If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Transbronchial real-time lung tumor localization with folate receptor–targeted near-infrared molecular imaging: A proof of concept study in animal models
Address for reprints: Kazuhiro Yasufuku, MD, PhD, Division of Thoracic Surgery, University Health Network, University of Toronto, Toronto General Hospital, 200 Elizabeth St, 9N-957, Toronto, Ontario M5G 2C4 Canada.
The diagnostic yield of bronchoscopy is not satisfactory, even with recent navigation technologies, especially for tumors located outside of the bronchial lumen. Our objective was to perform a preclinical assessment of folate receptor–targeted near-infrared imaging-guided bronchoscopy to detect peribronchial tumors.
Methods
Pafolacianine, a folate receptor-targeted molecular imaging agent, was used as a near-infrared fluorescent imaging agent. An ultra-thin composite optical fiberscope was used for laser irradiation and fluorescence imaging. Subcutaneous xenografts of KB cells in mice were used as folate receptor–positive tumors. Tumor-to-background ratio was calculated by the fluorescence intensity value of muscle tissues acquired by the ultra-thin composite optical fiberscope system and validated using a separate spectral imaging system. Ex vivo swine lungs into which pafolacianine-laden KB tumors were transplanted at various sites were used as a peribronchial tumor model.
Results
With the in vivo murine model, tumor-to-background ratio observed by ultra-thin composite optical fiberscope peaked at 24 hours after pafolacianine injection (tumor-to-background ratio: 2.56 at 0.05 mg/kg, 2.03 at 0.025 mg/kg). The fluorescence intensity ratios between KB tumors and normal mouse lung parenchyma postmortem were 6.09 at 0.05 mg/kg and 5.08 at 0.025 mg/kg. In the peribronchial tumor model, the ultra-thin composite optical fiberscope system could successfully detect fluorescence from pafolacianine-laden folate receptor–positive tumors with 0.05 mg/kg at the carina and those with 0.025 mg/kg and 0.05 mg/kg in the peripheral airway.
Conclusions
Transbronchial detection of pafolacianine-laden folate receptor–positive tumors by near-infrared imaging was feasible in ex vivo swine lungs. Further in vivo preclinical assessment is needed to confirm the feasibility of this technology.
Bronchoscopic tissue sampling for the diagnosis of lung lesions has an excellent safety profile, but diagnostic performance is unsatisfactory, even with the recent technologies, especially for peribronchial tumors. Molecular-targeted transbronchial imaging of peribronchial tumors may permit real-time and efficient tumor localization, facilitating more precise tissue sampling.
See Commentary on page e252.
The incidence of abnormal peripheral lung lesions has been increasingly found due to widespread use of chest computed tomography (CT) in routine medical care.
For tissue diagnosis of peripheral lung lesions, although CT-guided transthoracic needle aspiration is often performed because of its high diagnostic yield,
Establishing the diagnosis of lung cancer: diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelines.
Although the diagnostic yield of the transbronchial approach has improved due to recent technologies, including radial probe endobronchial ultrasound (RP-EBUS) and navigation tools, tissue biopsy for peripheral peribronchial lung tumors is still challenging. Because peribronchial lung tumors do not always completely surround the feeding bronchus, blind advancement of biopsy instruments may sample in locations different from the tumor location as identified by RP-EBUS, particularly because the radial probe must be withdrawn to perform the biopsy. This would fit with data showing significantly lower diagnostic yield when the RP-EBUS probe was adjacent to the lesion (ie, eccentric view) compared with when the probe was within it (ie, concentric view).
Navigation tools including virtual bronchoscopic navigation and electromagnetic navigation bronchoscopy (ENB) can guide the bronchoscopist to the target lesion using 3-dimensional reconstructions of CT images. However, CT-to-body divergence must be considered because those reconstructions are derived from CT data before bronchoscopy.
