PET/NIR-II fluorescence imaging and image-guided surgery of glioblastoma using a folate receptor α-targeted dual-modal nanoprobe.

PURPOSE: The surgery of glioblastoma (GBM) requires a maximal resection of the tumor when it is safe and feasible. The infiltrating growth property of the GBM makes it a challenge for neurosurgeons to identify the tumor tissue even with the assistance of the surgical microscope. This highlights the urgent requirement for imaging techniques that can differentiate tumor tissues during surgery in real time. Fluorescence image-guided surgery of GBM has been investigated using several non-specific fluorescent probes that emit light in the visible and the first near-infrared window (NIR-I, 700-900 nm), which limit the detection accuracy because of the non-specific targeting mechanism and spectral characteristics. Targeted NIR-II (1000-1700 nm) fluorescent probes for GBM are thus highly desired. The folate receptor (FR) has been reported to be upregulated in GBM, which renders it to be a promising target for specific tumor imaging. METHODS: In this study, the folic acid (FA) that can target the FR was conjugated with the clinically approved indocyanine green (ICG) dye and DOTA chelator for radiolabeling with 64Cu to achieve targeted positron emission tomography (PET) and fluorescence imaging of GBM. RESULTS: Surprisingly it was found that the resulted bioconjugate, DOTA-FA-ICG and non-radioactive natCu-DOTA-FA-ICG, were both self-assembled into nanoparticles with NIR-II emission signal. The radiolabeled DOTA-FA-ICG, 64Cu-DOTA-FA-ICG, was found to specifically accumulate in the orthotopic GBM models using in vivo PET, NIR-II, and NIR-I fluorescence imaging. The best time window of fluorescence imaging was demonstrated to be 24 h after DOTA-FA-ICG injection. NIR-II fluorescence image-guided surgery was successfully conducted in the orthotopic GBM models using DOTA-FA-ICG. All the fluorescent tissue was removed and proved to be GBM by the H&E examination. CONCLUSION: Overall, our study demonstrates that the probes, 64Cu-DOTA-FA-ICG and DOTA-FA-ICG, hold promise for preoperative PET examination and intraoperative NIR-II fluorescence image-guided surgery of GBM, respectively.

Novel multifunctional NIR-II aggregation-induced emission nanoparticles-assisted intraoperative identification and elimination of residual tumor.

Incomplete tumor resection is the direct cause of the tumor recurrence and metastasis after surgery. Intraoperative accurate detection and elimination of microscopic residual cancer improve surgery outcomes. In this study, a powerful D1-π-A-D2-R type phototheranostic based on aggregation-induced emission (AIE)-active the second near-infrared window (NIR-II) fluorophore is designed and constructed. The prepared theranostic agent, A1 nanoparticles (NPs), simultaneously shows high absolute quantum yield (1.23%), excellent photothermal conversion efficiency (55.3%), high molar absorption coefficient and moderate singlet oxygen generation performance. In vivo experiments indicate that NIR-II fluorescence imaging of A1 NPs precisely detect microscopic residual tumor (2 mm in diameter) in the tumor bed and metastatic lymph nodes. More notably, a novel integrated strategy that achieves complete tumor eradication (no local recurrence and metastasis after surgery) is proposed. In summary, A1 NPs possess superior imaging and treatment performance, and can detect and eliminate residual tumor lesions intraoperatively. This work provides a promising technique for future clinical applications achieving improved surgical outcomes.

Real-time intraoperative glioma diagnosis using fluorescence imaging and deep convolutional neural networks.

PURPOSE: Surgery is the predominant treatment modality of human glioma but suffers difficulty on clearly identifying tumor boundaries in clinic. Conventional practice involves neurosurgeon's visual evaluation and intraoperative histological examination of dissected tissues using frozen section, which is time-consuming and complex. The aim of this study was to develop fluorescent imaging coupled with artificial intelligence technique to quickly and accurately determine glioma in real-time during surgery. METHODS: Glioma patients (N = 23) were enrolled and injected with indocyanine green for fluorescence image-guided surgery. Tissue samples (N = 1874) were harvested from surgery of these patients, and the second near-infrared window (NIR-II, 1000-1700 nm) fluorescence images were obtained. Deep convolutional neural networks (CNNs) combined with NIR-II fluorescence imaging (named as FL-CNN) were explored to automatically provide pathological diagnosis of glioma in situ in real-time during patient surgery. The pathological examination results were used as the gold standard. RESULTS: The developed FL-CNN achieved the area under the curve (AUC) of 0.945. Comparing to neurosurgeons' judgment, with the same level of specificity >80%, FL-CNN achieved a much higher sensitivity (93.8% versus 82.0%, P < 0.001) with zero time overhead. Further experiments demonstrated that FL-CNN corrected >70% of the errors made by neurosurgeons. FL-CNN was also able to rapidly predict grade and Ki-67 level (AUC 0.810 and 0.625) of tumor specimens intraoperatively. CONCLUSION: Our study demonstrates that deep CNNs are better at capturing important information from fluorescence images than surgeons' evaluation during patient surgery. FL-CNN is highly promising to provide pathological diagnosis intraoperatively and assist neurosurgeons to obtain maximum resection safely. TRIAL REGISTRATION: ChiCTR ChiCTR2000029402. Registered 29 January 2020, retrospectively registered.

