Pre-clinical Evaluation of a Cyanine-Based SPECT Probe for Multimodal Tumor Necrosis Imaging

Purpose: Recently we showed that a number of carboxylated near-infrared fluorescent (NIRF) cyanine dyes possess strong necrosis avid properties in vitro as well as in different mouse models of spontaneous and therapy-induced tumor necrosis, indicating their potential use for cancer diagnostic- and prognostic purposes. In the previous study, the detection of the cyanines was achieved by whole body optical imaging, a technique that, due to the limited penetration of near-infrared light, is not suitable for investigations deeper than 1 cm within the human body. Therefore, in order to facilitate clinical translation, the purpose of the present study was to generate a necrosis avid cyanine-based NIRF probe that could also be used for single photon emission computed tomography (SPECT). For this, the necrosis avid NIRF cyanine HQ4 was radiolabeled with 111indium, via the chelate diethylene triamine pentaacetic acid (DTPA). Procedures: The necrosis avid properties of the radiotracer [111In]DTPA-HQ4 were examined in vitro and in vivo in different breast tumor models in mice using SPECT and optical imaging. Moreover, biodistribution studies were performed to examine the pharmacokinetics of the probe in vivo. Results: Using optical imaging and radioactivity measurements, in vitro, we showed selective accumulation of [111In]DTPA-HQ4 in dead cells. Using SPECT and in biodistribution studies, the necrosis avidity of the radiotracer was confirmed in a 4T1 mouse breast cancer model of spontaneous tumor necrosis and in a MCF-7 human breast cancer model of chemotherapy-induced tumor necrosis. Conclusions: The radiotracer [111In]DTPA-HQ4 possessed strong and selective necrosis avidity in vitro and in various mouse models of tumor necrosis in vivo, indicating its potential to be clinically applied for diagnostic purposes and to monitor anti-cancer treatment efficacy

Dual-Wavelength Imaging of Tumor Progression by Activatable and Targeting Near-Infrared Fluorescent Probes in a Bioluminescent Breast Cancer Model

Bioluminescence imaging (BLI) has shown its appeal as a sensitive technique for in vivo whole body optical imaging. However, the development of injectable tumor-specific near-infrared fluorescent (NIRF) probes makes fluorescence imaging (FLI) a promising alternative to BLI in situations where BLI cannot be used or is unwanted (e.g., spontaneous transgenic tumor models, or syngeneic mice to study immune effects)

The Necrosis-Avid Small Molecule HQ4-DTPA as a Multimodal Imaging Agent for Monitoring Radiation Therapy-Induced Tumor Cell Death

Purpose: Most effective antitumor therapies induce tumor cell death. Non-invasive, rapid and accurate quantitative imaging of cell death is essential for monitoring early response to antitumor therapies. To facilitate this, we previously developed a biocompatible necrosis-avid near-infrared fluorescence (NIRF) imaging probe, HQ4, which was radiolabeled with "'Indium-chloride el In-C13) via the chelate diethylene triamine pentaacetic acid (DTPA), to enable clinical translation. The aim of the present study was to evaluate the application of HQ4-DTPA for monitoring tumor cell death induced by radiation therapy. Apart from its NIRF and radioactive properties, HQ4-DTPA was also tested as a photoacoustic imaging probe to evaluate its performance as a multimodal contrast agent for superficial and deep tissue imaging. Materials and methods: Radiation-induced tumor cell death was examined in a xenograft mouse model of human breast cancer (MCF-7). Tumors were irradiated with three fractions of 9 Gy each. HQ4-DTPA was injected intravenously after the last irradiation, NIRF and photoacoustic imaging of the tumors were performed at 12, 20, and 40 h after injection. Changes in probe accumulation in the tumors were measured in vivo, and ex vivo histological analysis of excised tumors was performed at experimental endpoints. In addition, biodistribution of radiolabeled [In]DTPA-HQ4 was assessed using hybrid single-photon emission computed tomography computed tomography (SPECT CT) at the same time points. Results: In vivo NIRF imaging demonstrated a significant difference in probe accumulation between control and irradiated tumors at all time points after injection. A similar trend was observed using in vivo photoacoustic imaging, which was validated by ex vivo tissue fluorescence and photoacoustic imaging. Serial quantitative radioactivity measurements of probe biodistribution further demonstrated increased probe accumulation in irradiated tumors. Conclusion: HQ4-DTPA has high specificity for dead cells in vivo, potentiating its use as a contrast agent for determining the relative level of tumor cell death following radiation therapy using NIRF, photoacoustic imaging and SPECT in vivo. Initial preclinical results are promising and indicate the need for further evaluation in larger cohorts. If successful, such studies may help develop a new multimodal method for non-invasive and dynamic deep tissue imaging of treatment-induced cell death to quantitatively assess therapeutic response in patients