This has allowed accurate resection of tumors with adequate margins of normal tissue. A multicenter clinical trial was conducted to evaluate the efficacy of intraoperative molecular imaging using a near-infrared (NIR), folate receptor (FR)-targeted imaging agent, pafolacianine, for cancer in the lung.
Intraoperative fluorescence imaging was used to localize the tumor, confirm margins, and find occult synchronous lesions; fluorescence imaging prompted surgeons to modify their operation or upstage the patient's cancer in 26% of cases.
We have hypothesized that this fluorescence-guided approach for tumor localization could be modified to enable transbronchial localization of peribronchial tumors for diagnostic procedures. We used pafolacianine as a targeted imaging agent in combination with an ultra-thin laser fiberscope to irradiate and localize the tumor real-time. The presented study is the first data to support this principle.
Materials and Methods
Pafolacianine
Pafolacianine (OTL38, chemical formula C61H63N9Na4O17S4 [tetrasodium salt]; molecular weight, 1414.42 Da) is a folate analog conjugated with an indocyanine green-like dye.
Pafolacianine was used as a NIR fluorescent imaging agent in this study (peak excitation and emission wavelengths, 774-776/794-796 nm). Pafolacianine (>96% purity) was obtained in collaboration with On Target Laboratories. Pafolacianine was stored at 1508 μM in a –80 °C freezer. Dilutions of pafolacianine were made in 5% dextrose solution within 24 hours before used.
An ultra-thin composite optical fiberscope (UCF, OK Fiber Technology Co Ltd) with a 0.97-mm outer diameter tip was used for laser irradiation and imaging (white-light and NIR).
A preclinical research platform to evaluate photosensitizers for transbronchial localization and phototherapy of lung cancer using an orthotopic mouse model.
Laser excitation through the UCF was performed at 50 to 75 mW using a 776-nm laser source (CEL, Cateye diode laser, MOG Laboratories Pty Ltd). The UCF was connected to a charge-coupled device NIR camera system with an integrated 800 nm long-pass filter (FELH0800, Thorlabs) as the detector. The camera system was connected to a designated computer where image capture and image analysis software were used. Pylon Viewer (Basler) was used for capturing images to facilitate further image analysis, including semiquantitative fluorescence intensity assessment. OKFT Image Processing Software (OK Fiber Technology Co Ltd) was used to display the real-time white light and NIR images acquired by the camera system. In this software, a white-light image, a NIR image, an enhanced NIR image with pseudo-color overlay (green in this study), and an overlay image merging the white-light image and the pseudo-color overlay can be displayed simultaneously for review, image capture, or video capture. The exposure time, NIR image gain, and intensity threshold for the NIR signal pseudo-color overlay can be changed in the software.
Pafolacianine Fluorescence In Vitro
Pafolacianine was reconstituted in 5% dextrose solution at 4, 2, 1, 0.5, 0.25, 0.125, 0.063, and 0.031 μM concentrations (1 μM = 0.00132 mg/mL). Then, 1.5 mL of each solution was placed in a 24-well plate with a black flat bottom. The UCF was placed over each well at 3, 5, 7, 10, 15, and 20 mm from the solution surface. Semiquantitative analysis was performed on the acquired NIR images using ImageJ software (National Institutes of Health), where regions of interest were drawn with a circle over each well and the average intensities were recorded.
Animal Models
All animal studies were performed under an animal use protocol (AUP 4151) approved by the Animal Care Committee of the University Health Network (approved on January 12, 2021).
