99mTc-Labeled FAPI SPECT Imaging in Idiopathic Pulmonary Fibrosis: Preliminary Results.

AIM: Idiopathic pulmonary fibrosis (IPF) is associated with a poor prognosis, presenting the most aggressive form of interstitial lung diseases (ILDs). Activated fibroblasts are crucial for pathological processes. Fibroblast activation protein (FAP) inhibitor (FAPI) tracers would be promising imaging agents for these diseases. The purpose of this study was to evaluate a 99mTc-labeled FAPI tracer, 99mTc-HFAPI imaging in IPF patients. METHODS: Eleven IPF patients (nine males and two females; age range 55-75 year) were included in this pilot study. 99mTc-HFAPI serial whole-body scintigraphy at 5 min, 20 min, 40 min, 1 h, 2 h, 3 h, 4 h, and 6 h was acquired for dynamic biodistribution and dosimetry estimation in seven representative patients. SPECT/CT tomography fusion imaging of the chest region was performed in all patients at 4 h post-injection, which was considered as the optimal acquisition time. Dosimetry was calculated using OLINDA/EXM software (version 2.0; HERMES Medical Solutions). The quantified or semi-quantified standardized uptake values (SUVs) and lesion-to-background ratios (LBRs) of affected lung parenchyma were also calculated. The high-resolution CT (HRCT) stage was determined with visual evaluation, and the total HRCT score of each patient was measured using a weighting factor formula. Pulmonary function tests (PFTs) were recorded as well. Then, the relationships between the 99mTc-HFAPI results, disease extent on HRCT, and PFT results were investigated. RESULTS: Normal physiological uptake of 99mTc-HFAPI was observed mainly in the liver, intestinal tract, pancreas, gallbladder, and to a lesser extent in the spleen, kidneys, and thyroid, with no apparent retention in the blood circulation at the late time point. The mean injected activity of 99mTc-HFAPI was 813.4 MBq (range 695.6-888.0 MBq). No subjective side effects were noticed. The average whole-body effective dose was 0.0041 mSv/MBq per patient. IPF patients exhibited elevated pulmonary 99mTc-HFAPI uptake in abnormal lung regions, which was correlated with fibrotic regions on HRCT. Among different HRCT stage groups, both SUVmax and LBR showed significant differences (p < 0.001). The higher HRCT stage demonstrated significantly higher SUVmax and LBR. A linear correlation between 99mTc-HFAPI uptake and total HRCT score was observed for SUVmax (r = 0.7839, F = 54.41, p = 0.0094) and LBR (r = 0.7402, F = 56.33, p = 0.0092). 99mTc-HFAPI uptake also had moderate correlations with PFT results. CONCLUSIONS: Our preliminary data show that the 99mTc-HFAPI SPECT imaging is a promising new imaging modality in IPF patients. Investigations of its clinical value in monitoring disease progression and treatment response are needed in the future.

Myoglobin-loaded gadolinium nanotexaphyrins for oxygen synergy and imaging-guided radiosensitization therapy.

Gadolinium (Gd3+)-coordinated texaphyrin (Gd-Tex) is a promising radiosensitizer that entered clinical trials, but temporarily fails largely due to insufficient radiosensitization efficacy. Little attention has been given to using nanovesicles to improve its efficacy. Herein, Gd-Tex is transformed into building blocks "Gd-Tex-lipids" to self-assemble nanovesicles called Gd-nanotexaphyrins (Gd-NTs), realizing high density packing of Gd-Tex in a single nanovesicle and achieving high Gd-Tex accumulation in tumors. To elucidate the impact of O2 concentration on Gd-Tex radiosensitization, myoglobin (Mb) is loaded into Gd-NTs (Mb@Gd-NTs), resulting in efficient relief of tumor hypoxia and significant enhancement of Gd-Tex radiosensitization, eventually inducing the obvious long-term antitumor immune memory to inhibit tumor recurrence. In addition to Gd3+, the versatile Mb@Gd-NTs can also chelate 177Lu3+ (Mb@177Lu/Gd-NTs), enabling SPECT/MRI dual-modality imaging for accurately monitoring drug delivery in real-time. This "one-for-all" nanoplatform with the capability of chelating various trivalent metal ions exhibits broad clinical application prospects in imaging-guided radiosensitization therapy.

Smilagenin induces expression and epigenetic remodeling of BDNF in alzheimer's disease.

