Biological Evaluation of a Fluorescent-Imaging Agent for Medullary Thyroid Cancer in an Orthotopic Model

Abstract

Context: The primary and definitive treatment of medullary thyroid cancer (MTC) is surgical resection. Recurrent or residual disease is typically a result of incomplete surgical removal.

Objective: Our objective is to develop a compound that assists in intraoperative visualization of cancer, which would have the potential to improve surgical cure rates and outcomes.

Results: We report the biological characterization of Compound-17, which is labeled with IRdye800, allowing fluorescent visualization of MTC mouse models. We found that the agent has high affinity for two human MTC cell lines (TT and MZ-CRC1) in vitro and in vivo. We further tested the affinity of the compound in a newly developed MTC orthotopic xenograft model and found that Compound-17 produces fluorescent signals within MTC-derived orthotopic xenografts in comparison with a sequence-jumbled control compound and surrounding normal tissues.

Conclusions: Compound-17 is a unique and effective molecule for MTC identification that may have therapeutic potential.

Figure 1.

(a) Increased fluorescence is proportional to compound concentration. A scrambled control (Compound-83a) does not show fluorescence, indicating this compound does not interact with MTC cells in vitro. This increase in fluorescence in micromolar range suggests activity as a cell-penetrating peptide. Results shown are the mean of three independent experiments. (b) In vitro fluorescence of Compound-17 in MTC cells. Imaging of individual MTC cells indicates that Compound-17 staining overlaps with mitochondria in MTC cells. Fluorescence from Compound-83a cannot be detected in MTC cells (bottom).

Figure 2.

(a) Fluorescence of Compound-17 in MTC flank subcutaneous xenograft models. Forty-eight hours after injection, mice were euthanized, and an image was taken after skin removal (upper panel). The images were obtained on CRi Maestro white light excitation imager, as described in Materials and Methods. (b) Increased concentration of Compound-17 was observed in tumor (lower left) in comparison with muscle tissue. The highest concentration in organs was observed in kidneys (lower right panel), although some fluorescence could be observed in the lung and gastrointestinal tract. The images were obtained with FluobeamTM 800 NIR imaging system, as described in Materials and Methods.

Figure 3.

(a) TT orthotopic xenograft in situ. (b) TT orthotopic xenograft ex vivo. (c) MZ-CRC1 orthotopic xenograft in situ. (d) MZ-CRC1 orthotopic xenograft ex vivo. (e) Volumes of TT orthotopic xenografts as calculated by US imaging. (f) Volume comparison of TT orthotopic xenografts by US at 9 weeks and by caliper measurement at necropsy. (g) Volumes of MZ-CRC1 orthotopic xenografts as calculated by US imaging. (h) Volume comparison of MZ-CRC1 orthotopic xenografts by US at 9 weeks and by caliper measurement at necropsy.

Figure 4.

(a) Histological examination of orthotopic xenografts. Orthotopic xenografts developing from both MTC cell lines grow intrathyroidal (TT, 166 and 168; MZ-CRC1, 855) and adjacent to the thyroid (TT, 167; MZ-CRC1, 022 and 041). Calcitonin staining confirms origin of tumors as calcitonin-producing TT or MZ-CRC1 cells. Insets show that follicular cells are negative for calcitonin staining in the remaining normal tissue. Arrows within insets indicate unstained follicular cells. (b) Calcitonin concentration is increased in mice bearing MTC orthotopic xenografts. Wilcoxon rank test comparison showed increased serum calcitonin levels of orthotopic xenograft-bearing mice compared to controls. Necropsy tumor volumes also correlated with calcitonin levels. Spearman correlation was moderately positive for MZ-CRC1 tumors (0.57) and strongly positive for TT (0.8).

Figure 5.

High fluorescence observed in TT and MZ-CRC1 orthotopic xenografts. A total of 40 nmol Compound-83a (left, scramble control) or Compound-17 was injected by tail vein. The images were obtained following animal euthanasia and exposure of the orthotopic xenograft by neck incision 48 hours after injection. Images were obtained on CRi Maestro white light excitation imager, as described in Materials and Methods. Compound-17 allows imaging of orthotopic xenografts, whereas little to no fluorescence is observed in scrambled control or surrounding tissues of Compound-17–injected mice. The bottom panel includes bright field images of the orthotopic xenografts in situ following fluorescence imaging. White arrows indicate the location of the xenograft.