Is seeing believing?—signal differentiation in a preclinical transbronchial imaging study implementing a composite optical fiber bronchoscope to detect a folate receptor-targeted near-infrared fluorophore

Introduction

Background

Transbronchial imaging is a valuable clinical technique for diagnosis and local treatment of lung cancer, along with transthoracic puncture and surgical open-chest approaches (1). Real-time fluorescence imaging using exogeneous fluorescent agents (fluorophores) can provide valuable additional information by identifying multiple types of malignant lesions with higher specificity and/or sensitivity compared to conventional white-light imaging (2). Tumor specificity can be maximized by using fluorophore conjugated with tumor-specific ligands such as peptides, antibodies, affibodies, activatable tracers or small molecules (3-5). The use of near-infrared (NIR) fluorophores enables deeper penetration of the light in tissue as well as reduced tissue autofluorescence background compared with the visible range (5,6). Fluorescence endoscopic approaches to lung cancer diagnosis and treatment are receiving growing attention. For example, Zaric et al. reported that bronchoscopy utilizing tumor-specific endogenous mucosal fluorescence (autofluorescence) can improve assessment of central lung cancer extension (7). However, autofluorescence bronchoscopy is difficult for tumors located in peripheral bronchi or outside the bronchial wall, where direct visualization is not possible.

In general, fluorescence imaging has a wide range of applications in clinical sciences, including real-time intraoperative tumor localization, tumor margin detection and imaging of peritumoral vasculature (5,8-10). We previously reported preclinical transbronchial imaging using the folate receptor-targeting NIR fluorophore pafolacianine (11). Imaging with pafolacianine has been reported in several malignant and benign tumors, including lung and ovarian cancers, osteosarcoma and pituitary adenoma (11-14).

Rationale and knowledge gap objective

Fluorescent imaging of tumor tissue requires precise identification of the signal (14). In fluorescence-guided cancer surgery, for example, misjudging the tumor fluorescence may lead to a failure in obtaining appropriate resection margins. Similarly, fluorescence-guided biopsy requires detection of the tumor with high sensitivity and specificity. Anatomical structures also impact accurate guidance. For example, the endobronchial airways are narrow, particularly in the periphery, and imaging is performed in a space with only a few millimeters of bronchial inner diameter (15). As a result, the target (i.e., tumor containing the fluorophore) and the tip of the imaging fiber are in close proximity to the bronchial wall. It is also noteworthy that optical methods are highly sensitive and so can detect small changes in fluorescence signal intensity down to low concentrations.

The excitation light is typically several orders of magnitude more intense than the emitted fluorescence light, even with exogenous fluorophores of high fluorescence quantum yield, so that it is critical to remove the background of excitation light that is scattered by the tissue before it reaches the detector. This is usually done using one or more long-pass or band-pass optical filters (16,17). However, rejection of the excitation light comes at the cost of losing some of the fluorescence light, so that in practice total rejection of the excitation background is never achieved. Hence, it is important to understand the conditions under which excitation light leakage is likely to cause problems, including when imaging within a narrow lumen such as in fluorescence bronchoscopy. To our knowledge, this has not been explicitly investigated to date.

Objective

We aim to identify conditions under which the excitation and fluorescence emission signals cannot be fully separated and, conversely, the conditions that allow detection of only the tumor fluorophore signal without contamination by the laser excitation light. We also aim to inform clinicians about mis-interpretation of non-fluorophore related signals. We present this article in accordance with the ARRIVE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-613/rc).

Methods

Pafolacianine

Pafolacianine [Cytalux, OTL38, chemical formula C₆₁H₆₃N₉Na₄O₁₇S₄ (Tetrasodium salt)], kindly provided by On Target Laboratories (West Lafayette, IN, USA), is a folate analog conjugated with an indocyanine green-like dye with peak excitation and emission wavelengths at 774–776 and 794–796 nm, respectively (11,18). This was stored in the dark at –80 °C and, at 24 h prior to use, aliquots were thawed and diluted in 5% dextrose in Eppendorf tubes at a concentration of 0.0526 mg/mL for imaging/spectroscopic measurements. The dosages for in vivo studies are given below.