BACKGROUND: Smilagenin (SMI) is a lipid-soluble steroidal sapogenin, extracted from traditional Chinses medicinal herbs Radix Asparagi, which is extracted from the dry root of Asparagus cochinchinensis (Lour.) Merr. We previously found that SMI significantly increased brain-derived neurotrophic factor (BDNF) expression in Aß-intoxicated SH-SY5Y cells. METHODS: In this study, we performed behavioral tests to analyze cognitive function of WT and APP/PS1 mice treated with or without SMI, and found that SMI could significantly improve the learning and memory ability of APP/PS1 mice. Moreover, immunofluorescence and ELISA results showed that SMI pretreatment could effectively reduce the deposition of ß-amyloid plaques in the cortex and hippocampus of APP/PS1 mice (26 mg/kg/day for 60 days) and inhibit the secretion of Aß1-42 in N2a/APPswe cells (10 µM concentration for 24 hours). RESULTS: Mechanistically, SMI enhanced BDNF mRNA expression, elevated the global level of H3AC and H4AC, and increased the expression of P300 in AD models. Furthermore, chromatin immunoprecipitation results showed that SMI could increase the levels of H3AC and H4AC at the promoter of BDNF promoter Ⅱ and Ⅳ, indicating that SMI epigenetically regulates BDNF expression through HAT enhancement. To further verify the critical role of P300 by which SMI upregulated histone acetylation in BDNF, AD mice were treated with SMI and C646 simultaneously. Behavioral experiments showed that the improvement effects of SMI on cognitive impairment were abolished after P300 inhibition in APP/PS1 mice. CONCLUSIONS: Our research for the first time demonstrated that SMI showed neuroprotective effects by increasing the expression of P300 protein, thus upregulating histone acetylation levels in the promoter region of BDNF and promoting its transcription. Our findings provide an important theoretical basis for the treatment of Alzheimer's disease with SMI extracted from Asparagus cochinchinensis (Lour.) Merr.

Implication of 99mTc-sum IL-2 SPECT/CT in immunotherapy by imaging of tumor-infiltrating T cells.

BACKGROUND: Although immune checkpoint blockade (ICB) and adoptive T cell transfer (ACT) therapy have achieved impressive clinical outcomes, majority of patients do not respond to immunotherapy. Tumor-infiltrating T cells, a critical factor to immunotherapy, is dynamically changing. Therefore, a reliable real-time in vivo imaging system for tumor-infiltrating T cells, but not immunohistochemical analyses, will be more valuable to predict response and guide immunotherapy. In this study, we developed a new SPECT/CT imaging probe 99mTc-sum IL-2 targeting the IL-2Rß/IL-2Rγ (CD122/CD132) receptor on tumor-infiltrating T cells, and evaluated its application in predicting the immune response to anti-PD-L1 (αPD-L1) therapy as well as tracking infused T cells in ACT therapy. METHODS: The binding affinity of the super mutated IL-2 (sum IL-2) in various T cell subtypes was measured. Sum IL-2 was subsequently labeled with 99mTc through Sortase-A mediated site-specific transpeptidation. SPECT/CT imaging and biodistribution studies of 99mTc-sum IL-2 were performed in a MC38 mouse model. Wild type IL-2 (IL-2) was used as control in the above studies. Finally, we evaluated 99mTc-sum IL-2 SPECT/CT for the detection of tumor-infiltrating T cells in the context of αPD-L1 immunotherapy and ACT therapy. RESULTS: Sum IL-2 preferentially bound to CD8+ T cells, especially activated CD8+ T cells, while IL-2 showed biased binding to Treg cells. As a result, 99mTc-sum IL-2 could detect tumor-infiltrating T cells. In the MC38 tumor model, SPECT/CT imaging showed the increased tumor uptake of 99mTc-sum IL-2 after αPD-L1 treatment, suggesting that the treatment significantly increased tumor-infiltrating T cells, resulting in a correspondingly significant curative effect. In addition, 99mTc-sum IL-2 SPECT/CT could also track the infiltration of antigen-specific cytotoxic CD8+ T cells during ACT therapy. CONCLUSION: 99mTc-sum IL-2 has great clinical potential for non-invasive and specific SPECT/CT imaging of tumor-infiltrating T cells as well as for timely prediction and evaluation of the therapeutic efficacy of ICB and ACT therapy.

Molecular imaging of HER2 expression in breast cancer patients using a novel peptide-based tracer 99mTc-HP-Ark2: a pilot study.

