Fast and Durable Intraoperative Near-infrared Imaging of Ovarian Cancer Using Ultrabright Squaraine Fluorophores

Abstract

The residual tumor after surgery is the most significant prognostic factor of patients with epithelial ovarian cancer. Near-infrared (NIR) fluorescence-guided surgery is actively utilized for tumor localization and complete resection during surgery. However, currently available contrast-enhancing agents display low on-target binding, unfavorable pharmacokinetics, and toxicity, thus not ideal for clinical use. Here we report ultrabright and stable squaraine fluorophores with optimal pharmacokinetics by introducing an asymmetric molecular conformation and surface charges for rapid transporter-mediated cellular uptake. Among the tested, OCTL14 shows low serum binding and rapid distribution into cancer tissue via organic cation transporters (OCTs). Additionally, the charged squaraine fluorophores are retained in lysosomes, providing durable intraoperative imaging in a preclinical murine model of ovarian cancer up to 24 h post-injection. OCTL14 represents a significant departure from the current bioconjugation approach of using a non-targeted fluorophore and would provide surgeons with an indispensable tool to achieve optimal resection.

Keywords: Intraoperative Near-Infrared Fluorescence Imaging; Lysosome; Organic Cation Transporter; Ovarian Cancer; Squaraine.

Figure 1.

Chemical structures, physicochemical properties, and optical properties of the functionalized squaraines. (a) Chemical structures of the five asymmetric squaraine derivatives. (b) Quantitative calculation of physicochemical properties of the squaraine fluorophores. (c) Optical properties of the squaraine fluorophores in phosphate-buffered saline (PBS, pH 7.4) with 5% bovine serum albumin (BSA). MW, molecular weight; TPSA, topological polar surface area; HBD, hydrogen bond donors; HBA, hydrogen bond acceptors; Ext. Coeff., extinction coefficient; QY, quantum yield; Abs, absorbance; Fl, fluorescence.

Figure 2.. Tumor cell targetability and cytotoxicity of squaraines.

Murine and human ovarian cancer cell lines including ID8 cells, CAOV3 and SKOV3 cells, NIH3T3 fibroblast, and C2C12 muscle cells were cultured and incubated at 37°C for 30 min in 10% or 0% FBS media in the presence of 2 μM of OCTL12, OCTL13, OCTL14, OCTL15, and OCTL16 and imaged using an epifluorescence microscope. (a-d) Representative fluorescence images of cells in (a) 10% or (c) 0% FBS media. Scale bar = 100 μm. Quantitative measurements of the fluorescence intensity of cells in (b) 10% and (d) 0% FBS. The relative mean fluorescence intensity (MFI) of cells in each group was calculated by comparing the highest OCTL16 group of C2C12 cells in the presence of serum. *P < 0.05 by one-way ANOVA followed by Tukey’s multiple comparisons test (n = 3, mean ± s.e.m.). (e) Cytotoxicity of squaraines. Cells were treated with 2, 5, 10, and 20 μM of each compound for 24 h, followed by an assessment of cell viability using the Cell Counting Kit-8 (CCK-8). *P < 0.05 by two-way ANOVA followed by Tukey’s multiple comparisons test (n = 3, mean ± s.e.m.).

Figure 3.

In vivo evaluation of tumor cell targetability of squaraines in a mouse model of ovarian cancer. (a) Mice were injected with 100 nmol of each squaraine fluorophore 6 h prior to imaging. Scale bars = 5 mm. (b) Quantitative analysis of target-to-background ratio (TBR) of peritoneal tumors for each squaraine fluorophore (n = 3, mean ± s.e.m.). *P < 0.05 by one-way ANOVA followed by Tukey’s multiple comparisons test. (c) Tumor targetability and biodistribution of OCTL14. Intestine and liver were covered with black paper to block fluorescent signals from these organs. The graph shows the SBR of resected organs and tumors. Scale bar = 1 cm. (d) Pharmacokinetics of OCTL14. 100 nmol of OCTL14 was injected intravenously into CD-1 mice. Blood concentration (%ID/g) decay curve and distribution (t_1/2α) and elimination half-life (t_1/2β) values are shown (n = 3, mean ± s.e.m.). Urine samples were collected 4 h post-injection. %ID/g, percent injected dose per gram; AUC, the area under the curve. (e) Dose-response curve of OCTL14. Tumor-bearing mice were intravenously injected with 25, 50, 100, and 150 nmol of OCTL14. TBR was determined for tumor 6 h post-injection by NIR imaging. (f) Quantitative time-course assessment of TBR. Tumor-bearing mice were intravenously injected with 100 nmol of OCTL14. TBR was observed at 3, 10, 30, 60, 120, 240, 360, 480, and 600 min and 24 h post-injection by NIR imaging (n = 3, mean ± s.e.m.). The blue window shows the duration in which significantly higher TBR was obtained compared to that at 3 min. *P < 0.05 by one-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 4.

