Non-Small Cell Lung Cancer Imaging Using a Phospholipase A2 Activatable Fluorophore.

Lung cancer, the most common cause of cancer-related death in the United States, requires advanced intraoperative detection methods to improve evaluation of surgical margins. In this study we employed DDAO-arachidonate (DDAO-A), a phospholipase A2 (PLA2) activatable fluorophore, designed for the specific optical identification of lung cancers in real-time during surgery. The in vitro fluorescence activation of DDAO-A by porcine sPLA2 was tested in various liposomal formulations, with 100 nm extruded EggPC showing the best overall characteristics. Extruded EggPC liposomes containing DDAO-A were tested for their stability under various storage conditions, demonstrating excellent stability for up to 4 weeks when stored at -20 °C or below. Cell studies using KLN 205 and LLC1 lung cancer cell lines showed DDAO-A activation was proportional to cell number. DDAO-A showed preferential activation by human recombinant cPLA2, an isoform highly specific to arachidonic acid-containing lipids, when compared to a control probe, DDAO palmitate (DDAO-P). In vivo studies with DBA/2 mice bearing KLN 205 lung tumors recapitulated these results, with preferential activation of DDAO-A relative to DDAO-P following intratumoral injection. Topical application of DDAO-A-containing liposomes to human (n = 10) and canine (n = 3) lung cancers ex vivo demonstrated the preferential activation of DDAO-A in tumor tissue relative to adjacent normal lung tissue, with fluorescent tumor-to-normal ratios (TNR) of up to 5.2:1. The combined results highlight DDAO-A as a promising candidate for clinical applications, showcasing its potential utility in intraoperative and back-table imaging and topical administration during lung cancer surgeries. By addressing the challenge of residual microscopic disease at resection margins and offering stability in liposomal formulations, DDAO-A emerges as a potentially valuable tool for advancing precision lung cancer surgery and improving curative resection rates.

Dose optimization of second window indocyanine green in meningioma patients.

OBJECTIVE: Surgery remains the first line treatment for meningiomas and can benefit from fluorescence-guided surgical techniques such as second-window indocyanine green (SWIG). In the current study, we compared the use of the standard SWIG dose of 5.0 mg/kg relative to 2.5 mg/kg indocyanine green (ICG) in meningioma patients. METHODS: Patients were prospectively enrolled in an IRB-approved study of SWIG and received either the standard dose of 5.0 mg/kg or a reduced dose of 2.5 mg/kg of ICG around 24 h prior to their surgery. Intraoperative near-infrared fluorescence imaging was performed with exo- and endoscopic systems. Signal-to-background ratio (SBR) was calculated to quantify fluorescence and was compared between 5.0 mg/kg and 2.5 mg/kg ICG. All patients received pre-operative MRI and, in select cases, the pre-operative MRI was correlated to intraoperative fluorescence imaging. RESULTS/DISCUSSION: In the current study, we found no significant difference in the SBR of meningiomas in patients that were administered with either 5.0 mg/kg or 2.5 mg/kg ICG. However, in five patients that received the standard-dose SWIG regimen of 5.0 mg/kg ICG we observed dose-related fluorescence quenching - referred to as "inversion" - that interfered with tumor visualization during fluorescence-guided surgery (FGS). When correlated to pre-operative MRI, a similar rim pattern was observed around the primary tumor on T2 FLAIR, which, in retrospect, could be used as a predictor for inversion during FGS in meningioma patients receiving standard-dose ICG. CONCLUSION: This study demonstrated that a reduced ICG dose was as effective as standard-dose SWIG in meningioma patients. We therefore recommend to adjust the standard ICG dose for meningioma patients to 2.5 mg/kg particularly when rim enhancement is observed on pre-operative T2 FLAIR.

Second window ICG predicts postoperative MRI gadolinium enhancement in high grade gliomas and brain metastases.