4T1-luc2 tumor growth curve indicated by BLI and FLI.

<p>During tumor progression, from day 4 to day 18, there was a steady increase in both BLI and FLI signal intensities.</p

Immunohistological and fluorescent analysis of tumor sections.

<p>The upper panels display sections stained with antibodies against EGFR (A), GLUT-1 (B), MMP-9 (C) and CathepsinB (D). The lower panels show the corresponding sections with FLI signals from animals injected with IRDye 800CW EGF (E), IRDye 800CW 2-DG (F), MMPSense680 (G) or ProSense680 (H). Scale bar = 2 mm.</p

<i>In vivo</i> tumor cell detection limit studies.

<p>Different numbers of 4T1-luc2 cells were implanted orthotopically into nude mice. 1×10⁵, 5×10⁴, 2.5×10⁴ and 1.25×10⁴ cells were implanted at the upper left, upper right, lower left and lower right MFPs, respectively. BLI measurements at day 1 and 4 are shown in panels A and D. The corresponding IRDye CW800 EGF FLI measurements are shown in panels B and E, respectively. Quantitative analysis of the BLI and FLI measurements using both IRDye CW800 EGF and Prosense680 are shown in panels C and F.</p

<i>In vitro</i> NIRF probe activation/binding assays.

<p>4T1-luc2 breast cancer cells were seeded in 96-well plates and incubated with increasing concentrations of Prosense680 or MMPSense680 (A) and IRDye 800CW EGF or IRDye 800CW 2-DG (B) and were subsequently imaged. Intracellular accumulation of Prosense680 (45nM; 24 h incubation) was visualized by confocal microscopy (C). Intracellular accumulation of IRDye 800CW 2-DG (10 µM; 2 h incubation) was visualized by the Nuance multispectral imaging system (D). NIRF probes, cell nuclei and membranes are indicated in red, blue and green, respectively.</p

TBRs of 4T1-luc2 tumors exploiting four different NIRF probes.

<p>The TBRs, measured on the final day of the experiment, for ProSense680, IRDye 800CW 2-DG, MMPSense680 and IRDye 800CW EGF ranged from 2.29±0.38 to 4.02±0.24.</p

Schematic representation of the localization of the signal intensity areas used to calculate TBRs.

<p>TBRs were calculated by dividing the mean fluorescence intensity of four areas, of the same size, located inside the tumor region (1–4) by the mean fluorescence intensity of four areas located approximately 5 mm outside the tumor region (background fluorescence) (5–8).</p

<i>In vitro</i> tumor cell detection limit studies.

<p>4T1-luc2 cells were seeded in 96-well plates with various initial densities (39 to 2×10⁴ cells per well). The next day, cells were used for BLI measurement or incubated with either Prosense680 or with IRDye 800CW EGF and then used for FLI measurements. Statistic significance was calculated by determining the difference between optical signals from wells with and without cells. The star indicates the first significant difference. * = P<0.05; *** = P<0.0001.</p