Radiopharmaceutical and Eu3+ doped gadolinium oxide nanoparticles mediated triple-excited fluorescence imaging and image-guided surgery.

Cerenkov luminescence imaging (CLI) is a novel optical imaging technique that has been applied in clinic using various radionuclides and radiopharmaceuticals. However, clinical application of CLI has been limited by weak optical signal and restricted tissue penetration depth. Various fluorescent probes have been combined with radiopharmaceuticals for improved imaging performances. However, as most of these probes only interact with Cerenkov luminescence (CL), the low photon fluence of CL greatly restricted it's interaction with fluorescent probes for in vivo imaging. Therefore, it is important to develop probes that can effectively convert energy beyond CL such as ß and γ to the low energy optical signals. In this study, a Eu3+ doped gadolinium oxide (Gd2O3:Eu) was synthesized and combined with radiopharmaceuticals to achieve a red-shifted optical spectrum with less tissue scattering and enhanced optical signal intensity in this study. The interaction between Gd2O3:Eu and radiopharmaceutical were investigated using 18F-fluorodeoxyglucose (18F-FDG). The ex vivo optical signal intensity of the mixture of Gd2O3:Eu and 18F-FDG reached 369 times as high as that of CLI using 18F-FDG alone. To achieve improved biocompatibility, the Gd2O3:Eu nanoparticles were then modified with polyvinyl alcohol (PVA), and the resulted nanoprobe PVA modified Gd2O3:Eu (Gd2O3:Eu@PVA) was applied in intraoperative tumor imaging. Compared with 18F-FDG alone, intraoperative administration of Gd2O3:Eu@PVA and 18F-FDG combination achieved a much higher tumor-to-normal tissue ratio (TNR, 10.24 ± 2.24 vs. 1.87 ± 0.73, P = 0.0030). The use of Gd2O3:Eu@PVA and 18F-FDG also assisted intraoperative detection of tumors that were omitted by preoperative positron emission tomography (PET) imaging. Further experiment of image-guided surgery demonstrated feasibility of image-guided tumor resection using Gd2O3:Eu@PVA and 18F-FDG. In summary, Gd2O3:Eu can achieve significantly optimized imaging property when combined with 18F-FDG in intraoperative tumor imaging and image-guided tumor resection surgery. It is expected that the development of the Gd2O3:Eu nanoparticle will promote investigation and application of novel nanoparticles that can interact with radiopharmaceuticals for improved imaging properties. This work highlighted the impact of the nanoprobe that can be excited by radiopharmaceuticals emitting CL, ß, and γ radiation for precisely imaging of tumor and intraoperatively guide tumor resection.

NIR-II fluorescence-guided liver cancer surgery by a small molecular HDAC6 targeting probe.