In Vivo Mouse Xenograft Model
To establish optimal settings for in vivo use of the UCF system, subcutaneous xenografts were created in mice using a human FR-positive oral epidermal carcinoma cell line (KB). A total of 100 μL cell mixture (70% cell suspension of 1.0 × 106 cells in phosphate-buffered saline, 30% extracellular matrix [Matrigel]) was inoculated subcutaneously in the right thigh of 8- to 12-week-old female athymic nude mice (NCr-Foxn1nu; Taconic Farms Inc). All mice were fed with alfalfa-free diets. Subcutaneous tumors approximately 5 mm in diameter were used for assessment of NIR intensity in the time series. Mice were infused with 0.05, 0.025, or 0.0125 mg/kg pafolacianine (n = 3 per dose) or 5% dextrose solution (negative control; n = 1) and intermittently imaged at 6, 24, 48, and 72 hours after injection. Mice were anesthetized with isoflurane and placed in a lateral decubitus position. UCF was placed over the surface of the tumors (right thigh) and normal limb (left thigh, “background”) at a distance of 5, 10, and 15 mm for NIR image capture. Further NIR images were captured using an in vivo whole-mouse fluorescence imaging system (Maestro, CRi) with 710 to 760 nm excitation and greater than 800 nm detection. For biodistribution assessment of pafolacianine, mice with 5- to 10-mm diameter tumors infused with 0.05 or 0.025 mg/kg pafolacianine (n = 3 per dose) were killed at 24 hours postinfusion. The fluorescence intensities from tumors and organs were semiquantitatively assessed by the Maestro fluorescence imaging system. Additional images of the resected tumors and lungs were recorded by the UCF.
Ex Vivo Peribronchial Tumor Swine Model
To assess the feasibility of NIR-guided transbronchial tumor localization, we created a pseudotumor model using ex vivo swine lungs implanted with mouse xenograft tumors to mimic peribronchial tumors. Mice with KB tumors more than 10 mm in diameter were selected for pafolacianine administration. A dose of 0.025 mg/kg or 0.05 mg/kg pafolacianine was injected intravenously into the tail vein 24 hours before tumor resection (n = 3 per dose). Pafolacianine-laden KB tumors were transplanted at peribronchial sites (carina and peripheral airway) in 1 set of ex vivo swine lungs (body weight, 75 kg, Caughell Farms Inc). Mediastinal soft tissue was removed around the carina to allow placement of the tumor. To put tumors next to a peripheral airway, the lungs were incised longitudinally along the peripheral bronchus to expose the bronchial wall. Tumors were placed beside the exposed bronchus to mimic peribronchial lung tumors. A bronchoscope (BF-P190, Olympus Medical Systems Corporation) was inserted through an endotracheal tube and approached the sites where pafolacianine-laden tumors were placed. Subsequently, the UCF was inserted into the bronchoscope working channel to observe NIR fluorescence from the tumors. In anticipation of the impact of the bronchial wall thickness on laser light transmission, relative to mouse skin, laser power was increased to 75 mW for the ex vivo model. Before placing the tumors at peribronchial sites, the absence of autofluorescence signal from the respective bronchial wall was confirmed by UCF using the same settings as for subsequent imaging. All procedures were performed in a dark room.
Statistical Analysis
Fluorescence intensities were summarized as means with standard error. For in vivo murine studies, at least 3 samples were used per group unless noted. Statistics were calculated using GraphPad Prism 8 (GraphPad Software).
Results
In Vitro Evaluation of the Ultra-Thin Composite Optical Fiberscope's Ability to Detect Pafolacianine Fluorescence
A semiquantitative assessment of pafolacianine fluorescence with different concentrations was conducted using the UCF camera system at different exposure times and different distances between the pafolacianine fluid surface and the UCF tip. At a 5-mm distance, an exposure time of 640 ms was required to clearly visualize fluorescence at lower concentrations (Figure E1, A). At the same exposure time, higher concentration solutions (2 and 4 μM) could be detected at a 10-mm distance from the surface; intensity decreased with distance (Figure E1, B and C). With the fluorescence-enhancing function of the UCF camera software, faint fluorescence became easier to detect through display of a green pseudo-color overlay than that with standard NIR images (Figure E1, D).