A prospective trial evaluating the utility of second window indocyanine green (SWIG) in predicting postoperative MRI gadolinium enhancement was performed on high-grade gliomas (HGGs) and brain metastases. Compared to white light alone, SWIG demonstrated a higher sensitivity, negative predictive value, and accuracy in predicting residual neoplasm on MRI. The specificity of SWIG for predicting MRI enhancement was higher in HGGs than brain metastases. Clinically, near-infrared (NIR) imaging was better able to predict tumor recurrence than postoperative MRI. These results illustrate how SWIG is able to take advantage of gadolinium-like distribution properties to extravasate into the tumor microenvironment, enabling guidance in surgical resection. The video can be found here: https://stream.cadmore.media/r10.3171/2021.10.FOCVID21204.

Indocyanine green fluorescence-guided surgery in head and neck cancer: A systematic review.

OBJECTIVE: To assess the feasibility and effectiveness of indocyanine green (ICG) for image-guided resection of head and neck cancer (HNC). DATA SOURCES: PubMed, Embase, and Scopus databases. REVIEW METHODS: Searches were conducted from database inception to February 2022. Patient and study characteristics, imaging parameters, and imaging efficacy data were extracted from each study. RESULTS: Nine studies met inclusion criteria, representing 103 head and neck tumors. Weighted mean ICG dose and imaging time were 1.27 mg/kg and 11.77 h, respectively. Among the five studies that provided quantitative metrics of imaging efficacy, average ICG tumor-to-background ratio (TBR) was 1.56 and weighted mean ONM-100 TBR was 3.64. Pooled sensitivity and specificity across the five studies were 91.7 % and 71.9 %, respectively. CONCLUSION: FGS with ICG may facilitate real-time tumor-margin delineation to improve margin clearance rates and progression-free survival. Future studies with validated, quantitative metrics of imaging success are necessary to further evaluate the prognostic benefit of these techniques.

Design and evaluation of Raman reporters for the Raman-silent region.

Rationale: Surface enhanced Raman scattering (SERS) is proving to be a useful tool for biomedical imaging. However, this imaging technique can suffer from poor signal-to-noise ratio, as the complexity of biological tissues can lead to overlapping of Raman bands from tissues and the Raman reporter molecule utilized. Methods: Herein we describe the synthesis of triple bond containing Raman reporters that scatter light in the biological silent window, between 1750 cm-1 and 2750 cm-1. Results: Our SERS nanoprobes are comprised of uniquely designed Raman reporters containing either alkyne- or cyano-functional groups, enabling them to be readily distinguished from background biological tissue. Conclusion: We identify promising candidates that eventually can be moved forward as Raman reporters in SERS nanoparticles for highly specific contrast-enhanced Raman-based disease or analyte detection in biological applications.

Design and synthesis of gold nanostars-based SERS nanotags for bioimaging applications.

Surface-enhanced Raman spectroscopy (SERS) nanotags hold a unique place among bioimaging contrast agents due to their fingerprint-like spectra, which provide one of the highest degrees of detection specificity. However, in order to achieve a sufficiently high signal intensity, targeting capabilities, and biocompatibility, all components of nanotags must be rationally designed and tailored to a specific application. Design parameters include fine-tuning the properties of the plasmonic core as well as optimizing the choice of Raman reporter molecule, surface coating, and targeting moieties for the intended application. This review introduces readers to the principles of SERS nanotag design and discusses both established and emerging protocols of their synthesis, with a specific focus on the construction of SERS nanotags in the context of bioimaging and theranostics.

225Actinium-labeled prostate-specific membrane antigen targeting peptide induces complete response in a metastatic prostate cancer patient.

Targeted radionuclide therapy has emerged as a promising and potentially curative strategy for high-grade prostate cancer. However, limited data are available on efficacy, quality of life, and pretherapeutic biomarkers. Here, we highlight the case of a patient with prostate-specific membrane antigen (PSMA)-positive metastatic castrate-resistant prostate cancer who displayed complete response to 225Ac-PSMA-617 after having been resistant to standard-of-care therapy, then initially partially responsive but later resistant to subsequent immunotherapy, and resistant to successive 177Lu-PSMA-617. In addition, the patient's baseline germline mutation likely predisposed him to more aggressive disease.

Small Molecule, Multimodal, [18F]-PET and Fluorescence Imaging Agent Targeting Prostate-Specific Membrane Antigen: First-in-Human Study.

