Far-Red Spray-On Imaging Probes for FAP-Targeted Cancer Surgery.

Ovarian cancer is the most lethal gynecological cancer, with a survival rate of approximately 40% at five years from the diagno. The first-line treatment consists of cytoreductive surgery combined with chemotherapy (platinum- and taxane-based drugs). To date, the main prognostic factor is related to the complete surgical resection of tumor lesions, including occult micrometastases. The presence of minimal residual diseases not detected by visual inspection and palpation during surgery significantly increases the risk of disease relapse. Intraoperative fluorescence imaging systems have the potential to improve surgical outcomes. Fluorescent tracers administered to the patient may support surgeons for better real-time visualization of tumor lesions during cytoreductive procedures. In the last decade, consistent with the discovery of an increasing number of ovarian cancer-specific targets, a wide range of fluorescent agents were identified to be employed for intraoperatively detecting ovarian cancer. Here, we present a collection of fluorescent probes designed and developed for fluorescence-guided ovarian cancer surgery. Original articles published between 2011 and November 2022 focusing on fluorescent probes, currently under preclinical and clinical investigation, were searched in PubMed. The keywords used were targeted detection, ovarian cancer, fluorescent probe, near-infrared fluorescence, fluorescence-guided surgery, and intraoperative imaging. All identified papers were English-language full-text papers, and probes were classified based on the location of the biological target: intracellular, membrane, and extracellular.

Malignant gliomas are major causes of cancer-related mortality and morbidity. Traditional surgery usually leads to incomplete resection of gliomas resulting in the high incidence of tumor recurrence. Advanced medical imaging technology, such as fluorescence imaging-guided surgery, combined with tumor-specific imaging probes allows the identification of tumor margins and improved surgery. However, there are two pressing issues that need to be addressed: first, few fluorescence imaging probes can specifically target gliomas; second, fluorescence molecular imaging (FMI) cannot get the in-depth information of deep-seated gliomas; both of which affect the complete removal of the gliomas. In this study, the biodistribution of smart matrix metalloproteinase (MMP) targeting near-infrared (NIR) fluorescent probe MMPSense 750 FAST (MMP-750) was examined in both U87MG-GFP-fLuc glioma xenograft and orthotopic mouse models using FMI. Then, CT and FMI images of orthotopic gliomas were acquired for the reconstruction of fluorescence molecular tomography (FMT) using a randomly enhanced adaptive subspace pursuit (REASP) algorithm. Furthermore, the resection of orthotopic glioma was performed using the fluorescence surgical navigation system after the injection of the MMP-750 probe. After surgery, bioluminescence imaging (BLI) and hematoxylin and eosin staining were carried out to confirm the precision resection of the tumor. FMI results showed that the MMP-750 probe can specifically target U87MG glioma in vivo. FMT presented the spatial information of the orthotopic glioma using the REASP reconstruction algorithm. Furthermore, MMP-750 could effectively delineate the tumor margin during glioma surgery leading to a complete resection of the tumors. The smart MMP-750 specifically targets the glioma and FMT of MMP-750 provides 3D information for the spatial localization of the glioma. MMP-750 can work as an ideal fluorescence probe for guiding the intraoperative surgical resection of the glioma, possessing clinical translation.