In Vivo Characterization of Pafolacianine in Mice
To determine whether pafolacianine accumulates in FR expressing tumors in vivo and to reproduce previously published data,
we evaluated mice bearing KB subcutaneous xenografts. Peak tumor-to-background ratios (TBRs) assessed using the in vivo whole-mouse fluorescence imaging system were observed at 24 hours after injection of 0.05 mg/kg, 0.025 mg/kg, and 0.0125 mg/kg doses with an average TBR of 2.67, 2.59, and 2.16, respectively (Figure 1, A-C). At 24 hours after pafolacianine administration in vivo, fluorescence from subcutaneous KB tumors could be clearly detected by UCF, with strong signal compared with the contralateral normal thigh (background) (Figure 2, A). Peak tumor-to-muscle ratios assessed semiquantitatively on NIR images captured by the UCF system were 2.56, 2.03, and 1.19 at a 10-mm distance 24 hours after injection of 0.05 mg/kg, 0.025 mg/kg, and 0.0125 mg/kg pafolacianine, respectively (Figure 2, B). At an earlier timepoint (6 hours), although tumor fluorescence was strong, background fluorescence was also strong (Figure E2). Some lag in the capture and alignment of NIR signal relative to the white light image was seen due to the long exposure time (640 ms), but the alignment delay was thought to be manageable by ensuring the UCF was moved gradually (Video 1).
Figure 1Pafolacianine pharmacokinetics and biodistribution in mice bearing KB cell subcutaneous xenografts as assessed by an overhead whole-mouse spectral imaging system (Maestro). A, Fluorescence of athymic nude mice infused with pafolacianine at 3 different doses (0.05, 0.025, and 0.0125 mg/kg) was assessed serially until 72 hours. B, The intensity (average scaled counts/sec) and (C) tumor-to-muscle ratio were plotted over time (n = 3 per dose). D, For biodistribution assessment, mice infused with 0.05 and 0.025 mg/kg of pafolacianine were killed at 24 hours postinfusion. The fluorescence intensities from ex vivo subcutaneous xenografts and organs were semiquantitatively assessed. E, The values of tumor, thigh muscle, and lung were compared. Overall, pafolacianine fluorescence had similar TBR at 0.025 mg/kg and 0.05 mg/kg (n = 3 per dose), although with higher fluorescence intensities at 0.05 mg/kg. Red horizontal lines indicate the mean, and error bars represent the standard error. Different symbols represent different animals.
Figure 2Representative overlay images of fluorescence in KB tumors and background tissue observed by the UCF. A, Subcutaneous xenografts with KB cells and contralateral normal thighs (background) in mice were assessed at 24 hours after pafolacianine infusion at 3 different doses (0.05, 0.025, and 0.0125 mg/kg) (n = 3 per dose; n = 1 negative control injected with 5% dextrose solution). B, The TBR assessed by the UCF at different distances. Red horizontal lines indicate the mean and error bars represent standard error. Different symbols represent different animals. Laser power was 50 mW; exposure time, 640 ms; image gain, 30; threshold of pseudo-color, 25.
At 24 hours after injection, tumors and organs were resected, and the ex vivo fluorescence intensity ratios between KB tumors and lungs were 6.09 and 5.08 in mice dosed at 0.05 mg/kg and 0.025 mg/kg, respectively, by the Maestro fluorescence imaging system (Figure 1, D and E). Consistent with previous reports, pafolacianine was accumulated predominantly in the kidneys due to their FR expression and renal excretion of pafolacianine.
Display of the NIR fluorescence signal could be optimized by setting appropriate thresholds for the UCF to eliminate background signal in normal lung tissue while retaining signal intensity in ex vivo KB tumors (Video 2).
To assess the feasibility of a transbronchial NIR-guided approach to lung tumor localization, mouse xenografts were transplanted into ex vivo swine lungs in the region of the carina and peripheral airways. A total of 6 tumors administered with a dose of 0.05 mg/kg (n = 3) or 0.025 mg/kg (n = 3) were used in this assessment. All tumors were used within 90 minutes after resection and protected from light throughout. Tumor placement is summarized in Figure 3. Carinal tumors were placed approximately 0.5 cm below the tracheal bifurcation along the left side at the carina level. Peripheral airway tumors were placed adjacent to a peripheral bronchus (lumen diameter, 2.6 mm) of the right lower lobe at a location approximately 2.5 cm deep to the pleura, mimicking peripheral peribronchial lung lesions. Tumor NIR fluorescence was transbronchially detected by the UCF system at the carina using a 0.05 mg/kg dose, whereas tumor fluorescence with both 0.05 mg/kg and 0.025 mg/kg dosing was observed in the peripheral airway (Figure 3 and Video 3).