Composite optical fiberscope (COF)

A COF (OK Fiber Technology Co., Ltd., Kyoto, Japan) with a 0.97 mm outer diameter tip was used to deliver the fluorescence excitation light and for white-light and fluorescence imaging, as described previously (11). In brief, light from a broad-band source and from 776 nm CW diode laser (Cateye: MOG Laboratories Pty Ltd., Victoria, Australia) operating at 10–75 mW output power was couple into the COF, which was connected to an NIR Charge Coupled Device (CCD) camera system with an integrated 800 nm long-pass filter (FELH0800: Thorlabs, Newton, NJ, USA). This filter has 3.5 mm thickness, 25 mm diameter and cut-on wavelength of 800 nm [optical density (OD) >5 over 200–789 nm]. The experiments below were conducted with minimal ambient light for the mouse studies and under low ambient lighting for the swine studies. The camera was connected to a dedicated computer for capture, display and analysis of the real-time white light and NIR fluorescence images, which were displayed simultaneously for review. The exposure time, NIR image gain and threshold for an NIR signal pseudo-color overlay were all User controlled.

Spectrometry

The fluorescence spectra were measured using one of two commercial spectrometers (FLAME-S or USB2000+: Ocean Optics, Orlando, FL, USA). FLAME-S was used in baseline device characteristics experiments and mouse experiments. USB2000+ was used in swine experiments. For direct connection of the light source to the spectrometer without the COF, an integrating sphere (Ocean Optics) was used to reduce the light intensity on the detector. For spectral measurements the COF was connected to the video input (Figure S1A). The same long-pass filter was used in both the image acquisition and spectral measurements. The spectra were normalized to the signal integration time and expressed as counts per second (cps).

In vivo and ex vivo studies

A protocol was prepared before the study without registration. All animal experiments were performed under a project license (No. AUP 4150, 4151) granted by the Animal Care Committee of University Health Network, Toronto, Canada, in compliance with the institutional guidelines for the care and use of animals.

Mouse studies

Subcutaneous tumor xenografts were created as described previously (11). Briefly, 8–12-week-old female athymic nude mice (NCr-Foxn1^nu: Taconic Farms Inc., Germantown, NY, USA) were inoculated subcutaneously in the flank with 1×10⁶ cells human FR-positive oral epidermal carcinoma cells (KB) suspended in PBS and Matrigel (Corning, AZ, USA) (200 μL total, 70:30 by volume). Tumors were grown to 8–15 mm diameter. Pafolacianine was prepared at the dose used in human clinical trials (0.025 mg/kg), at a high dose (0.25 mg/kg) for the purpose of detecting a strong tumor fluorescence signal, and a negative control (5% dextrose) (n=1 each). Mice were injected via the tail vein 24 h prior to imaging/spectroscopy, during which time the mice were kept in dim ambient light and allowed food and water ad libitum.

The mice were anesthetized with isoflurane and placed prone on a heating pad (Figure S1B) and the following measurements were performed to investigate whether the scattered 776 nm laser excitation light could be distinguished from the pafolacianine fluorescence signal. For this, the COF was connected to the 776 mm laser and to the white-light source. After the in vivo imaging and spectroscopy were completed, the mice were euthanized by carbon dioxide and the tumors were excised together with contralateral thigh muscle as a tissue background reference for ex-vivo experiments in the same setup. The optical fiber bundle used for image collection was connected to either the CCD camera or the spectrometer. The COF was placed over the surface of the target (tumor or thigh muscle surrounded by skin) at a distance of 10 mm. The 776 nm laser excitation power was 50 mW.