BACKGROUND: Hepatocellular carcinoma (HCC) is the sixth most common malignancy globally and ranks third in terms of both mortality and incidence rates. Surgical resection holds potential as a curative approach for HCC. However, the residual disease contributes to a high 5-year recurrence rate of 70%. Due to their excellent specificity and optical properties, fluorescence-targeted probes are deemed effective auxiliary tools for addressing residual lesions, enabling precise surgical diagnosis and treatment. Research indicates histone deacetylase 6 (HDAC6) overexpression in HCC cells, making it a potential imaging biomarker. This study designed a targeted small-molecule fluorescent probe, SeCF3-IRDye800cw (SeCF3-IRD800), operating within the Second near-infrared window (NIR-II, 1000-1700 nm). The study confirms the biocompatibility of SeCF3-IRD800 and proceeds to demonstrate its applications in imaging in vivo, fluorescence-guided surgery (FGS) for liver cancer, liver fibrosis imaging, and clinical samples incubation, thereby preliminarily validating its utility in liver cancer. METHODS: SeCF3-IRD800 was synthesized by combining the near-infrared fluorescent dye IRDye800cw-NHS with an improved HDAC6 inhibitor. Initially, a HepG2-Luc subcutaneous tumor model (n = 12) was constructed to investigate the metabolic differences between SeCF3-IRD800 and ICG in vivo. Subsequently, HepG2-Luc (n = 12) and HCCLM3-Luc (n = 6) subcutaneous xenograft mouse models were used to assess in vivo targeting by SeCF3-IRD800. The HepG2-Luc orthotopic liver cancer model (n = 6) was employed to showcase the application of SeCF3-IRD800 in FGS. Liver fibrosis (n = 6) and HepG2-Luc orthotopic (n = 6) model imaging results were used to evaluate the impact of different pathological backgrounds on SeCF3-IRD800 imaging. Three groups of fresh HCC and normal liver samples from patients with liver cancer were utilized for SeCF3-IRD800 incubation ex vivo, while preclinical experiments illustrated its potential for clinical application. FINDINGS: The HDAC6 inhibitor 6 (SeCF3) modified with trifluoromethyl was labeled with IRDy800CW-NHS to synthesize the small-molecule targeted probe SeCF3-IRD800, with NIR-II fluorescence signals. SeCF3-IRD800 was rapidly metabolized by the kidneys and exhibited excellent biocompatibility. In vivo validation demonstrated that SeCF3-IRD800 achieved optimal imaging within 8 h, displaying high tumor fluorescence intensity (7658.41 ± 933.34) and high tumor-to-background ratio (5.20 ± 1.04). Imaging experiments with various expression levels revealed its capacity for HDAC6-specific targeting across multiple HCC tumor models, suitable for NIR-II intraoperative imaging. Fluorescence-guided surgery experiments were found feasible and capable of detecting sub-visible 2 mm tumor lesions under white light, aiding surgical decision-making. Further imaging of liver fibrosis mice showed that SeCF3-IRD800's imaging efficacy remained unaffected by liver pathological conditions. Correlations were observed between HDAC6 expression levels and corresponding fluorescence intensity (R2 = 0.8124) among normal liver, liver fibrosis, and HCC tissues. SeCF3-IRD800 identified HDAC6-positive samples from patients with HCC, holding advantages for perspective intraoperative identification in liver cancer. Thus, the rapidly metabolized HDAC6-targeted small-molecule NIR-II fluorescence probe SeCF3-IRD800 holds significant clinical translational value. INTERPRETATION: The successful application of NIR-II fluorescence-guided surgery in liver cancer indicates that SeCF3-IRD800 has great potential to improve the clinical diagnosis and treatment of liver cancer, and could be used as an auxiliary tool for surgical treatment of liver cancer without being affected by liver pathology. FUNDING: This paper is supported by the National Natural Science Foundation of China (NSFC) (92,059,207, 62,027,901, 81,930,053, 81,227,901, 82,272,105, U21A20386 and 81,971,773), CAS Youth Interdisciplinary Team (JCTD-2021-08), the Zhuhai High-level Health Personnel Team Project (Zhuhai HLHPTP201703), and Guangdong Basic and Applied Basic Research Foundation under Grant No. 2022A1515011244.

NIR-II fluorescence imaging-guided colorectal cancer surgery targeting CEACAM5 by a nanobody.