BACKGROUND: A first-in-human study of [18F]-BF3-Cy3-ACUPA, a small-molecule imaging agent that can be unimolecularly both positron emitting and fluorescent, is conducted to determine its safety, biodistribution, radiation dosimetry, feasibility in tumor detection by preoperative positron emission tomography (PET), as well as its intraoperative fluorescence imaging utility in patients with prostate-specific membrane antigen positive (PSMA+) tumors. METHODS: Ten patients aged 66 ± 7 years received a 6.5 ± 3.2 mCi intravenous injection of [18F]-BF3-Cy3-ACUPA and underwent PET/computed tomography (CT) imaging. Radiation dosimetry of [18F]-BF3-Cy3-ACUPA, normal organ biodistribution, and tumor uptakes were examined. Two patients were prescheduled for radical prostatectomy (RP) with extended pelvic lymphadenectomy approximately 24 hours following [18F]-BF3-Cy3-ACUPA injection and imaging. Without reinjection, intraoperative fluorescence imaging was performed on freshly excised tissue during RP. Frozen sections of excised tissue during RP were submitted for confirmatory histopathology and multiphoton fluorescence and brightfield microscopy. RESULTS: Absorbed doses by organs including the kidneys and salivary glands were similar to 68Ga-PSMA-11 imaging. [18F]-BF3-Cy3-ACUPA physiologic radiotracer accumulation and urinary/biliary excretion closely resembled the distribution of other published PSMA tracers including [18F]-JK-PSMA-7, [18F]-PSMA-1007, [18F]-DCFPyL, and [18F]-DCFBC. 19F-BF3-Cy3-ACUPA was retained in PSMA+ cancer tissues in patients for at least 24 hours, allowing for intraoperative fluorescence assessment of the prostate and of the embedded prostate cancer without contrast reinjection. After 24 hours, the imaging agent mostly decayed or cleared from the blood pool. Preoperative PET and fluorescence imaging findings were confirmed with final histopathology and multiphoton microscopy. CONCLUSION: Our first-in-human results demonstrate that [18F]-BF3-Cy3-ACUPA is safe and feasible in humans. Larger trials with this PET tracer are expected to further define its capabilities and its clinical role in the management of PSMA+ tumors, especially in prostate cancer.

Somatostatin Receptor as a Molecular Imaging Target in Human and Canine Cushing Disease.

OBJECTIVES: Fluorescence-guided surgery may improve completeness of resection in transsphenoidal surgery for Cushing disease (CD) by enabling visualization of residual tumor tissue at the margins. In this review we discuss somatostatin receptors (SSTRs) as targets for fluorescence-guided surgery and overview existing SSTR-specific imaging agents. We also compare SSTR expression in normal pituitary and corticotrophinoma tissues from human and canine CD patients to assess canines as a translational model for CD. METHODS: A PubMed literature search was conducted for publications containing the terms canine, somatostatin receptor, Cushing's disease, and corticotroph adenoma. SSTR expression data from each study was documented as the presence or absence of expression or, when possible, the number of tumors expressing a given SSTR subtype within a group of tumors being studied. Studies that used reverse transcription polymerase chain reaction to quantify SSTR expression were selected for additional comparative analysis. RESULTS: SSTR5 is strongly expressed in human corticotroph adenomas and weakly expressed in surrounding pituitary parenchyma, a pattern not conclusively observed in canine patients. SSTR2 mRNA expression is similar in human normal pituitary and corticotrophinoma cells but may be significantly higher in canine normal pituitary tissue than in corticotroph tumoral tissue. Limited data were available on SSTR subtypes 1, 3, and 4. CONCLUSIONS: Further studies must fill the knowledge gaps related to species-specific SSTR expression, so using canine CD as a translational model may be premature. We do conclude that the expression profile of SSTR5 (i.e., high local expression in pituitary adenomas relative to normal surrounding tissues) makes SSTR5 a promising molecular target for FGS.

Nanoscopic subcellular imaging enabled by ion beam tomography.