BACKGROUND: Due to the temporal and spatial heterogeneity of human epidermal growth factor receptor 2 (HER2) expression in breast tumors, immunohistochemistry (IHC) cannot accurately reflect the HER2 status in real time, which may cause misguided treatment decisions. HER2-specific imaging can noninvasively determine HER2 status in primary and metastatic tumors. In this study, HER2 expression in breast cancer patients was determined in vivo by SPECT/CT of 99mTc-HP-Ark2, comparing with PET/CT of 18F-FDG lesion by lesion. METHODS: A novel HER2-targeted peptide probe 99mTc-HP-Ark2 was constructed. Biodistribution and nanoScan SPECT/CT imaging were performed in mice models. The correlation between the quantified tumor uptake and HER2 expression in tumor cells was analyzed. In the pilot clinical study, a total of 34 breast cancer patients (mean age ± SD: 49 ± 10 y) suspected of having breast cancer according to mammography or ultrasonography were recruited at Peking Union Medical College Hospital, and 99mTc-HP-Ark2 SPECT/CT and 18F-FDG PET/CT were carried out with IHC and fluorescence in situ hybridization as validation. RESULTS: Small animal SPECT/CT of 99mTc-HP-Ark2 clearly identified tumors with different HER2 expression. The quantified tumor uptake and tumor HER2 expression showed a significant linear correlation (r = 0.932, P < 0.01). Among the 36 primary lesions in the 34 patients, when IHC (2 +) or IHC (3 +) was used as the positive evaluation criterion, 99mTc-HP-Ark2 SPECT/CT imaging with a tumor-to-background ratio of 1.44 as the cutoff value reflected the HER2 status with sensitivity of 89.5% (17/19), specificity of 88.2% (15/17) and accuracy of 88.9% (32/36), while the 18F-FDG PET/CT showed sensitivity of 78.9% (15/19), specificity of 70.6% (12/17) and accuracy of 75.0% (27/36). In particular, 100% of IHC (3 +) tumors were all identified by 99mTc-HP-Ark2 SPECT/CT imaging. CONCLUSION: 99mTc-HP-Ark2 SPECT/CT can provide a specific, noninvasive evaluation of HER2 expression in breast cancer, showing great potential to guide HER2-targeted therapies in clinical practice. CLINICALTRIALS: gov Trial registration: NCT04267900. Registered 11th February 2020. Retrospectively registered, https://www. CLINICALTRIALS: gov/ct2/results?pg=1&load=cart&id=NCT04267900 .

Palmitic Acid-Conjugated Radiopharmaceutical for Integrin αvß3-Targeted Radionuclide Therapy.

Peptide receptor radionuclide therapy (PRRT) is an emerging approach for patients with unresectable or metastatic tumors. Our previously optimized RGD peptide (3PRGD2) has excellent targeting specificity for a variety of integrin αvß3/αvß5-positive tumors and has been labeled with the therapeutic radionuclide [177Lu]LuCl3 for targeted radiotherapy of tumors. However, the rapid clearance of [177Lu]Lu-DOTA-3PRGD2 (177Lu-3PRGD2) in vivo requires two doses of 111 MBq/3 mCi to achieve effective tumor suppression, limiting its further clinical application. Albumin binders have been attached to drugs to facilitate binding to albumin in vivo to prolong the drug half-life in plasma and obtain long-term effects. In this study, we modified 3PRGD2 with albumin-binding palmitic acid (Palm-3PRGD2) and then radiolabeled Palm-3PRGD2 with 177Lu. [177Lu]Lu-DOTA-Palm-3PRGD2 (177Lu-Palm-3PRGD2) retained a specific binding affinity for integrin αvß3/αvß5, with an IC50 value of 5.13 ± 1.16 nM. Compared with 177Lu-3PRGD2, the 177Lu-Palm-3PRGD2 circulation time in blood was more than 6 times longer (slow half-life: 73.42 min versus 11.81 min), and the tumor uptake increased more than fivefold (21.34 ± 4.65 %IA/g and 4.11 ± 0.70 %IA/g at 12 h post-injection). Thus, the significant increase in tumor uptake and tumor retention resulted in enhanced efficacy of targeted radiotherapy, and tumor growth was completely inhibited by a single and relatively lowdose of 18.5 MBq/0.5 mCi. Thus, 177Lu-Palm-3PRGD2 shows great potential for clinical application.

Immunotherapy for Alzheimer's disease: targeting ß-amyloid and beyond.

