. (A) Fluorescence intensity image of Pan-IR700 solution: Fluorescence intensities were decreased according to decrease of concentration of Pan-IR700. (B) Fluorescence lifetime (FLT) image of Pan-IR700 solution: The FLT at various concentrations of Pan-IR700 solutions was almost the same value, 3.56 +/− 0.081 ns; 3.62 (2.5 µg/mL), 3.58 (5 µg/mL), 3.44 (20 µg/mL), 3.60ns (40 µg/mL). (C) LED light-irradiation for A431 cell pellets changes FLTs. A431 cell line incubated with Pan-IR700 for 24 hours were treated with PIT at doses of 0, 8, 15 and 30 J/cm². FLT shortened to 3.09, 2.94 and 2.85ns, compared with 3.28ns before light exposure. These represented shortenings of 9.1, 10.1 and 13.1%, respectively. (D) FLT of A431 pellets depends on the incubation time with Pan-IR700. FLT values escalate with incubation time with Pan-IR700. FLT changes from 2.98ns (1 hour) to 3.42ns (24 hours).

Figure 2

Serial fluorescence (lower) and differential interference contrast (DIC) microcopic images (upper) of A431 cells, which were pre-incubated with Pan-IR700 (10 µg/mL) at 37°C for 24 h, 5, 15, 60 and 90 sec after start exposing NIR light. . (Pan-IR700 gradually internalized into cytoplasm in A431 cells after bound to cell membrane up to 24h post-injection.) Morphological changes on DIC become severer by exposing more dose of NIR light. Scale bars, 50 µm.

Figure 3

Comparison with FLTs of irradiated tumors (dark gray bar) and non-irradiated tumors (bright gray bar). (A) FLT images before and after PIT at the dose of 10, 30, 50 J/cm² in the same mouse which was inoculated with A431 cells on both sides of the mouse dorsum. Right-sided tumor was treated by PIT whereas the left was covered. FLTs of A431 tumors treated with PIT with 50 J/cm² (B), 30 J/cm² (C) and 10 J/cm², (D) were plotted. PIT with the NIR light dose of 30 and 50 J/cm² demonstrated significant (P<0.05) shortenings in FLT immediately, compared with non-irradiated tumors. However, FLT did not significantly shorten at a low dose of 10 J/cm². Transient prolongations of FLTs were observed at 6 hours after PIT likely due to uptake by reactive macrophages. Mann-Whitney's U test was used for the statistical analysis.

Figure 4

(A) FLT in PIT treated tumors with 50 and 30 J/cm² shortened significantly (p < 0.01) compared with no treatment control (0 J/cm²) (control). FLTs were immediately shortened to 69.1 +/− 10.9% and 61.5 +/− 5.1 % by PIT with 50 and 30 J/cm², respectively. A431 tumors irradiated with only 10 J/cm² showed no significant shortens of FLT were seen immediately. FLT shortened by only 7.7% at 48 hours after PIT compared with no treatment control. (B) FLT of non-irradiated tumors in PIT treated mice shortened slightly more than that in the untreated mice over time, but these changes were not significant. They may be caused by a small amount of light diffusing through tissue from the irradiated side, even though the surface of the tumor was covered. Student’s t test was used for the statistical analysis. (C) Histological specimens of A431 tumors, which were treated with PIT at 0, 10, 30, and 50 J/cm², are shown. All specimens are stained with Hematoxylin and Eosin. Microscopic evaluation of treated tumors revealed various degrees of necrosis and micro-hemorrhage with clusters of healthy or damaged tumor cells after PIT. Necrotic damage was diffuse and intense and fewer surviving tumor cells are seen, when 30 and 50 J/cm² of NIR light was administered. In contrast, when only 10 J/cm² of NIR light was administered, necrotic cell damage was found in only limited areas within the tumor while substantial amounts of healthy cancer foci remained. Scale indicates 50µm.