Early detection and complete resection of oral squamous cell carcinoma (OSCC) are crucial to improving patient survival and prognosis. However, specifically targeted imaging probes for OSCC detection are limited. Our study aimed to synthesize a novel near-infrared fluorescence (NIRF) probe for precision detection and fluorescence image-guided surgery in OSCC. Bioinformatics data indicated that glucose transporter 1 (GLUT1) is highly expressed in patients with OSCC. We demonstrated high and specific GLUT1 expression upon immunohistochemical staining of samples from 20 patients with OSCC. The specific expression of GLUT1 was further validated in both human OSCC cell lines and OSCC tumor xenografts. Based on these findings, the GLUT1 inhibitor WZB117 was utilized to synthesize a novel NIRF imaging probe, WZB117-IR820. The fluorescence molecular imaging data revealed that WZB117-IR820 could specifically bind to the tumor areas in an orthotopic OSCC mouse model after intravenous injection and could be further applied for precision fluorescence image-guided surgery with no residual tumor in the orthotopic CAL27-fLUC mouse tumor model. For further clinical translational application in patients with OSCC, precise delineation of OSCC tumor areas was achieved after topical application of the WZB117-IR820 imaging probe and was validated by histopathological and immunohistochemical analyses. In conclusion, we synthesized a novel fluorescent imaging probe, WZB117-IR820, which has potential clinical applications for early detection and fluorescence image-guided surgery in OSCC with no observable toxicity.

Recent developments in multimodality fluorescence imaging probes

An advanced organic molecular probe for multimodal fluorescence imaging of cellular lipid droplets

IntroductionPositive resection margins occur in about 2.8%-8.2% gastric cancer surgeries and is associated with poor prognosis. Intraoperative guidance using Nearinfrared (NIR) fluorescence imaging is a promising technique for tumor detection and margin assessment. The goal of this study was to develop a tumor-specific probe for real-time intraoperative NIR fluorescence imaging guidance.MethodsThe tumor vascular homing peptide specific for gastric cancer, GEBP11, was conjugated with a near-infrared fluorophore, Cy5.5. The binding specificity of the GEBP11 probes to tumor vascular endothelial cells were confirmed by immunofluorescent staining. The ability of the probe to detect tumor lesions was evaluated in two xenograft models. An orthotopic gastric cancer xenograft model was used to evaluate the efficacy of the GEBP11 NIR probes in real-time surgical guidance.ResultsIn vitro assay suggested that both mono and dimeric GEBP11 NIR probes could bind specifically to tumor vascular epithelial cells, with dimeric peptides showed better affinity. In tumor xenograft mice, live imaging suggested that comparing with free Cy5.5 probe, significantly stronger NIR signals could be detected at the tumor site at 24-48h after injection of mono or dimeric GEBP11 probes. Dimeric GEBP11 probe showed prolonged and stronger NIR signals than mono GEBP11 probe. Biodistribution assay suggested that GEBP11 NIR probes were enriched in gastric cancer xenografts. Using dimeric GEBP11 NIR probes in real-time surgery, the tumor margins and peritoneal metastases could be clearly visualized. Histological examination confirmed the complete resection of the tumor.Conclusion(GEBP11)2-ACP-Cy5.5 could be a potential useful probe for intraoperative florescence guidance in gastric cancer surgery.