Alzheimer's disease (AD) is the most common neurodegenerative disease in the elderly worldwide. However, the complexity of AD pathogenesis leads to discrepancies in the understanding of this disease, and may be the main reason for the failure of AD drug development. Fortunately, many ongoing preclinical and clinical studies will continually open up avenues to unravel disease mechanisms and guide strategies for AD diagnosis and drug development. For example, immunotherapeutic strategies targeting amyloid-ß (Aß) and tau proteins were once deemed almost certainly effective in clinical treatment due to the excellent preclinical results. However, the repeated failures of clinical trials on vaccines and humanized anti-Aß and anti-tau monoclonal antibodies have resulted in doubts on this strategy. Recently, a new anti-Aß monoclonal antibody (Aducanumab) has been approved by the US Food and Drug Administration, which brings us back to the realization that immunotherapy strategies targeting Aß may be still promising. Meanwhile, immunotherapies based on other targets such as tau, microglia and gut-brain axis are also under development. Further research is still needed to clarify the forms and epitopes of targeted proteins to improve the accuracy and effectiveness of immunotherapeutic drugs. In this review, we focus on the immunotherapies based on Aß, tau and microglia and their mechanisms of action in AD. In addition, we present up-to-date advances and future perspectives on immunotherapeutic strategies for AD.

Circular RNA Cwc27 contributes to Alzheimer's disease pathogenesis by repressing Pur-α activity.

Circular RNAs (circRNAs) have gained growing attention in participating in various biological processes and referring to multiply kinds of diseases. Although differentially expressed circRNA profiling in Alzheimer's disease (AD) has been established, little is known about the precise characteristic and functions of key circRNAs with direct relevance to AD in gene expression and disease-related cognition. Herein, we screened and identified circCwc27 as a novel circRNA implicated in AD. CircCwc27 was a neuronal-enriched circRNA that abundantly expressed in the brain and significantly upregulated in AD mice and patients. Knockdown of circCwc27 markedly improved AD-related pathological traits and ameliorated cognitive dysfunctions. Mechanistically, we excluded the miRNA decoy mechanism and focused on the important function of circRNA-RNA-binding protein (RBP) interaction in AD. CircCwc27 directly bound to purine-rich element-binding protein A (Pur-α), increased retention of cytoplasmic Pur-α, and suppressed Pur-α recruitment to the promoters of a cluster of AD genes, including amyloid precursor protein (APP), dopamine receptor D1 (Drd1), protein phosphatase 1, regulatory inhibitor subunit1B (Ppp1r1b), neurotrophic tyrosine kinase, receptor, type 1 (Ntrk1), and LIM homeobox 8 (Lhx8). Downregulation of circCwc27 enhanced the affinity of Pur-α binding to these promoters, leading to altered transcription of Pur-α targets. Moreover, Pur-α overexpression largely phenocopied circCwc27 knockdown in preventing Aß deposition and cognitive decline. Together, our findings suggest significant functional consequences of a circRNA-protein interaction, that circCwc27, by associating with the regulatory protein Pur-α, may act as a crucial player in AD pathogenesis and represent a promising AD therapeutic target with clinical translational potential.

An Integrin-αvß6/α5ß1-Bitargeted Probe for the SPECT Imaging of Pancreatic Adenocarcinoma in Preclinical and Primary Clinical Studies.

Pancreatic adenocarcinoma (PA) is one of the deadliest human malignancies. However, early detection, prediction of surgical resectability, and prognosis of PA are challenging with current conventional imaging technologies in the clinic. Molecular imaging technologies combined with novel imaging probes could be useful for early detection and accurate staging of PA. Integrin αvß6 and α5ß1 are found to be overexpressed in PA. In this study, integrin αvß6/α5ß1-bitargeted probes 99mTc-HYNIC-isoDGR (99mTc-isoDGR) and 99mTc-HYNIC-PEG4-PisoDGR2 (99mTc-3PisoDGR2) were prepared and evaluated in the BxPC-3 human pancreatic tumor model. Both subcutaneous and in situ BxPC-3 tumors could be clearly visualized by 99mTc-isoDGR nanoScan SPECT/CT imaging with a high ratio of tumor to background. The blocking study with excess nonradioactive peptide showed a significantly reduced tumor uptake, which confirmed the specificity of 99mTc-isoDGR. Biodistribution results confirmed the imaging results. The dimer tracer 99mTc-3PisoDGR2 significantly enhanced tumor uptake compared with 99mTc-isoDGR, and the spontaneous PA lesion in the mouse model could be clearly visualized by 99mTc-3PisoDGR2. The primary clinical study also verified the ability of 99mTc-3PisoDGR2 for detection of PA. Therefore, SPECT/CT imaging using the integrin αvß6/α5ß1-bitargeted 99mTc-3PisoDGR2 provided a potential approach for the noninvasive detection of PA.