The mechanism of cellular uptake for OCTL14. (a) Histological analysis of OCTL14 injected tumor. Hematoxylin and Eosin (H&E), NIR fluorescence, and merged images of resected tumors are shown. Scale bars = 200 μm. (b) Inhibition assay of cellular uptake of OCTL14 in ID8 cells. Cells were pre-blocked with bromsulphthalein (BSP), cyclosporin A, MK-571, D22, 2DG, oligomycin, Dyngo 4a, Pitstop 2 for 5-30 min or incubated and then incubated with 2 μM of OCTL14 for 30 min. Alternatively, cells were incubated with 2 μM of OCTL14 at 4 °C for 30 min. Scale bar = 100 μm. *P < 0.05 by one-way ANOVA followed by Tukey’s multiple comparisons test (n = 3, mean ± s.e.m.). (c) Immunohistochemical analysis of OCTN1, OCTN2, OCT1, OCT2, OCT3, and OCT6 expression in the target tissue. Tumors and adnexa were stained with anti- OCTN1, OCTN2, OCT1, OCT2, OCT3, and OCT6, followed by appropriate secondary antibody using avidin-biotin complex/diaminobenzidine histochemistry. Scale bar = 50 μm. (d) Subcellular localization of OCTL14 (10 μM) in ID8 cells was determined after 30 min of incubation at 37 °C. Nuclei, mitochondria, or lysosomes were stained with NucBlue™, MitoTracker Green FM, or LysoTrackerTM Red DND-99 L7528 and imaged using an epifluorescence microscope. Scale bar = 20 μm. (e) Co-localization index of OCTL14 with MitoTracker Green or LysoTracker Red. The index was calculated by dividing the area of overlap between OCTL14 and MitoTracker or LysoTracker by the total area of MitoTracker or LysoTracker, respectively. The Co-localization index was determined in 6 photographic areas (70 × 92 μm² each) for each probe (n = 3 ± s.d.). ****P < 0.0001 by two-tailed Student’s t-test.

Figure 5.

Intraoperative imaging of OCTL14 for FGS. (a) FGS using the squaraine. Tumor-bearing mice were intravenously injected with 100 nmol of OCTL14. FGS was performed 6 h post-injection. The yellow circle indicates the lesions for which intraoperative image-guided surgeries were performed. The middle panels show the tumor locations before/after surgery and resected tumors of different sizes. Scale bars = 1 cm. (b) Clinical impact of the use of OCTL14. The number of tumors detected using color images only was compared to those detected using NIR imaging by experienced surgeons (n = 3, mean ± s.e.m.). *P < 0.05 by two-way ANOVA followed by Tukey’s multiple comparisons test. (c) Dual imaging of tumors and ureters. 100 nmol of OCTL14 and 25 nmol of ZW800-PEG were injected intravenously into tumor mice 6 h and 0.5 h prior to imaging, respectively. The 700-nm channel image shows the accumulation of OCTL14 in tumors, while the 800-nm channel image shows the ureteral signals. Arrows indicate the tumor locations. In color–NIR merged image, 700-nm fluorescence (OCTL14) and 800-nm fluorescence (ZW800-PEG) were pseudo-colored red and lime green, respectively, and overlaid onto the color image in real-time. Scale bars = 5 mm. (d) Quantitative time-course assessment of SBR for the ureters (n = 3, mean ± s.e.m.). Scale bar = 5 mm. (e) A summary schematic of the study. A squaraine fluorophore displays ultrabright optical properties and optimal pharmacokinetics, allowing high contrast and durable near-infrared imaging for fluorescence-guided surgery of ovarian cancer. The primary mechanisms of the tumor targetability of OCTL14 involve its rapid diffusion across tumor vasculature and cellular uptake via organic cation transporters and retention in the lysosome.