Figure 3Representative images of fluorescence from pafolacianine-laden folate receptor--positive tumors transbronchially captured by the composite optical fiberscope in a peribronchial tumor swine model. Left: KB tumors from athymic mice infused with 0.025 or 0.05 mg/kg pafolacianine (n = 3 per dose) were transplanted into ex vivo swine lungs to mimic peribronchial tumors at the carina (top) and peripheral airway (bottom). Access to the peribronchial space was obtained by incising the lungs longitudinally along the peripheral bronchus in the right lower lobe to expose the bronchial wall. Green ellipses show the location where tumors were placed. Tumors were held gently with forceps. Yellow arrowheads show the location of the exposed peripheral bronchus. Right: Representative images from corresponding white light, NIR enhanced with pseudo-color, and merged overlay (merge) views are shown for each dose. Fluorescence from tumors with 0.025 mg/kg of pafolacianine was not detected at the carina level. Laser power, 75 mW; exposure time, 640 ms; image gain 36; threshold value for pseudo-color, 25. WL, White light; NIR, near-infrared.
In this study, we provide preliminary evidence supporting the feasibility of fluorescence-guided transbronchial lung tumor localization using an FR-targeted NIR agent, pafolacianine, and the UCF system (Figure 4). We demonstrated the feasibility of transbronchial NIR-guided tumor localization in a peribronchial lung tumor model. This work may provide fundamental data for transbronchial real-time tumor detection and localization by fluorescence guidance in future human clinical trials.
Figure 4Overview of a proof of concept study of FR-targeted NIR-guided transbronchial tumor detection. In this study, pafolacianine was used as an FR-targeted NIR agent, and the UCF was deployed through the bronchoscope working channel to capture images/videos of the NIR fluorescence. Pafolacianine was administered intravenously into athymic nude mice bearing subcutaneous tumors created with FR-positive KB cells. To create a peribronchial lung tumor model, xenograft tumors were resected and transplanted into ex vivo swine lungs in the region of the carina and peripheral airways. Fluorescence from tumors was transbronchially detected by the UCF system at the carina level using a 0.05 mg/kg dose, whereas tumors with both 0.05 mg/kg and 0.025 mg/kg doses were observed in the peripheral airway. Molecular-targeted transbronchial imaging of peribronchial tumors may permit real-time and efficient tumor localization, facilitating more precise tissue sampling. This preclinical study may provide fundamental data for future human clinical trials.
For visualizing peribronchial tumors beyond the bronchial wall, significant tissue penetration depth is required. NIR fluorescence, as is provided by pafolacianine, has the advantage of improved tissue penetration compared with fluorescence within the visible spectrum, as is provided by agents such as 5-aminolevulonic acid and fluorescein.
indocyanine green is thought to accumulate into tumors via the enhanced permeability and retention effect, which lacks a degree of tumor specificity compared with FR-targeted agents.
However, as our study showed in the mouse model, the relative difference in fluorescence intensity between the tumor and the lung is high (ratio, 6.1 with 0.05 mg/kg and 5.1 with 0.025 mg/kg). The specificity of pafolacianine for tumors provided sufficiently high TBR to localize lung tumors during video-assisted surgery in clinical trials.
Thus, pafolacianine might be a suitable fluorescence agent for transbronchial detection of peribronchial tumors considering NIR light's tissue penetration and pafolacianine's high specificity for FR-positive tumors.