Post-transcriptional regulation of α7 nAChR expression by miR-98-5p modulates cognition and neuroinflammation in an animal model of Alzheimer's disease.

Alzheimer's disease (AD) is a complicated neurodegenerative disease and therefore addressing multiple targets simultaneously has been believed as a promising therapeutic strategy against AD. α7 nicotinic acetylcholine receptor (nAChR), which plays an important role in improving cognitive function and alleviating neuroinflammation in central nervous system (CNS), has been regarded as a potential target in the treatment of AD. However, the regulation of α7 nAChR at post-transcriptional level in mammalian brain remains largely speculated. Herein, we uncovered a novel post-transcriptional regulatory mechanism of α7 nAChR expression in AD and further demonstrated that miR-98-5p suppressed α7 nAChR expression through directly binding to the 3'UTR of mRNA. Knockdown of miR-98-5p activated Ca2+ signaling pathway and consequently reversed cognitive deficits and Aß burden in APP/PS1 mice. Furthermore, miR-98-5p downregulation increased α7 nAChR expression, and ameliorated neuroinflammation via inhibiting NF-κB pathway and upregulating Nrf2 target genes. Our findings illustrate a prominent regulatory role of miR-98-5p in targeting inflammation and cognition, and provide an insight into the potential of miR-98-5p/α7 nAChR axis as a novel therapeutic strategy for AD.

A Dual pH-Responsive DOX-Encapsulated Liposome Combined with Glucose Administration Enhanced Therapeutic Efficacy of Chemotherapy for Cancer.

BACKGROUND: The acidic microenvironment of cancer can promote tumor metastasis and drug resistance. Acidic tumor microenvironment-targeted therapy is currently an important means for treating tumors, inhibiting metastasis, and overcoming drug resistance. In this study, a dual pH-responsive DOX-encapsulated liposome (DOPE-DVar7-lip@DOX) was designed and fabricated for targeting the acidic tumor microenvironment. On the one hand, the response of acid-sensitive peptide (DVar7) to the acidic tumor microenvironment increased the uptake of liposomes in tumors and prolonged the retention time; on the other hand, the response of acid-sensitive phospholipid (DOPE) to the acidic tumor microenvironment improved the controlled release of DOX in tumors. METHODS: The acid-sensitive peptide DVar7 modified liposomes can be obtained by simple incubation of DSPE-DVar7 with DOX-loaded DOPE liposomes (DOPE-lip@DOX). The tumor targeting of the dual pH-responsive liposome was investigated in vitro and in vivo by near-infrared fluorescence imaging. The tumor therapeutic efficacy of DOPE-DVar7-lip@DOX was evaluated in breast cancer mouse model using the traditional liposome as a control. Moreover, we regulated the tumor microenvironment acidity by injecting glucose to further enhance the therapeutic efficacy of cancer. RESULTS: DVar7 can allosterically insert into the tumor cell membrane in the acidic tumor microenvironment to enhance the tumor uptake of liposomes and prolong the retention time of liposomes in tumor. In addition, the therapeutic efficacy of pH-responsive liposomes can be further enhanced by glucose injection regulating the acidity of tumor microenvironment. DISCUSSION: DVar7 modified acid-sensitive nanocarriers combined with acidity regulation have great potential to improve drug resistance in clinical practice, thus improving the response rate and therapeutic effect of chemotherapy.

An efficient method for the site-specific 99mTc labeling of nanobody.

Recently, there has been a lot of interest by using nanobodies (heavy chain-only antibodies produced naturally from the Camelidae) as targeting molecules for molecular imaging, especially for the nuclear medicine imaging. A radiolabeled method that generates a homogeneous product is of utmost importance in radiotracer development for the nuclear medicine imaging. The conventional method for the radiolabeling of nanobodies is non-specifically, which conjugates the radioisotope chelating group to the side chain ɛ-amine group of lysine or sulfhydryl of cysteine of nanobodies, with a shortcoming of produce of the heterogeneous radiotracer. Here we describe a method for the site-specific radioisotope 99mTc labeling of nanobodies by transpeptidase Sortase A. The radiolabeling process includes two steps: first step, NH2-GGGGK(HYNIC)-COOH peptide (GGGGK = NH2-Gly-Gly-Gly-Gly-Lys-COOH, HYNIC = 6-hydrazinonicotinyl) was labeled with 99mTc to obtain GGGGK-HYNIC-99mTc; second step, Sortase A catalyzes the formation of a new peptide bond between the peptide motif LPETG (NH2-Leu-Pro-Glu-Thr-Gly-COOH) expressed C-terminally on the nanobody and the N-terminal of GGGGK-HYNIC-99mTc. After a simple purification process, homogeneous single-conjugated and stable 99mTc-labeled nanobodies were obtained in >50% yield. This approach demonstrates that the Sortase A-mediated conjugation is a valuable strategy for the development of site-specifically 99mTc-labeled nanobodies.

