Evaluation of optical imaging agents in a fluorescence-guided surgical model of head and neck cancer

Abstract

Background: Tumor proliferation often occurs from pathologic receptor upregulation. These receptors provide unique targets for near-infrared (NIR) probes that have fluorescence-guided surgery (FGS) applications. We demonstrate the use of three smart-targeted probes in a model of head and neck squamous cell carcinoma.

Methods: A dose escalation study was performed using IntegriSense750, ProSense750EX, and ProSense750FAST in mice (n = 5) bearing luciferase-positive SCC-1 flank xenograft tumors. Whole body fluorescence imaging was performed serially after intravenous injection using commercially available open-field (LUNA, Novadaq, Canada) and closed-field NIR systems (Pearl, LI-COR, Lincoln, NE). An ex vivo, whole-body biodistribution was conducted. Lastly, FGS was performed with IntegriSense750 to demonstrate orthotopic and metastatic disease localization.

Results: Disease fluorescence delineation was assessed by tumor-to-background fluorescence ratios (TBR). Peak TBR values were 3.3 for 1 nmol ProSense750EX, 5.5 for 6 nmol ProSense750FAST, and 10.8 for 4 nmol IntegriSense750 at 5.5, 3, and 4 d post administration, respectively. Agent utility is unique: ProSense750FAST provides sufficient contrast quickly (TBR: 1.5, 3 h) while IntegriSense750 produces strong (TBR: 10.8) contrast with extended administration-to-resection time (96 h). IntegriSense750 correctly identified all diseased nodes in situ during exploratory surgeries. Ex vivo, whole-body biodistribution was assessed by tumor-to-tissue fluorescence ratios (TTR). Agents provided sufficient fluorescence contrast to discriminate disease from background, TTR>1. IntegriSense750 was most robust in neural tissue (TTR: 64) while ProSense750EX was superior localizing disease against lung tissue (TBR: 13).

Conclusion: All three agents appear effective for FGS.

Keywords: Fluorescence imaging; Head and neck cancer; Optical guided surgery; Surgical oncology.

Figure 1

displays results from the dose-time ranging study. (A) Quantification of the tumor-to-background (TBR) mean fluorescence intensity for IntegriSense750 across 96h. (B) Illustrates visually the change in tumor-to-background fluorescence contrast for the 4nmol dose at 6h, 48h, and 96h. (C) Shows the temporal effect of ProSense750EX across 132h along with (D) qualitative images of the 1nmol dose at the 12h, 72h, and 132h time points. (E) TBR values for ProSense750FAST through 72h (F) Images from the 6nmol ProSense750FAST dose at 3h, 36h, and 72h post injection. Data are average TBR ± SD.

Figures 2

illustrate results from a 12 tissue, whole body, ex vivo biodistribution for IntegriSense750. (A) Qualitative assessment of the fluorescence signal produced in each of the 12 dissected tissues as well as tumor (left) for the 4nmol dose; bright field images detail tissue shape (right). (B) Quantification of mean fluorescence intensity per milligram tissue (MFI/mg) ± SD for each dose for all 12 dissected tissues as well as tumor. (C) Table of tumor-to-tissue MFI/mg ratios (TTR) ± SD for all 12 dissected tissues.

Figures 3

illustrate results from a 12 tissue, whole body, ex vivo biodistribution for ProSense750EX. (A) Qualitative assessment of the fluorescence signal produced in each of the 12 dissected tissues as well as tumor (left) for the 1nmol dose; bright field images detail tissue shape (right). (B) Quantification of mean fluorescence intensity per milligram tissue (MFI/mg) ± SD for each dose for all 12 dissected tissues as well as tumor. (C) Table of tumor-to-tissue MFI/mg ratios (TTR) ± SD for all 12 dissected tissues.

Figures 4

illustrate results from a 12 tissue, whole body, ex vivo biodistribution for ProSense750FAST. (A) Qualitative assessment of the fluorescence signal produced in each of the 12 dissected tissues as well as tumor (left) for the 6nmol dose; bright field images detail tissue shape (right). (B) Quantification of mean fluorescence intensity per milligram tissue (MFI/mg) ± SD for each dose for all 12 dissected tissues as well as tumor. (C) Table of tumor-to-tissue MFI/mg ratios (TTR) ± SD for all 12 dissected tissues.

Figure 5

features images illustrating the tumor mass detection threshold for each agent at the corresponding optimal dose - 4nmol IntegriSense750, 1nmol ProSense750EX, and 6nmol ProSense750FAST. Bright field, bioluminescence, and fluorescence (closed-field) are shown with inset tumor fragment weight and corresponding TBR according to fluorescence. Red arrows indicate the tumor fragment.

Figure 6

displays the utility of the 4nmol IntegriSense750 dose for fluorescence-guided surgery (FGS) in an orthotopic, metastatic model of head and neck squamous cell carcinoma. Column 1 contains bright field images; while Column 2 the biolumninescence images, Column 3 the closed-field fluorescence images, and Column 4 the open-field fluorescence images. (A) Removal of overlaying skin. (B) Illustrates the utility of FGS to remove the diseased lymph node, and (C) demonstrates the function of FGS for removing the primary tumor en bloc. White and red arrows indicate primary and metastatic disease, respectively.