Multiplexed ion beam imaging (MIBI) has been previously used to profile multiple parameters in two dimensions in single cells within tissue slices. Here, a mathematical and technical framework for three-dimensional (3D) subcellular MIBI is presented. Ion-beam tomography (IBT) compiles ion beam images that are acquired iteratively across successive, multiple scans, and later assembled into a 3D format without loss of depth resolution. Algorithmic deconvolution, tailored for ion beams, is then applied to the transformed ion image series, yielding 4-fold enhanced ion beam data cubes. To further generate 3D sub-ion-beam-width precision visuals, isolated ion molecules are localized in the raw ion beam images, creating an approach coined as SILM, secondary ion beam localization microscopy, providing sub-25 nm accuracy in original ion images. Using deep learning, a parameter-free reconstruction method for ion beam tomograms with high accuracy is developed for low-density targets. In cultured cancer cells and tissues, IBT enables accessible visualization of 3D volumetric distributions of genomic regions, RNA transcripts, and protein factors with 5 nm axial resolution using isotope-enrichments and label-free elemental analyses. Multiparameter imaging of subcellular features at near macromolecular resolution is implemented by the IBT tools as a general biocomputation pipeline for imaging mass spectrometry.

A protease-activated, near-infrared fluorescent probe for early endoscopic detection of premalignant gastrointestinal lesions.

Fluorescence imaging is currently being actively developed for surgical guidance; however, it remains underutilized for diagnostic and endoscopic surveillance of incipient colorectal cancer in high-risk patients. Here we demonstrate the utility and potential for clinical translation of a fluorescently labeled cathepsin-activated chemical probe to highlight gastrointestinal lesions. This probe stays optically dark until it is activated by proteases produced by tumor-associated macrophages and accumulates within the lesions, enabling their detection using an endoscope outfitted with a fluorescence detector. We evaluated the probe in multiple murine models and a human-scale porcine model of gastrointestinal carcinogenesis. The probe provides fluorescence-guided surveillance of gastrointestinal lesions and augments histopathological analysis by highlighting areas of dysplasia as small as 400 µm, which were visibly discernible with significant tumor-to-background ratios, even in tissues with a background of severe inflammation and ulceration. Given these results, we anticipate that this probe will enable sensitive fluorescence-guided biopsies, even in the presence of highly inflamed colorectal tissue, which will improve early diagnosis to prevent gastrointestinal cancers.

A dual-modal PET/near infrared fluorescent nanotag for long-term immune cell tracking.

Adoptive cell transfer of targeted chimeric antigen receptor (CAR) T cells has emerged as a highly promising cancer therapy. The pharmacodynamic action or CAR T cells is closely related to their pharmacokinetic profile; because of this as well as the risk of non-specific action, it is important to monitor their biodistribution and fate following infusion. To this end, we developed a dual-modal PET/near infrared fluorescent (NIRF) nanoparticle-based imaging agent for non-genomic labeling of human CAR T cells. Since the PET/NIRF nanoparticles did not affect cell viability or cytotoxic functionality and enabled long-term whole-body CAR T cell tracking using PET and NIRF in an ovarian peritoneal carcinomatosis model, this platform is a viable imaging technology to be applied in other cancer models.

Applications of indocyanine green in brain tumor surgery: review of clinical evidence and emerging technologies.

Indocyanine green (ICG) is a water-soluble dye that was approved by the FDA for biomedical purposes in 1956. Initially used to measure cardiocirculatory and hepatic functions, ICG's fluorescent properties in the near-infrared (NIR) spectrum soon led to its application in ophthalmic angiography. In the early 2000s, ICG was formally introduced in neurosurgery as an angiographic tool. In 2016, the authors' group pioneered a novel technique with ICG named second-window ICG (SWIG), which involves infusion of a high dose of ICG (5.0 mg/kg) in patients 24 hours prior to surgery. To date, applications of SWIG have been reported in patients with high-grade gliomas, meningiomas, brain metastases, pituitary adenomas, craniopharyngiomas, chordomas, and pinealomas.The applications of ICG have clearly expanded rapidly across different specialties since its initial development. As an NIR fluorophore, ICG has advantages over other FDA-approved fluorophores, all of which are currently in the visible-light spectrum, because of NIR fluorescence's increased tissue penetration and decreased autofluorescence. Recently, interest in the latest applications of ICG in brain tumor surgery has grown beyond its role as an NIR fluorophore, extending into shortwave infrared imaging and integration into nanotechnology. This review aims to summarize reported clinical studies on ICG fluorescence-guided surgery of intracranial tumors, as well as to provide an overview of the literature on emerging technologies related to the utility of ICG in neuro-oncological surgeries, including the following aspects: 1) ICG fluorescence in the NIR-II window; 2) ICG for photoacoustic imaging; and 3) ICG nanoparticles for combined diagnostic imaging and therapy (theranostic) applications.