High Intensity Focused Ultrasound-Responsive and Ultrastable Cerasomal Perfluorocarbon Nanodroplets for Alleviating Tumor Multidrug Resistance and Epithelial-Mesenchymal Transition.

Hypoxia is a hostile hallmark of most solid tumors, which often leads to multidrug resistance (MDR) and causes the failure of chemotherapy. Hypoxia also promotes epithelial-mesenchymal transition (EMT), leading to acceleration of tumor metastasis. Many chemotherapeutic drugs can further exacerbate hypoxia and thus promote metastasis. Therefore, relieving hypoxia is necessary for chemotherapy to inhibit both MDR and EMT. Herein, highly stable cerasomal perfluorocarbon nanodroplets with an atomic layer of polyorganosiloxane surface and pH-sensitive tumor-targeting peptide (D-vPCs-O2) were fabricated to co-deliver oxygen and therapeutic drug, doxorubicin. High-intensity focused ultrasound (HIFU) was utilized to trigger the co-release of doxorubicin and oxygen and simultaneously enhance ultrasound imaging, therefore achieving imaging-guided drug delivery. Mild-temperature HIFU (M-HIFU) not only triggered oxygen release from nanodroplets but also slightly elevated tumor temperature to accelerate tumor blood flow. The oxygen release and temperature elevation jointly relieved tumor hypoxia and alleviated MDR, which greatly enhanced the drug therapeutic efficacy as compared to clinically used doxorubicin and Doxil. Overall side effects were also largely reduced owing to the ultrastable drug loading of cerasome. The improvement of insufficient chemotherapy and the relief of tumor hypoxia corporately down-regulated TGF-ß1, leading to the alleviation of EMT, and therefore significantly inhibited tumor metastasis. When "D-vPCs-O2 + M-HIFU" was utilized as a neoadjuvant chemotherapy, nanodroplets down-regulated heat shock proteins, reducing tumor relapse after the high-temperature HIFU (H-HIFU)-mediated hyperthermia ablation. The chemo-hyperthermia therapy totally eradicated tumors without any relapse or metastasis, providing a promising way to treat the triple-negative breast cancer, which is highly malignant, easily metastatic, and lacks effective treatments.

Nuclear imaging-guided PD-L1 blockade therapy increases effectiveness of cancer immunotherapy.

OBJECTIVES: Strategies to improve the responsiveness of programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) checkpoint blockade therapy remain an essential topic in cancer immunotherapy. In this study, we developed a new radiolabeled nanobody-based imaging probe 99mTc-MY1523 targeting PD-L1 for the enhanced therapeutic efficacy of PD-L1 blockade immunotherapy by the guidance of 99mTc-MY1523 SPECT/CT imaging. METHODS: The binding affinity and specificity of nanobody MY1523 were measured in vitro. MY1523 was radiolabeled with 99mTc by a site-specific transpeptidation of Sortase-A, and the biodistribution and single photon emission CT (SPECT)/CT were performed in mice bearing different tumors. We used interferon-γ (IFN-γ) as an intervention means to establish animal models with different levels of PD-L1 expression, then investigated the ability of 99mTc-MY1523 SPECT/CT for the in vivo non-invasive measurement of PD-L1 expression in tumors. Finally, the PD-L1 blockade immunotherapies guided by 99mTc-MY1523 SPECT/CT were carried out in MC-38, A20, and 4T1 tumor-bearing mouse models, followed by the testing of tumor infiltration T cells. RESULTS: MY1523 exhibited a high binding affinity and specificity to PD-L1 and had no competitive binding with the therapeutic antibody. 99mTc-MY1523 was prepared with high specific activity and radiochemical purity. It was found that tumor PD-L1 expression was dynamically upregulated by IFN-γ intervention in MC-38, A20, and 4T1 tumor-bearing mouse models, as indicated by 99mTc-MY1523 SPECT/CT. The PD-L1 blockade therapy initiated during the therapeutic time window determined by 99mTc-MY1523 SPECT/CT imaging significantly enhanced the therapeutic efficacy in all animal models, while the tumor growth was effectively suppressed, and the survival time of mice was evidently prolonged. A correlation between dynamically upregulated PD-L1 expression and improved PD-L1 blockade therapy effectiveness was revealed, and the markedly increased infiltration of effector T cells into tumors was verified after the imaging-guided therapy. CONCLUSION: Our results demonstrated that 99mTc-MY1523 SPECT/CT allowed a real-time, quantitative and dynamic mapping of PD-L1 expression in vivo, and the imaging-guided PD-L1 blockade immunotherapy significantly enhanced the therapeutic efficacy. This strategy merits translation into clinical practice for the better management of combination therapies with radiotherapy or chemotherapy.