Transbronchial fluorescence-guided real-time tumor localization may be more beneficial when targeting peribronchial tumors compared with endobronchial tumors. Because of recent technological advancements, robotic bronchoscopy has been adopted in some institutions. These platforms can enter more distal bronchi, to the 8th to 10th generation depending on the lobe, with continuous visualization due to its small tip diameter and with stable positioning due to its complex mechanics.
Endobronchial tumors can be readily identified with direct vision as well as RP-EBUS. However, peribronchial tumors without endoluminal invasion are difficult to localize precisely, in turn impairing sampling. Even with ENB, navigation accuracy may not be sufficient. A systematic review found an overall pooled CT-to-body divergence, also known as “average fiducial target registration error,” of 5.1 mm with ENB.
When aiming at small peripheral nodules less than 1 cm in size, even a 5-mm registration error would significantly affect localization. Precise localization is essential for accurate deployment of the biopsy needle for tumors located outside the bronchial lumen. Our technique in this study, using a combination of an ultra-thin fiberscope and fluorescence guidance, may be helpful to confirm the location of targets in small airways in a real-time manner. The size of the peripheral bronchus where we placed tumors in this study was 2.6 mm, which corresponds with the sixth to seventh bronchial generation in humans.
Assessment of bronchial wall thickness and lumen diameter in human adults using multi-detector computed tomography: comparison with theoretical models.
If the UCF is used in combination with a robotic bronchoscope, there is an opportunity to perform fluorescence-guided localization followed immediately by accurate needle biopsy by virtue of the robotic platform's stable positioning and orientation, even in small airways.
Study Limitations
There are some limitations in this study. First, this is a proof of concept study of transbronchial fluorescence-guided tumor detection. In the ex vivo swine lung model, only the target tumors had fluorescent pafolacianine, whereas pafolacianine was not infused into the pig before lung resection. Therefore, the effect of background intensity or the circulation of pafolacianine in the blood was not assessed in this study. Our next step would be transplanting pafolacianine-laden tumors from mice into live pigs infused with pafolacianine. Second, fluorescence from the tumor cannot be definitively distinguished from reflected laser light with the camera system used in this study, particularly when the fiberscope is extremely close to the bronchial wall. In this study, obvious reflected light was not observed because the fiberscope was kept approximately 3 to 5 mm from the mucosal surface. An integrated spectrometer would be required to accurately distinguish true tumor fluorescence from false-positive laser reflection. Third, pafolacianine-laden tumors were placed adjacent to the carina or bronchus without intervening tissues. For the carina, mediastinal tissues were removed in the ex vivo swine lungs to evaluate the effect purely from the thickness of the airway wall. In some clinical cases, intrapulmonary tumors are separated from the closest feeding bronchus by a few millimeters. At present, our study best reflects peribronchial tumors immediately adjacent to the bronchus. It is possible that performance may decrease slightly in the presence of such intervening tissues. Fourth, target tumors used in this study were created with strongly FR-positive KB cells. We need to verify this technique using representative lung cancer cell lines in future experiments.
Conclusions
Pafolacianine-laden FR-positive tumors were transbronchially detected by NIR fluorescence imaging in real time in ex vivo swine lungs. Molecular-targeted NIR-guided bronchoscopy may enhance the transbronchial localization of certain tumors, which may facilitate more accurate bronchoscopic sampling. Further in vivo preclinical assessment is needed to better characterize the feasibility of this approach.
K.Y. is a consultant for Olympus Medical Systems Corporation and a recipient of research funding from Johnson & Johnson Enterprise Innovation. Ultra-thin composite fiberscope access was provided by Johnson & Johnson Enterprise Innovation and OK Fiber Technology Co Ltd. Pafolacianine access was provided by On Target Laboratories. All other authors reported no conflicts of interest.
The Journal policy requires editors and reviewers to disclose conflicts of interest and to decline handling or reviewing manuscripts for which they may have a conflict of interest. The editors and reviewers of this article have no conflicts of interest.