BACKGROUND: Surgery is the cornerstone of colorectal cancer (CRC) treatment, yet complete removal of the tumour remains a challenge. The second near-infrared window (NIR-II, 1000-1700 nm) fluorescent molecular imaging is a novel technique, which has broad application prospects in tumour surgical navigation. We aimed to evaluate the ability of CEACAM5-targeted probe for CRC recognition and the value of NIR-II imaging-guided CRC resection. METHODS: We constructed the probe 2D5-IRDye800CW by conjugated anti-CEACAM5 nanobody (2D5) with near-infrared fluorescent dye IRDye800CW. The performance and benefits of 2D5-IRDye800CW at NIR-II were confirmed by imaging experiments in mouse vascular and capillary phantom. Then mouse colorectal cancer subcutaneous tumour model (n = 15), orthotopic model (n = 15), and peritoneal metastasis model (n = 10) were constructed to investigate biodistribution of probe and imaging differences between NIR-I and NIR-II in vivo, and then tumour resection was guided by NIR-II fluorescence. Fresh human colorectal cancer specimens were incubated with 2D5-IRDye800CW to verify its specific targeting ability. FINDINGS: 2D5-IRDye800CW had an NIR-II fluorescence signal extending to 1600 nm and bound specifically to CEACAM5 with an affinity of 2.29 nM. In vivo imaging, 2D5-IRDye800CW accumulated rapidly in tumour (15 min) and could specifically identify orthotopic colorectal cancer and peritoneal metastases. All tumours were resected under NIR-II fluorescence guidance, even smaller than 2 mm tumours were detected, and NIR-II had a higher tumour-to-background ratio than NIR-I (2.55 ± 0.38, 1.94 ± 0.20, respectively). 2D5-IRDye800CW could precisely identify CEACAM5-positive human colorectal cancer tissue. INTERPRETATION: 2D5-IRDye800CW combined with NIR-II fluorescence has translational potential as an aid to improve R0 surgery of colorectal cancer. FUNDINGS: This study was supported by Beijing Natural Science Foundation (JQ19027), the National Key Research and Development Program of China (2017YFA0205200), National Natural Science Foundation of China (NSFC) (61971442, 62027901, 81930053, 92059207, 81227901, 82102236), Beijing Natural Science Foundation (L222054), CAS Youth Interdisciplinary Team (JCTD-2021-08), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16021200), the Zhuhai High-level Health Personnel Team Project (Zhuhai HLHPTP201703), the Fundamental Research Funds for the Central Universities (JKF-YG-22-B005) and Capital Clinical Characteristic Application Research (Z181100001718178). The authors would like to acknowledge the instrumental and technical support of the multi-modal biomedical imaging experimental platform, Institute of Automation, Chinese Academy of Sciences.

Near-Infrared Window II Fluorescence Image-Guided Surgery of High-Grade Gliomas Prolongs the Progression-Free Survival of Patients.

OBJECTIVE: This translational study aims to investigate the clinical benefits of indocyanine green (ICG) based near-infrared window II (NIR-II) fluorescence image-guided surgery (FGS) on high-grade glioma (HGG) patients. METHODS: Patients were randomly assigned to receive FGS or traditional white light image-guided surgery (WLS). The detection rate of NIR-II fluorescence was observed. Complete resection rate, progression-free survival (PFS), overall survival (OS), and neurological status were compared. Tissue samples were obtained from the FGS group, with the diagnosis based on the surgeons and the fluorescence recorded for comparison of diagnostic capability. Patients with WHO grade III gliomas or glioblastomas (GBM) were analyzed separately. RESULTS: 15 GBM and 4 WHO grade III glioma patients in the FGS group and 18 GBM and 4 WHO grade III glioma patients in the WLS group were enrolled. The detection rate of NIR-II fluorescence was 100% for GBM. The complete resection rate was significantly increased by the FGS for GBM (FGS, 100% [95% CI 73.41-100] vs. WLS, 50% [95% CI 29.03-70.97], P = 0.0036). The PFS and OS of the FGS group were also significantly prolonged (Median PFS: FGS, 9.0 months vs. WLS, 7.0 months, P < 0.0001; Median OS: FGS, 19.0 months vs. WLS, 15.5 months, P = 0.0002). No recurrence was observed in WHO grade III glioma patients. CONCLUSIONS: NIR-II FGS achieves a much better complete resection rate of GBM than conventional WLS, leading to greatly improved survival of GBM patients. SIGNIFICANCE: NIR-II FGS is a highly promising technique worthy of exploring more clinical applications.

Excitation-based fully connected network for precise NIR-II fluorescence molecular tomography.

Fluorescence molecular tomography (FMT) is a novel imaging modality to obtain fluorescence biomarkers' three-dimensional (3D) distribution. However, the simplified mathematical model and complicated inverse problem limit it to achieving precise results. In this study, the second near-infrared (NIR-II) fluorescence imaging was adopted to mitigate tissue scattering and reduce noise interference. An excitation-based fully connected network was proposed to model the inverse process of NIR-II photon propagation and directly obtain the 3D distribution of the light source. An excitation block was embedded in the network allowing it to autonomously pay more attention to neurons related to the light source. The barycenter error was added to the loss function to improve the localization accuracy of the light source. Both numerical simulation and in vivo experiments showed the superiority of the novel NIR-II FMT reconstruction strategy over the baseline methods. This strategy was expected to facilitate the application of machine learning in biomedical research.