Developing PEGylated Reversed D-Peptide as a Novel HER2-Targeted SPECT Imaging Probe for Breast Cancer Detection.

Human epidermal growth factor receptor-2 (HER2)-enriched breast cancer is characterized by strong invasiveness, high recurrence rate, and poor prognosis. HER2-specific imaging can help screening right patients for appropriate HER2-targeted therapies. Previously, we have developed a 99mTc-labeled HER2-targeted H6 peptide for SPECT imaging of breast cancer. However, the poor metabolic stability and high gallbladder uptake hamper its clinical application. In this study, a retro-inverso D-peptide of H6 (RDH6) was designed to increase the metabolic stability. PEGylation was used to improve its water solubility and in vivo pharmacokinetics. The results showed that the D-amino acids in 99mTc-PEG4-RDH6 brought better metabolic stability than 99mTc-PEG4-H6, thus achieving higher tumor uptake. As the length of the PEG chain increases, the hydrophilicity of the probes gradually increased, which may also be the main cause for the decreased liver uptake. Compared with radiotracers modified by PEG4 and PEG12, 99mTc-PEG24-RDH6 had a comparable tumor uptake and the lowest liver radioactivity. The SPECT imaging demonstrated that 99mTc-PEG24-RDH6 could specifically distinguish HER2-positive tumors from HER2-negative tumors with better imaging contrast, which thus has the potential for clinical screening of HER2-positive breast patients.

An Integrin Alpha 6-Targeted Radiotracer with Improved Receptor Binding Affinity and Tumor Uptake.

In this study, we reported a 99mTc-labeled integrin α6-targeted peptide as the molecular imaging probe for tumor imaging by single-photon emission computed tomography (SPECT). We found that replacing Cys-Cys cyclized RWY peptide (sequence: cCRWYDENAC) with lactam-bridged cyclic cKiE peptide (sequence: cKRWYDENAisoE) did not sacrifice the integrin α6-binding affinity and specificity of cKiE radiotracer. To further improve the radiotracer's tumor targeting capability, the dimerized cKiE peptide (termed cKiE2) was designed, and the corresponding radiotracer 99mTc-cKiE2 was evaluated for tumor uptake and in vivo pharmacokinetics properties in tumor models. We found that cKiE2 showed higher binding affinity to integrin α6 than did monomeric RWY or cKiE peptide. The biodistribution results showed that the tumor uptake of 99mTc-cKiE2 was twice higher than that of 99mTc-RWY (3.20 ± 0.12 vs 1.26 ± 0.06 %ID/g, P < 0.001) at 0.5 h postinjection. The tumor to nontargeting tissue ratios were also enhanced in most normal organs. Specificity of 99mTc-cKiE2 for integrin α6 was demonstrated by competitive blocking of tumor uptake with excess cold peptide (3.20 ± 0.24 to 1.38 ± 0.23 %ID/g, P < 0.001). The integrin α6-positive tumors were clearly visualized by 99mTc-cKiE2/SPECT with low background except with a relatively high kidney uptake. The tumor uptake of 99mTc-cKiE2 correlates well with the tumor integrin α6 expression levels in a linear fashion (R2 = 0.9623). We also compared 99mTc-cKiE2 with an integrin αvß3-targeted radiotracer 99mTc-3PRGD2 in the orthotopic hepatocellular carcinoma tumor models. We found that the orthotopic tumor was clearly visualized with 99mTc-cKiE2. 99mTc-3PRGD2 imaging did not show tumor contours in situ as clearly as 99mTc-cKiE2. The tumor-to-liver ratios of 99mTc-cKiE2 and 99mTc-3PRGD2 were 2.20 ± 0.17 and 0.85 ± 0.20. In conclusion, 99mTc-cKiE2 is an improved SPECT radiotracer for imaging integrin α6-positive tumors and has great potential for further clinical application.