The authors thank Silvia Chen, Hannah McEwen, Benjamin Lundgren, Sumith Kularatne, Kiyoshi Oka, and Sachiko Minakawa for their collaboration, and Judy McConnell for research coordination.
Fluorescence of in vivo FR-positive tumors and background observed with the UCF after pafolacianine infusion. Athymic mice bearing KB cell subcutaneous xenografts infused with 0.05 or 0.025 mg/kg pafolacianine were imaged using the UCF. Corresponding white light and the overlay images (merging the white light and NIR pseudo-color enhancement) are shown (n = 3 per dose). The UCF was moved continuously during video capture keeping the distance between the tissue surface and UCF tip approximately 10 mm. Laser power, 50 mW; exposure time, 640 ms; image gain, 30; threshold of pseudo-color, 25. FR, Folate receptor; NIR, near-infrared; UCF, ultra-thin composite optical fiberscope. Video available at: https://www.jtcvs.org/article/S0022-5223(22)01034-0/fulltext.
Fluorescence of in vivo FR-positive tumors and background observed with the UCF after pafolacianine infusion. Athymic mice bearing KB cell subcutaneous xenografts infused with 0.05 or 0.025 mg/kg pafolacianine were imaged using the UCF. Corresponding white light and the overlay images (merging the white light and NIR pseudo-color enhancement) are shown (n = 3 per dose). The UCF was moved continuously during video capture keeping the distance between the tissue surface and UCF tip approximately 10 mm. Laser power, 50 mW; exposure time, 640 ms; image gain, 30; threshold of pseudo-color, 25. FR, Folate receptor; NIR, near-infrared; UCF, ultra-thin composite optical fiberscope. Video available at: https://www.jtcvs.org/article/S0022-5223(22)01034-0/fulltext.
Fluorescence of ex vivo FR-positive tumors and lungs observed with the UCF after pafolacianine infusion. Athymic mice bearing KB cell subcutaneous xenografts infused with 0.05 or 0.025 mg/kg pafolacianine were killed at 24 hours postinfusion for resection of the tumors and lungs. Corresponding white light and the overlay images (merging the white light and NIR pseudo-color enhancement) are shown. The distance between the tissue surface and the UCF tip was 10 mm. Laser power, 50 mW; exposure time, 640 ms; image gain, 30; threshold of pseudo-color, 25. FR, Folate receptor; NIR, near-infrared; UCF, ultra-thin composite optical fiberscope. Video available at: https://www.jtcvs.org/article/S0022-5223(22)01034-0/fulltext.
Fluorescence of ex vivo FR-positive tumors and lungs observed with the UCF after pafolacianine infusion. Athymic mice bearing KB cell subcutaneous xenografts infused with 0.05 or 0.025 mg/kg pafolacianine were killed at 24 hours postinfusion for resection of the tumors and lungs. Corresponding white light and the overlay images (merging the white light and NIR pseudo-color enhancement) are shown. The distance between the tissue surface and the UCF tip was 10 mm. Laser power, 50 mW; exposure time, 640 ms; image gain, 30; threshold of pseudo-color, 25. FR, Folate receptor; NIR, near-infrared; UCF, ultra-thin composite optical fiberscope. Video available at: https://www.jtcvs.org/article/S0022-5223(22)01034-0/fulltext.
Representative videos of pafolacianine-laden FR-positive tumor fluorescence captured by the UCF imaging system in a swine peribronchial tumor model. Each quadrant is from a different pafolacianine dose and lung location. The image software magnifies the merge image mode (white light and NIR pseudo-color overlay) on the left, whereas on the right corresponding alternative modes are shown (top to bottom: white light, raw NIR, merge mode, enhanced NIR with pseudo-color overlay). Laser power, 75 mW; exposure time, 640 ms; image gain 36; threshold for pseudo-color, 25. FR, Folate receptor; NIR, near-infrared; UCF, ultra-thin composite optical fiberscope. Video available at: https://www.jtcvs.org/article/S0022-5223(22)01034-0/fulltext.