Isotopically Encoded Nanotags for Multiplexed Ion Beam Imaging.

High-dimensional profiling of markers and analytes using approaches, such as barcoded fluorescent imaging with repeated labeling and mass cytometry has allowed visualization of biological processes at the single-cell level. To address limitations of sensitivity and mass-channel capacity, a nanobarcoding platform is developed for multiplexed ion beam imaging (MIBI) using secondary ion beam spectrometry that utilizes fabricated isotopically encoded nanotags. Use of combinatorial isotope distributions in 100 nm sized nanotags expands the labeling palette to overcome the spectral bounds of mass channels. As a proof-of-principle, a four-digit (i.e., 0001-1111) barcoding scheme is demonstrated to detect 16 variants of 2H, 19F, 79/81Br, and 127I elemental barcode sets that are encoded in silica nanoparticle matrices. A computational debarcoding method and an automated machine learning analysis approach are developed to extract barcodes for accurate quantification of spatial nanotag distributions in large ion beam imaging areas up to 0.6 mm2. Isotopically encoded nanotags should boost the performance of mass imaging platforms, such as MIBI and other elemental-based bioimaging approaches.

Detection of Premalignant Gastrointestinal Lesions Using Surface-Enhanced Resonance Raman Scattering-Nanoparticle Endoscopy.

Cancers of the gastrointestinal (GI) tract are among the most frequent and most lethal cancers worldwide. An important reason for this high mortality is that early disease is typically asymptomatic, and patients often present with advanced, incurable disease. Even in high-risk patients who routinely undergo endoscopic screening, lesions can be missed due to their small size or subtle appearance. Thus, current imaging approaches lack the sensitivity and specificity to accurately detect incipient GI tract cancers. Here we report our finding that a single dose of a high-sensitivity surface-enhanced resonance Raman scattering nanoparticle (SERRS-NP) enables reliable detection of precancerous GI lesions in animal models that closely mimic disease development in humans. Some of these animal models have not been used previously to evaluate imaging probes for early cancer detection. The studies were performed using a commercial Raman imaging system, a newly developed mouse Raman endoscope, and finally a clinically applicable Raman endoscope for larger animal studies. We show that this SERRS-NP-based approach enables robust detection of small, premalignant lesions in animal models that faithfully recapitulate human esophageal, gastric, and colorectal tumorigenesis. This method holds promise for much earlier detection of GI cancers than currently possible and could lead therefore to marked reduction of morbidity and mortality of these tumor types.

Biodegradable fluorescent nanoparticles for endoscopic detection of colorectal carcinogenesis.

Early and comprehensive endoscopic detection of colonic dysplasia - the most clinically significant precursor lesion to colorectal adenocarcinoma - provides an opportunity for timely, minimally-invasive intervention to prevent malignant transformation. Here, the development and evaluation of biodegradable near-infrared fluorescent silica nanoparticles (FSN) is described that have the potential to improve adenoma detection during fluorescence-assisted white-light colonoscopic surveillance in rodent and human-scale models of colorectal carcinogenesis. FSNs are biodegradable (t1/2 of 2.7 weeks), well-tolerated, and enable detection and delineation of adenomas as small as 0.5 mm2 with high tumor-to-background ratios. Furthermore, in the human-scale, APC 1311/+ porcine model, the clinical feasibility and benefit of using FSN-guided detection of colorectal adenomas using video-rate fluorescence-assisted white-light endoscopy is demonstrated. Since nanoparticles of similar size (e.g., 100-150-nm) or composition (i.e., silica, silica/gold hybrid) have already been successfully translated to the clinic, and, clinical fluorescent/white light endoscopy systems are becoming more readily available, there is a viable path towards clinical translation of the proposed strategy for early colorectal cancer detection and prevention in high-risk patients.