Author Correction: A next-generation tumor-targeting IL-2 preferentially promotes tumor-infiltrating CD8+ T-cell response and effective tumor control.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Imaging and monitoring HER2 expression in breast cancer during trastuzumab therapy with a peptide probe 99mTc-HYNIC-H10F.

PURPOSE: The novel molecular imaging probe 99mTc-HYNIC-H10F was developed for patient screening and efficacy monitoring of trastuzumab therapy by SPECT imaging of HER2 expression in breast cancer. METHODS: 99mTc-HYNIC-H10F was developed by labeling H10F peptide with 99mTc following an optimized protocol. Biodistribution and SPECT/CT were performed in mouse models bearing HER2-positive SK-BR3 and HER2-negative MDA-MB-231 human breast cancer xenografts, respectively. The treatment response to trastuzumab was monitored and quantified by SPECT/CT in two HER2-positive breast cancer models (SK-BR3 and MDA-MB-361). The preliminary clinical study was performed in two patients with breast cancer. RESULTS: SPECT/CT with 99mTc-HYNIC-H10F showed that the SK-BR3 tumors were clearly visualized, while the signals from MDA-MB-231 tumors were much lower. The tumor uptake of 99mTc-HYNIC-H10F could be blocked by excess unlabeled H10F peptide but not by excess trastuzumab. The growth of two HER2-positive tumors was prominently suppressed at day 11 post-treatment. However, SPECT/CT reflected much earlier therapy response at day 4 post-treatment. The HER2 expression in tumors of breast cancer patients could be detected by 99mTc-HYNIC-H10F SPECT/CT imaging. CONCLUSIONS: 99mTc-HYNIC-H10F specifically accumulates in HER2-positive tumors. Compared with trastuzumab, 99mTc-HYNIC-H10F binds to a different domain of HER2 antigen, providing new opportunities to monitor HER2 expression levels before/during/after trastuzumab treatment for more effective personalized treatment.

A 64 Cu-porphyrin-based dual-modal molecular probe with integrin αv ß3 targeting function for tumour imaging.

Pyropheophorbide-a (Pyro) is a promising multifunctional molecule for multimodal tumour imaging and photodynamic therapy, but its clinical applications are seriously restricted by the limited tumour accumulation capability. Here, we designed and synthesized a small-molecule probe that achieved specific dual-modal tumour imaging based on Pyro. Briefly, a novel molecule combining Pyro, an RGD dimer peptide (3PRGD2 ) and 64 Cu, was designed and synthesized, and the obtained molecule, 64 Cu-Pyro-3PRGD2 , exhibited high tumour specificity in both positron emission tomography and optical imaging in vivo. c (RGDfk) peptide blocking significantly reduced the efficacy of the probe, which confirmed the integrin αV ß3 targeting of this molecular probe. 64 Cu-Pyro-3PRGD2 had very low accumulation in normal organs and could be rapidly cleared through kidney metabolism, which prevented the potential damage to adjacent normal tissues. Overall, combining tumour targeting, dual-modal imaging, and biosafety, 64 Cu-Pyro-3PRGD2 has the potential for clinical use as a molecular imaging probe for tumour diagnosis.

Blood-triggered generation of platinum nanoparticle functions as an anti-cancer agent.

Since the discovery of metal nanoparticles (NPs) in the 1960s, unknown toxicity, cost and the ethical hurdles of research in humans have hindered the translation of these NPs to clinical use. In this work, we demonstrate that Pt NPs with protein coronas are generated in vivo in human blood when a patient is treated with cisplatin. These self-assembled Pt NPs form rapidly, accumulate in tumors, and remain in the body for an extended period of time. Additionally, the Pt NPs are safe for use in humans and can act as anti-cancer agents to inhibit chemotherapy-resistant tumor growth by consuming intracellular glutathione and activating apoptosis. The tumor inhibitory activity is greatly amplified when the Pt NPs are loaded in vitro with the chemotherapeutic drug, daunorubicin, and the formulation is effective even in daunorubicin-resistant models. These in vivo-generated metal NPs represent a biocompatible drug delivery platform for chemotherapy resistant tumor treatment.