There has been a dramatic increase in the use of fluorescence imaging for visualizing biological structures during surgery; a MEDLINE search with the keywords “fluorescence imaging” and “surgery” yielded year-on-year increases in the number of publications, which reached more than 1,200 articles in 2012 (fig. ​(fig.1).1). Use of fluorescence imaging in surgical settings started at the beginning of the 21st century with angiography using indocyanine green (ICG) as a source of fluorescence during coronary artery bypass grafting [1], and this was followed by sentinel node navigation in breast cancer surgery [2] and intraoperative angiography for cerebral aneurysm [3] in the mid 2000s. Since near-infrared imaging systems became commercially available for open surgery in 2005 and for laparoscopic surgery in 2011, the application of fluorescence-guided surgery has further accelerated. Fig. 1 Annual number of publications (N) on fluorescence imaging in the field of surgical treatment. A MEDLINE search was carried out using the keywords “fluorescence imaging” and “surgery” (accessed on 19 December 2013). The ... Among the numerous fluorescent probes available for in vivo imaging, ICG remains the mainstay in the clinical setting. The safety of ICG has been established over more than 50 years of clinical usage. Its fluorescent properties (excitation 750-810 nm, emission around 830 nm) mean that the signal is not absorbed by hemoglobin or water, which confers advantages in visualizing deep-lying structures. In the field of hepatobiliary surgery, the fact that ICG is excreted in bile is a very useful pharmacological characteristic. For example, ICG-fluorescence imaging enables delineation of the bile ducts following intravenous ICG injection (fluorescence cholangiography) [4, 5, 6]. Using ICG, intraoperative identification of both primary and secondary hepatic malignancies is possible through visualization of the biliary excretion disorders that exist in hepatocellular carcinoma tissues and in non-cancerous hepatic parenchyma compressed by metastatic tumors [7, 8, 9, 10]; this approach may lead to the development of photodynamic treatment [11]. Currently available techniques can also be used to assess portal uptake in hepatic segments with venous occlusion during liver resection or transplantation [12]. Another clinically available fluorescent probe is 5-aminolevulinic acid; however, its application to digestive surgery has rarely been evaluated. In basic and preclinical studies, many novel fluorescent probes have been developed to enhance ICG-based fluorescence imaging to delineate biliary and vascular anatomy and to detect cancerous tissues other than hepatocellular carcinoma. Among these techniques, intraoperative fluorescence imaging of pancreatic leaks has the potential to reduce the incidence and severity of postoperative pancreatic fistulas [13]. Novel fluorescence imaging systems are also being actively developed to allow simultaneous identification of two or more structures with different fluorescence probes [14]; furthermore, fluorescence goggle systems worn by the operating surgeon should soon be able to provide real-time visual information on the surgical field [15]. The history and techniques of fluorescence-guided surgery are well-summarized in a recent review article by Vahrmeijer et al. [16] and are also described in detail in the book Fluorescent Imaging: Treatment of Hepatobiliary and Pancreatic Diseases[17]. In addition, the first international symposium on fluorescence-guided surgery, held in February 2014, promoted the sharing of cutting-edge knowledge for further development of these exciting new techniques. Conventionally, intraoperative diagnosis and decision-making have been based on surgeons' visual inspection and palpation, with the aid of ultrasonography and radiation. Fluorescence imaging will open a new era of surgery, in which more detailed information on vascular anatomy and cancer status will be rapidly available for surgeons to view when needed, enhancing the safety and efficacy of digestive surgery.

Accurate delineation of tumor margins and maximal safe resection are critical for successful curative oncologic surgery. However, fibroblast activation protein (FAP)-targeted probes suitable for fluorescence imaging remain limited. Here, we developed novel FAP-targeted fluorescent probes to accurately delineate tumor margins and enable rapid intraoperative identification of tumor boundaries in resected specimens. DOTA chelator for radiolabelling with gallium-68 was incorporated into dual-modality FAP-targeted probes synthesised by conjugating FAP-2286 and 3BP-3940 with the near-infrared (NIR) fluorophore IRDye800CW. These probes were evaluated both in vitro and in vivo using HEK293T-FAP cells stably expressing FAP and xenograft mouse models. Positron emission tomography (PET) and (NIR-II) fluorescence imaging assessed the specificity and ability of probes to delineate tumor margins. For clinical validation, resected lung tissue was incubated ex vivo with the probes. Tumor regions and margins were identified using fluorescence imaging and subsequently validated by haematoxylin and eosin (H&E) staining and FAP immunohistochemistry. Both IRDye800CW-FAP-2286 and IRDye800CW-3BP-3940 exhibited high affinity for FAP-positive cells in vitro. PET imaging revealed high tumor specificity for both probes in vivo. In vivo and ex vivo NIR-II fluorescence imaging enabled accurate visualisation of tumor margins, with IRDye800CW-3BP-3940 exhibiting superior performance compared with IRDye800CW-FAP-2286. In the clinical specimen, IRDye800CW-3BP-3940 successfully delineated tumor regions with strong concordance to histopathological findings. We developed and validated a novel dual-modality molecular probe, IRDye800CW-3BP-3940, which integrated PET and NIR fluorescence imaging capabilities. This probe enabled highly specific detection of FAP-positive tumors and precise delineation of tumor margins in resected specimens.