Swine peribronchial tumor model

For the ex vivo swine model, one set of adult swine lungs and trachea (Caughell Farms Inc., Fingal, ON, Canada) was mechanically ventilated (Figure S1C). For the in vivo studies an adult Yorkshire pig (30 kg) was sedated by intramuscular injection (ketamine 20 mg/kg, midazolam 0.3 mg/kg, atropine 0.04 mg/kg and buprenorphine 0.01 mg/kg). General anesthesia was induced and maintained by isoflurane. The pig was placed supine and a longitudinal chest incision was made. Mediastinal soft tissue was removed around the bronchus to allow placement of the tumor. A pafolacianine- or negative control-infused KB cell xenograft tumor (“Mouse studies” section) was placed manually on the outer wall of the bronchus by forceps. The COF was connected to the 776 mm laser and white-light source. The COF was inserted into the working channel of a clinical 1.7 mm diameter bronchoscope (BF-MP190, Olympus Medical Systems Corporation, Tokyo, Japan), which was then inserted through an endotracheal tube and navigated close to where the tumors were placed. Laser excitation through the COF was performed at 50–125 mW. The distance between the COF tip and the bronchial mucosa was measured by marking the COF at the insertion point of the bronchoscope working channel, keeping the bronchoscope position constant. Transbronchial COF imaging and spectroscopic measurements were acquired separately while changing the distance from the COF tip to the bronchial mucosa.

Statistical analysis

Since the number of animals used in this study was n=1 for all experimental series, statistical analysis was not required.

Results

Baseline device characteristics

The spectrum from the LED white-light source (without long-pass filtering) was measured using the integrating sphere and FLAME-S spectrometer (Figure S1A,S1D). This showed 3 peaks in the visible region (Figure S1E, E1), while the incandescent source had a smooth waveform peaking at 600–700 nm (Figure S1E, E2). The laser source, operating at the lowest power of 10 mW, showed only the 776 nm peak with no background signal (Figure S1E, E3). When the spectra from the light sources were measured with the long-pass (>800 nm) filter in place (Figure S1F), the white light showed no peak (Figure S1G, G1) and the incandescent source peaked above 800 nm (Figure S1G, G2).

When the spectrum from control solution or pafolacianine solution was measured using COF with the long-pass filter present (Figure 1A), there was no detectable signal from the control solution (Figure 1B, B1), while a spectral peak around 810–830 nm was detected in the pafolacianine solution (Figure 1B, B2).

Figure 1 Instrument setup, spectral measurement, and fluorescence imaging. (A) Schematic diagram showing the 776 nm diode laser and white-light source connected to the COF, and the imaging device and spectrometer. (B) Measured spectra and NIR images (B1) in control phantom without fluorophore and (B2) in pafolacianine solution (0.0526 mg/mL). COF, composite optical fiberscope; cps, counts per second; F, filter; NIR, near-infrared; SM, spectrometer; WL, white light.

Mouse model

In the mouse model setup in vivo (Figure 2A and Figure S1B), no 776 nm peak was observed at either pafolacianine concentration (Figure 2B). No 810 nm peak was seen in the negative control tumor, while clear peaks were observed at the two injected doses, with the 0.25 mg/kg signal being stronger. The ex vivo samples showed similar results (Figure S2).

Figure 2 Studies in athymic nude mice. (A) Subcutaneous tumor induction and fluorophore administration (0.025 or 0.25 mg/kg Pafolacianine in 5% dextrose). The photo shows the unilateral tumor and normal tissue area probed. (B) Spectroscopy showing absence of the 776 nm peak, independent of the presence of fluorophore at either concentration, together with the true fluorophore peaks, and corresponding images. cps, counts per second.

Swine peribronchial tumor model

In the porcine lungs with pafolacianine-infused KB tumor with a dose of 0.25 mg/kg (Figure 3A and Figure S3A), when the distance from the COF tip to the bronchial mucosa was 10 mm, a clear 810 nm peak was observed (Figure 3B). This signal was also observed from within the bronchi (Figure 3B, B2, Video S1). When the pafolacianine-infused KB tumor from the mouse given a dose of 0.025 mg/kg, the signal was faint but still observable in the pig (Figure S3B).

Figure 3 Studies in the swine transbronchial tumor model. (A) Transplantation of tumor from mouse model at 24 h after i.v. administration of 0.25 mg/kg pafolacianine. For implantation of tumor next to a peripheral airway, an incision was made along the peripheral bronchus to expose the bronchial wall, the bronchoscope was inserted through an endotracheal tube and navigated close to the implantation site outside the bronchus, and the tumor was placed manually adjacent to/outside of the bronchus. (B) Spectra (B1) and images (B2) from pafolacianine-infused tumor at 10 mm distance. (C) Spectra (C1) and images (C2) in the lung from negative control tumor at 10 mm distance. (D) Spectra (D1) and images (D2) in the lung from negative control tumor at 1 mm distance. cps, counts per second; FR, folate receptor; i.v., intravenous.