Dual-Modality Surface-Enhanced Resonance Raman Scattering and Multispectral Optoacoustic Tomography Nanoparticle Approach for Brain Tumor Delineation.

Difficulty in visualizing glioma margins intraoperatively remains a major issue in the achievement of gross total tumor resection and, thus, better clinical outcome of glioblastoma (GBM) patients. Here, the potential of a new combined optical + optoacoustic imaging method for intraoperative brain tumor delineation is investigated. A strategy using a newly developed gold nanostar synthesis method, Raman reporter chemistry, and silication method to produce dual-modality contrast agents for combined surface-enhanced resonance Raman scattering (SERRS) and multispectral optoacoustic tomography (MSOT) imaging is devised. Following intravenous injection of the SERRS-MSOT-nanostars in brain tumor bearing mice, sequential MSOT imaging is performed in vivo and followed by Raman imaging. MSOT is able to accurately depict GBMs three-dimensionally with high specificity. The MSOT signal is found to correlate well with the SERRS images. Because SERRS enables uniquely sensitive high-resolution surface detection, it could represent an ideal complementary imaging modality to MSOT, which enables real-time, deep tissue imaging in 3D. This dual-modality SERRS-MSOT-nanostar contrast agent reported here is shown to enable high precision depiction of the extent of infiltrating GBMs by Raman- and MSOT imaging in a clinically relevant murine GBM model and could pave new ways for improved image-guided resection of brain tumors.

MUC1 Aptamer Targeted SERS Nanoprobes.

Recently, surface-enhanced Raman scattering (SERS) nanoprobes (NPs) have shown promise in the field of cancer imaging due to their unparalleled signal specificity and high sensitivity. Here we report the development of a DNA aptamer targeted SERS NP. Recently, aptamers are being investigated as a viable alternative to more traditional antibody targeting due to their low immunogenicity and low cost of production. We developed a strategy to functionalize SERS NPs with DNA aptamers, which target Mucin1 (MUC1) in human breast cancer (BC). Thorough in vitro characterization studies demonstrated excellent serum stability and specific binding of the targeted NPs to MUC1. In order to test their in vivo targeting capability, we co-injected MUC1-targeted SERS NPs, and as controls non-targeted and blocked MUC1-targeted SERS NPs in BC xenograft mouse models. A two-tumor mouse model with differential expression of MUC1 (MDA-MB-468 and MDA-MB-453) was used to control for active versus passive targeting in the same animals. The results showed that the targeted SERS NPs home to the tumors via active targeting of MUC1, with low levels of passive targeting. We expect this strategy to be an advantageous alternative to antibody-based targeting and useful for targeted imaging of tumor extent, progression, and therapeutic response.

Chelator-Free Radiolabeling of SERRS Nanoparticles for Whole-Body PET and Intraoperative Raman Imaging.

A single contrast agent that offers whole-body non-invasive imaging along with the superior sensitivity and spatial resolution of surface-enhanced resonance Raman scattering (SERRS) imaging would allow both pre-operative mapping and intraoperative imaging and thus be highly desirable. We hypothesized that labeling our recently reported ultrabright SERRS nanoparticles with a suitable radiotracer would enable pre-operative identification of regions of interest with whole body imaging that can be rapidly corroborated with a Raman imaging device or handheld Raman scanner in order to provide high precision guidance during surgical procedures. Here we present a straightforward new method that produces radiolabeled SERRS nanoparticles for combined positron emission tomography (PET)-SERRS tumor imaging without requiring the attachment of molecular chelators. We demonstrate the utility of these PET-SERRS nanoparticles in several proof-of-concept studies including lymph node (LN) tracking, intraoperative guidance for LN resection, and cancer imaging after intravenous injection. We anticipate that the radiolabeling method presented herein can be applied generally to nanoparticle substrates of various materials by first coating them with a silica shell and then applying the chelator-free protocol.