BackgroundEarly detection and treatment are critical for improving the survival and prognosis of patients with cervical cancer. However, there is a notable scarcity of targeted imaging probes specifically designed to detect high-grade squamous intraepithelial lesions (HSIL) and cervical cancer. Our study aimed to address this gap by identifying and validating a targeted imaging probe for these conditions.ResultsUsing bioinformatics data, we identified galectin-7 (GAL7) as highly expressed in patients with cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC). Immunohistochemical staining of biopsy samples from 30 HSIL and cervical cancer patients verified the high and specific expression of GAL7. Further validation was performed using mouse and human CESC cell lines and tumor xenografts, confirming the consistent expression of GAL7. Based on this finding, we synthesized a GAL7-specific antibody conjugated with FITC, creating the GAL7-FITC fluorescence imaging probe. Fluorescence molecular imaging revealed that GAL7-FITC exhibited specific binding to various CESC cell lines and xenograft mouse models. Additionally, the diagnostic capability of GAL7-FITC was demonstrated in fresh HSIL specimens from cervical cone excisions, validated through histopathology and immunohistochemical analysis.ConclusionsOur study identified GAL7 as a specific target for CESC and successfully developed the GAL7-FITC fluorescence imaging probe. GAL7-FITC has shown promising potential for clinical application in the early detection of HSIL and CESC, providing rapid fluorescence imaging diagnosis without observable toxicity. This advancement may significantly enhance the accuracy and speed of cervical cancer diagnostics, ultimately improving patient outcomes.

Fluorescence-guided surgery (FGS) was pioneered for glioma and is now established as the standard of care. Gliomas are infiltrative tumours with diffuse margins. FGS provides improved intra-operative identification of tumour margins based on tumour-specific emission visible to the operating surgeon, resulting in increased rates of gross total resection. Multiple fluorescence agents may be used including 5-ALA, fluorescein sodium, and indocyanine green (ICG). This review details the indication, required equipment, mechanism of action, evidence base, limitations, and regulatory issues for each fluorophore as utilised in current clinical practice. FGS for glioma is limited by a reliance on subjective interpretation of visible fluorescence, which is often not present in low-grade glioma (LGG) or at the infiltrative tumour margin. Consequently, there has been a drive to develop enhanced, objective FGS techniques utilising both quantitative fluorescence (QF) imaging systems and novel fluorophores. This review provides an overview of emerging QF imaging systems for FGS. The pipeline for novel fluorophore development is also summarised.

Background: Peritoneal dissemination is common in advanced ovarian cancer. The completeness of cytoreduction is an independent prognostic factor. The intraoperative fluorescence imaging via tumor-specific near-infrared fluorophore might improve staging and surgical completeness. A promising target for ovarian cancer is the gonadotropin-releasing hormone receptor (GnRHR). This study aimed to develop a GnRHR-targeted near-infrared imaging probe for the detection of peritoneal metastases of ovarian cancer.Methods: Indocyanine green (ICG) was conjugated with GnRH antagonist peptide to develop an ovarian cancer-selective fluorescence probe GnRHa-ICG. GnRHR expression was detected in ovarian cancer tissues. The binding capacity of GnRHa-ICG and ICG was detected in both cancer cell lines and mouse models of peritoneal metastatic ovarian cancer using fluorescence microscopy, flow cytometry, and near-infrared fluorescence imaging.Results: Tissue microarray analysis revealed the overexpression of GnRHR in ovarian cancer. GnRH-ICG exhibited the binding capacity in a panel of cancer cell lines with different expression levels of GnRHR. In ovarian cancer mouse models, GnRHa-ICG signals were detected in peritoneal tumor lesions rather than normal peritoneal and intestines tissues. ICG showed intensive fluorescence signals in intestines. The tumor-to-muscle ratio and tumor-to-intestine ratio of GnRHa-ICG was 7.41 ± 2.82 and 4.37 ± 1.66, higher than that of ICG (4.60 ± 0.50 and 0.57 ± 0.06) at 2 h post administration. The fluorescence signal of peritoneal metastases peaked in intensity at 2 h and maintained for up to 48 h. ICG also showed a weak signal in the tumor lesions due to the enhanced permeability and retention effect, but the intensity decreased quickly within 48 h.Conclusions: The developed GnRHR-targeted imaging agent GnRHa-ICG could specifically detected peritoneal tumor lesions from normal peritoneal and intestines tissues because of the modification of GnRHa to ICG. The plateau period of GnRHa-ICG accumulation may be feasible for clinical applications in fluorescence-guided surgery. Our GnRHR imaging concept may be effective in other hormone-related tumors with upregulated GnRHR expression.