There was no fluorescence signal when negative control tumors were placed outside the bronchi (Figure 3C; Video S2). These findings indicated that the pafolacianine fluorescence was observed without significant interference from the excitation light leakage.

However, when the COF tip was placed in close proximity (~1 mm) to the bronchial mucosa and negative control tumor was placed outside the subcarinal bifurcation (Figure S3C), we observed a 776 nm peak (Figure 3D, D1) and signal (Figure 3D, D2; Video S3), indicating a significant signal from the excitation light leakage. With the COF tip placed at 1 mm from the mucosa at 75 or 125 mW laser power, the 776 nm signal was clearly seen (increasing with laser power) but was not seen at 10 mm distance (Figure S3D). Similarly, for control tumor (no fluorophore) and at COF-to-mucosa distances of 1, 5 or 10 mm and 50 mW laser power, we observed the 775 nm signal only at the shortest (1 mm) distance (Figure S3E).

Discussion

The baseline spectra with the 776 nm laser connected to the COF and the long-pass filter in place showed a clear peak around 810–830 nm for the pafolacianine solution and no detectable signal in the negative-control solution (Figure 1B), indicating effective filtering of the excitation light in this configuration. Figure S4 demonstrates the corresponding expected pafolacianine emission spectrum (19). No 776 nm laser light leakage was observed in either the in vivo or ex vivo mouse tumor model studies. No 810 nm signal was seen in the non-pafolacianine-infused negative control tumor but a dose-dependent signal was clearly seen in the infused tumors (Figure 2B, Figure S2). In the swine peribronchial tumor model, we investigated whether the observed fluorescence was indeed derived from the tumor. Thus, when the COF tip was positioned 10 mm from the bronchial tissue, pafolacianine-specific fluorescence of infused tumor placed outside the bronchial wall could be observed (Figure 3B). The 776 nm signal was seen only when the COF tip was extremely close (~1 mm) to the bronchus (Figure 3D; Figure S3D,S3E).

This study is the first to report detection of transbronchial NIR fluorophore-infused tumor by both fluorescence imaging and spectroscopy in the same setup in lung tumor models. Although the results are not fully generalizable and the instrumentation is still in the prototype version, the possibility for misinterpreting scattered laser excitation light when the instrument tip is very close to the bronchus should be considered, since this could lead to serious false-positive diagnostic errors and risk in fluorescence-guided interventions. As is well known, light absorption and scattering vary markedly with the tissue under investigation and are also strongly wavelength dependent (20,21). Transbronchoscopic imaging may be particularly susceptible to this artefact due to the narrow lumen, especially towards the lung periphery (15). Hence, it is important to be aware of and check for this potential artefact and to avoid very close proximity between the mucosal surface and the fluorescence bronchoscope tip, especially when using prototype equipment. We note that other tissue factors such as tissue curvature and reflectivity (which could be affected by mucosal secretions), as well as the endoscope optics (e.g., numerical aperture), could alter the critical distance for excitation light contamination. These aspects will be investigated in future studies. Also, in the event that fluorescence is observed when the bronchoscope tip is in close proximity to the mucosa, clinicians should test the veracity of the signal by imaging at the same proximity in a known fluorescence-negative region of tissue. The system used here is a prototype but similar configurations are used in other clinical research devices. Future comparisons will be required to assess the presence and magnitude of the light-leakage artefact in these systems and, ultimately, commercial fluorescence bronchoscopes.