Representative videos of pafolacianine-laden FR-positive tumor fluorescence captured by the UCF imaging system in a swine peribronchial tumor model. Each quadrant is from a different pafolacianine dose and lung location. The image software magnifies the merge image mode (white light and NIR pseudo-color overlay) on the left, whereas on the right corresponding alternative modes are shown (top to bottom: white light, raw NIR, merge mode, enhanced NIR with pseudo-color overlay). Laser power, 75 mW; exposure time, 640 ms; image gain 36; threshold for pseudo-color, 25. FR, Folate receptor; NIR, near-infrared; UCF, ultra-thin composite optical fiberscope. Video available at: https://www.jtcvs.org/article/S0022-5223(22)01034-0/fulltext.
Appendix E1
Figure E1Semiquantitative assessment of pafolacianine fluorescence by the UCF. A, NIR images of serially diluted pafolacianine solution at an exposure time of 40, 80, 160, 320, or 640 ms at a 5-mm distance. Laser power was 50 mW. B, NIR images of serially diluted pafolacianine solution at a distance of 3, 5, 7, 10, 15, 20 mm from the surface of the solution to the tip of the UCF at an exposure time of 640 ms. Laser power was 50 mW. C, Fluorescent intensities from NIR images at different distances as a function of pafolacianine concentration, measured in arbitrary units. D, Representative standard NIR images and enhanced NIR images with green pseudo-color by the UCF camera system (OKFT Image Processing Software). Laser power was 50 mW; exposure time, 640 ms; image gain, 30; threshold of pseudo-color, 25. NIR, Near-infrared.
Figure E2Representative overlay images of fluorescence in KB tumors and background tissue observed by the UCF across timepoints from a 10-mm distance. Fluorescence of subcutaneous xenografts with KB cells and contralateral normal thigh (“background”) were imaged by the UCF camera system, with corresponding NIR and overlay images shown at 6, 24, 48, and 72 hours after pafolacianine administration (0.05, 0.025, and 0.0125 mg/kg) (n = 3 per dose; n = 1 negative control injected with 5% dextrose solution). Laser power was 50 mW; exposure time, 640 ms; image gain, 30; threshold of pseudo-color, 25. NIR, Near-infrared.
Establishing the diagnosis of lung cancer: diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelines.
A preclinical research platform to evaluate photosensitizers for transbronchial localization and phototherapy of lung cancer using an orthotopic mouse model.
Assessment of bronchial wall thickness and lumen diameter in human adults using multi-detector computed tomography: comparison with theoretical models.
Johnson & Johnson Enterprise Innovation financially supported this study. A.G. is supported by a CIHR Frederick Banting and Charles Best Doctoral Award (FRN 170883) and a University of Toronto Hold'em for Life Oncology Clinician Scientist Award. Additional funding was provided through the William Coco Chair in Surgical Innovation for Lung Cancer.
Ishiwata and colleagues at the University of Toronto report their preclinical experiences involving a novel application of intraoperative molecular imaging (IMI) to improve yield of transbronchial biopsy for peribronchial lung lesions.1 IMI is an emerging technique that involves systemic delivery of optical contrast agents that preferentially accumulate in tumors. During procedures, these agents emit light when stimulated with a known quantity of energy (in this case, near infrared light). IMI has been evaluated by thoracic surgeons for several applications both during open and video-assisted thoracoscopic surgery/robotic-assisted thoracoscopic surgery; however, this report provides some of the first data supporting a role during endoscopic procedures.
01034-0&id=fx1.jpg){kind=link}
01034-0&id=fx2.jpg){kind=link}
01034-0&id=gr1.jpg){kind=link}
01034-0&id=gr2.jpg){kind=link}
01034-0&id=gr3.jpg){kind=link}
01034-0&id=gr4.jpg){kind=link}
01034-0&id=fx3.jpg){kind=link}
01034-0&id=fx7.jpg){kind=link}
01034-0&id=fx8.jpg){kind=link}