Small molecule interactions with amyloid proteins have had a huge impact in Alzheimer's disease (AD), especially in three specific areas: amyloid folding, metabolism and brain imaging. Amyloid plaque amelioration or prevention have, until recently, driven drug development, and only a few drugs have been advanced for use in AD. Amyloid proteins undergo misfolding and oligomerization via intermediates, eventually forming protease resistant amyloid fibrils. These fibrils accumulate to form the hallmark amyloid plaques and tangles of AD. Amyloid binding compounds can be grouped into three categories, those that: i) prevent or reverse misfolding, ii) halt misfolding or trap intermediates, and iii) accelerate the formation of stable and inert amyloid fibrils. Such compounds include hydralazine, glycosaminoglycans, curcumin, beta sheet breakers, catecholamines, and ATP. The versatility of amyloid binding compounds suggests that the amyloid structure may serve as a scaffold for the future development of sensors to detect such compounds. Metabolic dysfunction is one of the earliest pathological features of AD. In fact, AD is often referred to as type 3 diabetes due to the presence of insulin resistance in the brain. A recent study indicates that altering metabolism improves cognitive function. While metabolic reprogramming is one therapeutic avenue for AD, it is more widely used in some cancer therapies. FDA approved drugs such as metformin, dichloroacetic acid (DCA), and methylene blue can alter metabolism. These drugs can therefore be potentially applied in alleviating metabolic dysfunction in AD. Brain imaging has made enormous strides over the past decade, offering a new window to the mind. Recently, there has been remarkable development of compounds that have the ability to image both types of pathological amyloids: tau and amyloid beta. We have focused on the low cost, simple to use, near infrared fluorescence (NIRF) imaging probes for amyloid beta (Aβ), with specific attention on recent developments to further improve contrast, specificity, and sensitivity. With advances in imaging technologies, such fluorescent imaging probes will open new diagnostic avenues.

EphA2-targeted NIR-I/II fluorescent probe for specific imaging of colorectal cancer.