There are some limitations in this study, as follows. Firstly, we investigated the conditions under which laser excitation light leaks into the pafolacianine-infused tumor fluorescence signal, but the experiments involved only one specific instrument and fluorophore, so that this potential problem needs to be investigated for other scenarios. In general, the strongest optical filtering (i.e., the highest out-of-band OD) should be used to minimize the leakage: Even though an OD 5 filter was used (rejection factor of 100,000), we still observed detectable excitation light at close proximity to the tissue surface. Increasing the OD of the filter might reduce this, although the rejection efficiency is ultimately limited by transmission of off-axis light. Higher OD would also reduce the fluorescence signal strength, depending on the performance of the filter used. Ultimately, the optimum trade-off must be determined through continued testing of the system design (16,17). Secondly, the spectroscopic measurements were performed under the same setup as the imaging but were not acquired simultaneously in the mouse or swine models (Figures S1A,S1B,S3A); so that the conditions were not identical considering the respiratory motion of the lungs in the swine experiments. Thirdly, in the swine model the fluorophore-infused tumor was implanted in normal non-infused lung tissue, so that the tumor-to-background ratio may not be representative of systemically-administered fluorophore in patients. In contrast, in the mouse model, a pafolacianine dose of 0.25 mg/kg (10 times the clinical dosage concentration) showed clear fluorescence signal even in non-tumor-bearing contralateral muscle. This false-positivity is a separate issue from the problem of excitation light leakage. However, this would not alter the impact of the excitation light leakage into the fluorescence image. Fourth, given the exploratory aim of identifying potential imaging artefacts, n=1 was used for each condition. These findings will require further validation in larger cohorts. Lastly, the tumor cell line KB is derived from an epidermal oral carcinoma and expresses high folate receptor (18,22), resulting in stronger pafolacianine fluorescence signal than in other lung cancer cell lines investigated previously (23). Hence, the tumor fluorescence may be weaker in clinical cases than observed here, so that the effect of any laser light leakage would become even more pronounced. Furthermore, in lung cancer, FR heterogeneity will impact the interpretation of the results here in clinical practice (24,25).

Having the ability to measure fluorescence spectra during clinical endoscopy would alleviate the risk of confusing the excitation light leakage and true fluorescence signals, thereby increasing reliability. However, the potential benefits must be weighed against the increased instrument cost and procedure time, considering that the leakage laser light detected in the fluorescence channel was seen only when the probe tip was very close to the bronchial mucosa (A 3d). In most clinical settings endoscopists generally maintain proper patient positioning and avoid close proximity in order to maintain an unobstructed field-of-view (26,27), so that this capability would be “overkill”. Nevertheless, it is important to be aware of this potential artefact when using new fluorescence endoscopy equipment or procedures.

Conclusions

We have identified a potential artefact in fluorescence endoscopy in which the visualized fluorescence can be corrupted by excitation light leakage when the endoscope tip is close to the tissue surface, as is often the case in bronchoscopy, particularly in the peripheral lung. It is important to be aware of this potential false-positive signal and to check for it, for example, by imaging a region of tissue that is known to be lacking exogenous fluorophore uptake, especially when working with new equipment or performing new procedures where this situation may arise.

Acknowledgments

The authors would like to thank Judy McConnell (Toronto General Hospital) for laboratory management and Teesha Komal (University Health Network-STTARR) for technical assistance with imaging, as well as the University Health Network (UHN) Animal Resources Centre.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-613/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-613/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-613/prf

Funding: This work was supported by the William Coco Chair in Surgical Innovation for Lung Cancer, a joint Hospital-University Chair between University Health Network, the University of Toronto, Toronto General and Western Hospital Foundation, and the Terry Fox Research Institute New Frontiers Program (No. #1137).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-613/coif). Y.H. received a scholarship from the Uehara Foundation Overseas Research Fellowships (201940067). K.Y. was financially supported by collaboration with Johnson & Johnson Enterprise Innovation Inc. K.Y. also declared that COF access was provided in-kind by Johnson & Johnson Enterprise Innovation, Inc., and OK Fiber Technology Co., Ltd., and declared that pafolacianine was provided by On Target Laboratories. All authors declared that the manuscript was reviewed by Johnson & Johnson Enterprise Innovation, Inc., OK Fiber Technology Co., Ltd., and On Target Laboratories, Inc. before submission. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license (No. AUP 4150, 4151) granted by the Animal Care Committee of University Health Network, Toronto, Canada, in compliance with the institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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