First Clinical Investigation of Near-Infrared Window IIa/IIb Fluorescence Imaging for Precise Surgical Resection of Gliomas.

OBJECTIVE: The near-infrared window II (NIR-II, 1000-1700 nm) imaging, including NIR-IIa (1300-1400 mm) and NIR-IIb (1500-1700 mm), outperforms the near-infrared window I (NIR-I, 700-900 nm) imaging in biological researches. However, the advantages of NIR-IIa/IIb imaging in human study are ambiguous. This study aims to apply the NIR-IIa/IIb imaging to glioma resection and evaluate their performance by using the developed imaging instrument and intraoperative image fusion method. METHODS: A multispectral fluorescence imaging instrument that integrated NIR-I/II/IIa/IIb fluorescence imaging and an intraoperative image fusion method have been developed. Seven patients with grade III/IV glioma have been enrolled. NIR-I/II images of the tumor and NIR-I/II/IIa/IIb images of cerebral vessels were acquired with the administration of indocyanine green. Images were fused using the specialized fusion method to synchronously provide the distribution of the vessels and the surgical boundaries. RESULTS: The NIR-IIa/IIb imaging was successfully applied to the clinic. High imaging resolution and contrast have been attained in the NIR-IIa/IIb spectra. Besides, capillaries with an apparent diameter as small as 182 µm were acquired using NIR-IIb imaging. Tumor-feeding arteries were precisely blocked and tumors were excised to the maximum extent for all patients. The blood loss volume during surgery was significantly reduced compared with the control group. CONCLUSION: The multispectral fluorescence imaging showed high performance, which led to a significant reduction in blood loss volume. SIGNIFICANCE: The novel multispectral fluorescence imaging technology can assist surgeons in other vascular surgeries in the future.

First-in-human liver-tumour surgery guided by multispectral fluorescence imaging in the visible and near-infrared-I/II windows.

The second near-infrared wavelength window (NIR-II, 1,000-1,700 nm) enables fluorescence imaging of tissue with enhanced contrast at depths of millimetres and at micrometre-scale resolution. However, the lack of clinically viable NIR-II equipment has hindered the clinical translation of NIR-II imaging. Here, we describe an optical-imaging instrument that integrates a visible multispectral imaging system with the detection of NIR-II and NIR-I (700-900 nm in wavelength) fluorescence (by using the dye indocyanine green) for aiding the fluorescence-guided surgical resection of primary and metastatic liver tumours in 23 patients. We found that, compared with NIR-I imaging, intraoperative NIR-II imaging provided a higher tumour-detection sensitivity (100% versus 90.6%; with 95% confidence intervals of 89.1%-100% and 75.0%-98.0%, respectively), a higher tumour-to-normal-liver-tissue signal ratio (5.33 versus 1.45) and an enhanced tumour-detection rate (56.41% versus 46.15%). We infer that combining the NIR-I/II spectral windows and suitable fluorescence probes might improve image-guided surgery in the clinic.

Radiopharmaceuticals and Fluorescein Sodium Mediated Triple-Modality Molecular Imaging Allows Precise Image-Guided Tumor Surgery.

Radical resection is the most effective method for malignant tumor treatments. However, conventional imaging cannot fully satisfy the clinical needs of surgical navigation. This study presents a novel triple-modality positron emission tomography (PET)-Cerenkov radiation energy transfer (CRET)-confocal laser endomicroscopy (CLE) imaging strategy for intraoperative tumor imaging and surgical navigation. Using clinical radiopharmaceuticals and fluorescein sodium (FS), this strategy can accurately detect the tumor and guide the tumor surgery. The FS emission property under Cerenkov radiation excitation is investigated using 2-deoxy-2-18F-fluoroglucose and 11C-choline. Performances of the PET-CRET-CLE imaging and the CRET-CLE image-guided surgery are evaluated on mouse models. The CRET signal at 8 mm depth is stronger than the Cerenkov luminescence at 1 mm depth in phantoms. In vivo experiments indicate that 0.5 mL kg-1 of 10% FS generates the strongest CRET signal, which can be observed immediately after FS injection. A surgical navigation study shows that the tumors are precisely detected and resected using intraoperative CRET-CLE. In summary, a PET-CRET-CLE triple-modality imaging strategy is developed. This strategy can detect the tumors and precisely guide the tumor resection using clinical pharmaceuticals. This triple-modality imaging shows high potential in surgical navigation research and clinical translation.