Multimodality imaging of tumor response to doxil

Abstract

Purpose: Early assessment of tumor responses to chemotherapy could enhance treatment outcomes by ensuring that, from the beginning, treatments meet the individualized needs of patients. In this study, we applied multiple modality molecular imaging techniques to pre-clinical monitoring of early tumor responses to Doxil, focusing on imaging of apoptosis.

Methods: Mice bearing UM-SCC-22B human head and neck squamous cancer tumors received either PBS or 1 to 2 doses of Doxil® (doxorubicin HCl liposome injection) (10 mg/kg/dose). Bioluminescence signals from an apoptosis-responsive reporter gene were captured for apoptosis evaluation. Tumor metabolism and proliferation were assessed by( 18)F-FDG and 3'-(18)F-fluoro-3'-deoxythymidine ((18)F-FLT) positron emission tomography. Diffusion-weighted magnetic resonance imaging (DW-MRI) was performed to calculate averaged apparent diffusion coefficients (ADCs) for the whole tumor volume. After imaging, tumor samples were collected for histological evaluation, including terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), anti-CD31, and Ki-67 immunostaining.

Results: Two doses of Doxil significantly inhibited tumor growth. Bioluminescence imaging (BLI) indicated apoptosis of tumor cells after just 1 dose of Doxil treatment, before apparent tumor shrinkage. (18)F-FDG and (18)F-FLT PET imaging identified decreased tumor metabolism and proliferation at later time points than those at which BLI indicated apoptosis. MRI measurements of ADC altered in response to Doxil, but only after tumors were treated with 2 doses. Decreased tumor proliferation and increased apoptotic cells were confirmed by changes of Ki-67 index and apoptotic ratio.

Conclusion: Our study of tumor responses to different doses of Doxil demonstrated that it is essential to combine apoptosis imaging strategies with imaging of other critical biological or pathological pathways, such as metabolism and proliferation, to improve clinical decision making in apoptosis-related diseases and interventions.

Keywords: Apoptosis; Cancer Therapy; Doxil; Multimodality Imaging; Response Monitoring.

Figure 1

(A) Antitumor activity of Doxil in UM-SCC-22B tumor model. The mice were implanted subcutaneously with UM-SCC-22B cells expressing apoptosis-responsive cyclic firefly luciferase (cFluc). Two doses of Doxil (10 mg/kg on day 1, and day 3, i.v.) inhibited tumor growth. (B) In vivo bioluminescence imaging of tumors treated with Doxil (10 mg/kg) (top) or PBS (bottom) in the UM-SCC-22B tumor bearing mice. (C) Quantification of tumor BLI intensity (**, p < 0.01).

Figure 2

(A) Representative decay-corrected whole-body coronal microPET images of mice bearing UM-SCC-22B tumors at 1 h after intravenous injection of ¹⁸F-FDG (1.85 MBq/mouse) after Doxil or PBS treatment. The tumors are indicated by arrows. (B) Tumor uptake of ¹⁸F-FDG quantified by ROI analysis (**, p < 0.01).

Figure 3

(A) Representative decay-corrected whole-body coronal microPET images of mice bearing UM-SCC-22B tumors at 1 h after intravenous injection of ¹⁸F-FLT (1.85 MBq/mouse) after Doxil or PBS treatment. The tumors are indicated by arrows. (B) Tumor uptake of ¹⁸F-FLT quantified by ROI analysis (*, p < 0.05; **, p < 0.01).

Figure 4

(A) Representative T1WI, T2WI and ADC maps of tumors treated with PBS, 1 dose or 2 doses of Doxil. (B) Tumor ADC quantified by region-of-interest (ROI) analysis (**, p < 0.01).

Figure 5

Histologic analyis of tumor response to Doxil. Tumor samples treated with 2 doses of Doxil (10 mg/kg) or PBS were harvested for histological analysis. (A) Immunofluorescence staining of CD31and fluorescence from Doxorubicin in Doxil treated tumors. (B) Proliferation of UM-SCC-22B tumor cells after Doxil treatment as assessed by Ki-67 immunofluorescence staining. (C) Ki67 positive cells were counted and the Ki-67 staining index (SI) was calculated by plotting Ki-67 positive cell number against total cell number. A significant decrease in Ki67 SI was observed after Doxil treatment, compared to the control. Three samples were used from each group, and results were confirmed with a duplicate experiment. (D) TUNEL staining of UM-SCC-22B tumor section after treatment with Doxil. Apoptotic nuclei are shown in green. Normal cell nuclei are shown in blue, stained by DAPI. (E) Apoptotic cells are counted by positive TUNEL staining, and apoptotic ratio is calculated by plotting TUNEL positive cell number against total cell number. **, p < 0.01.