Background: Surgical resection remains the most promising potentially curative treatment strategy for many forms of cancer1. Residual malignant tissue after surgery, a consequence in part due to positive margins, contributes to the high mortality and disease recurrence that is further exacerbated by the aggressive nature of cancer2. In this study, multimodal contrast agents for integrated preoperative magnetic resonance imaging (MRI) and intraoperative fluorescence image-guided surgery (FIGS) are developed. Leveraging the strengths of preoperative MRI and intraoperative FIGS in tandem could provide superior guidance during surgery, potentially improving surgical resection outcome and reducing recurrence. However, integration of MRI and FIGS requires an imaging agent advanced beyond the current standard. To address this challenge, the imaging agent should consist of: (1) a fluorophore lacking photobleaching and toxicity while demonstrating a high quantum yield for FIGS and (2) an effective MRI contrast agent. In order to accommodate these requirements, a novel nanoparticle was synthesized based on hyaluronic acid (HA), a biopolymer that our group has demonstrated is a suitable carrier of fluorophores for FIGS3. Experimental Procedures: Self-assembled multimodal imaging nanoparticles (SAMINs) were developed as a mixed micelle formulation using HA chains functionalized with either gadolinium, the optimal contrast agent for MRI, or Cy7.5, a fluorescent dye we have demonstrated as a fluorophore for FIGS. Synthesis was confirmed through Raman and fluorescence spectroscopy and NMR. SAMIN size and charge were characterized by dynamic light scattering and zeta potential, and SAMIN gadolinium content was determined via inductively-coupled plasma mass spectrometry. Relaxivity studies were performed on a Bruker Biospec MRI scanner to determine efficacy as an MRI contrast agent. To evaluate the relationship between MR and fluorescence signal from SAMINs, we employed a simulated surgical phantom that we routinely use to evaluate the depth at which NIR imaging agents can be detected by FIGS. Results: Physicochemical characterization of SAMINs confirmed synthesis and stability. The T 1 and T 2 relaxivities of SAMINs were found to be R 1 = 5.5 mM-1s-1 and the R 2 = 10.7 mM-1s-1. After determining the relaxivity of the SAMINs, they were imaged using a LI-COR Trilogy fluorescence imaging unit to confirm fluorescence was retained. As the T1 and T2 signal increase and decrease, respectively, with increasing concentration of Gd3+, the fluorescence intensity increases with concentration of nanoparticles as expected. Tissue phantom studies showed the SAMINs to have high contrast for pre-operative MRI, and then demonstrated high contrast for fluorescence imaging and margin identification during FIGS. Conclusions: Tumor margin identification during surgery remains a barrier in improving surgical resection of cancer. The SAMINs developed in this study demonstrate high MRI and fluorescence contrast in phantom models, providing the basis for use as an imaging agent for both preoperative MRI and intraoperative FIGS. These imaging agents show promise for guiding surgeons during surgery by providing enhanced contrast between the tumor and surrounding tissue, helping to improve resection by defining tumor margins more clearly. 1. Hartwig W, Werner J, Jäger D, Debus J, Büchler MW. Improvement of surgical results for pancreatic cancer. Lancet Oncol . 2013;14(11):e476-e485. doi:10.1016/S1470-2045(13)70172-4. 2. Ethun CG, Kooby DA. The importance of surgical margins in pancreatic cancer. J Surg Oncol . November 2015. doi:10.1002/jso.24092. 3. Hill TK, Abdulahad A, Kelkar SS, et al. Indocyanine Green-Loaded Nanoparticles for Image-Guided Tumor Surgery. Bioconjug Chem . 2015;26(2):294-303. doi:10.1021/bc5005679. Citation Format: William M. Payne, Denis A. Svechkarev, Michael D. Boska, Aaron M. Mohs. Multimodal contrast agents for integrated preoperative and intraoperative imaging of cancer. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B19.

Fluorescence imaging in the second near-infrared window (NIR-II, 1,000-1,700 nm) demonstrates unique advantages of high sensitivity, high temporal and spatial resolution, and excellent image quality in comparison to that in the first near-infrared window (NIR-I, 650-950 nm) due to low scattering of photons and zero background fluorescence in the NIR-II region. Currently, a variety of NIR-II fluorescence imaging probes, such as single-wall carbon nanotubes, quantum dots, rare-earth-doped nanoparticles, conjugated polymer nanoparticles, small molecules, and AIEgens, have been developed as significant imaging tools for basic and clinical research. In this report, we begin with briefly introducing the advances of NIR-II fluorescence molecular probes in the last 10 years, including the design, preparation, and NIR-II fluorescence molecular imaging applications of AIEgens and indocyanine green (ICG). Subsequently, we highlight the large-scale preparation of NIR-II imaging probes and their applications in large animals, including New Zealand white rabbits and cynomolgus monkeys. Finally, we prospect the opportunities and challenges of NIR-II fluorescent molecular imaging probes in basic and translational studies.