Theranostics

Cerebrospinal fluid (CSF) is drained into the systemic lymphatics via paravertebral lymph nodes. Superficial and deep cervical lymph nodes collect CSF in mice, but the exact and quantified routes are unknown. Recently, we simultaneously visualized cervical, sacral and iliac lymph nodes via serial imaging on the intrathecal [64Cu]Cu-albumin positron emission tomography. Paravertebral lymph nodes might act as sentinels to monitor the CSF, brain, and spinal cord. We used 64Cu-labeled Escherichia coli extracellular vesicles, outer membrane vesicles (OMVs), as lymph node seekers for intrathecal administration with an optimized volume/rate and quantified the differential amounts of various paravertebral lymph nodes along the axis of the brain and spinal cord in mice. The quantified results revealed 77.3% in superficial and deep cervical lymph nodes, 11.4% in abdominal/pelvic lymph nodes and 11.3% in sacral lymph nodes. Click-labeled [64Cu]Cu-OMVs were drained to reach and stop at the lymph nodes on serial quantification. The cervical lymph nodes drained most of the OMV-laden CSF, which is proportional to the surface areas of the brain (70%) and spinal cord in mice. We propose that all paravertebral lymph nodes monitor the segmental regions of the brain and spinal cord as immediate sentinel lymph nodes against the central nervous system.

Incomplete extent of tumor resection is the major factor of glioblastoma recurrence, leading to its poor prognosis. Developing therapies that enable the minimal tumor cell remnant during surgical resection is a major challenge to combat this aggressive cancer. This study develops a glioblastoma integrin 5{beta}1-selective peptide binder through series strategy screening and labeling it with 68Ga, indocyanine green (ICG), or 177Lu for efficacious multimodal treatment. This strategy combines positron emission tomography (PET) imaging for preoperative diagnosis, intraoperative NIR-II fluorescence-guided surgery for maximal tumor resection, and followed by intracavity targeted radionuclide therapy for elimination of residual tumor cells. We identified GR, with five arginine mutation, exhibiting superior integrin 5{beta}1 binding affinity and brain penetration. [68Ga]GR demonstrated substantial tumor uptake and prolonged retention time in both mouse models and tissues from glioblastoma patients. The guidance of ICG-GR achieved accurately intraoperative tumor imaging and maximal tumor resection in orthotopic mouse glioblastoma models. Notably, combining intracavity administration of [177Lu]GR following ICG-GR guiding resection markedly inhibited tumor growth and reduced possibility of tumor recurrence compared with surgery alone (unguided or ICG-GR-guided) or unguided surgery followed by [177Lu]GR. Therefore, we have reported the integrin 5{beta}1-targeted radionuclide and optical agents for maximal elimination of tumor cells during surgical resection in glioblastoma patients. HIGHLIGHTSO_LIGR is a glioblastoma-specific peptide with both favorable binding affinity superior brain penetration. C_LIO_LIICG-GR is a glioblastoma-targeted NIR probe enabling maximal surgical resection. C_LIO_LIICG-GR-guided surgery with [177Lu]GR-based adjuvant therapy allows robust elimination of tumor cells. C_LI

The spleen, as the largest immune organ, plays a pivotal role in modulating immune responses, particularly in the context of carcinogenesis and tumor progression. Non-pharmacological manipulation, particularly splenic ultrasound stimulation (SUS), has demonstrated significant immunomodulatory efficacy in alleviating chronic inflammatory diseases, suggesting its potential to revitalize splenic immunocompetence suppressing tumor proliferation, yet remains underexplored. This study applied low-frequency pulsed focused ultrasound (FUS) noninvasively stimulating the spleen (FUS sti. spleen) to investigate the efficacy in enhancing antitumor immunity and suppressing hepatocellular carcinoma (HCC). The results showed that FUS sti. spleen significantly suppressed tumor proliferation, achieving a suppression rate of >70% for H22-HCC and >83% for Hepa1-6-HCC, along with significantly prolonged survival. Comprehensive flow cytometry, single-cell RNA sequencing (scRNA-seq) and cytokine analyses demonstrated that SUS profoundly reshaped the splenic and intratumoral immune landscape, specifically activating cytotoxic CD8+ T cells and NK cells while suppressing immunosuppressive cell populations. Mechanistically, FUS facilitated calcium influx in splenic immunocytes, activating multiple signaling pathways, such as TNF, NF{kappa}B, MAPK, HIF-1, and ErbB, thereby counteracting tumor-driven immunosuppressive polarization while potentiating robust immune activation that impedes malignant progression and neoplastic proliferation. Leveraging above insights, we developed spleen-targeted nanodroplets encapsulating bioavailable calcium ions (STNDs@Ca{superscript 2}), which, upon FUS stimulation, undergo cavitation-mediated controlled release of Ca{superscript 2}, further amplifying immunocyte activation and tumor suppression, achieving a remarkable H22-HCC suppression rate of over 90%. This study highlights the therapeutic potential of ultrasound-mediated splenic immunomodulation, both as a standalone intervention and in synergy with STNDs@Ca{superscript 2}, as a novel and noninvasive strategy for cancer immunotherapy.

PurposeThe limited availability of molecularly targeted low-molecular-weight imaging agents for monitoring multiple myeloma (MM)-targeted therapies has been a significant challenge in the field. In response, we developed [68Ga]Ga-AJ206, a peptide-based radiotracer that can be seamlessly integrated into the standard clinical workflow and is specifically designed to non-invasively quantify CD38 levels and pharmacodynamics by positron emission tomography (PET). Experimental designWe synthesized a high-affinity binder for quantification of CD38 levels. Affinity was tested using surface plasmon resonance, and In vitro specificity was evaluated using a gallium-68-labeled analog. Distribution, pharmacokinetics, and CD38 specificity of the radiotracer were assessed in MM cell lines and in primary patient-derived myeloma cells and xenografts (PDX) with cross-validation by flow cytometry and immunohistochemistry. Furthermore, we investigated the radiotracers potential to quantify CD38 pharmacodynamics induced by all-trans retinoic acid therapy (ATRA). Results[68Ga]Ga-AJ206 exhibited high CD38 binding specificity (KD: 19.1{+/-}0.99 nM) and CD38-dependent In vitro binding. [68Ga]Ga-AJ206-PET showed high contrast within 60 minutes and suitable absorbed dose estimates for clinical use. Additionally, [68Ga]Ga-AJ206 detected CD38 expression in xenografts, PDXs and disseminated disease models in a manner consistent with flow cytometry and immunohistochemistry findings. Moreover, [68Ga]Ga-AJ206-PET successfully quantified CD38 pharmacodynamics in PDXs, revealing increased CD38 expression in the tumor following ATRA therapy. Conclusions[68Ga]Ga-AJ206 exhibited the salient features required for clinical translation, providing CD38-specific high contrast images in multiple models of MM. [68Ga]Ga-AJ206-PET could be useful for quantifying total CD38 levels and pharmacodynamics during therapy to evaluate approved and new therapies in MM and other diseases with CD38 involvement. STATEMENT OF TRANSLATIONAL RELEVANCEThere is an unmet need for functional imaging agents to monitor the pharmacodynamic effects of new therapeutics targeting multiple myeloma (MM). MM is a challenging bone marrow plasma cell cancer that is associated with heterogenous responses and universal recurrence. While minimal residual disease monitoring by blood and invasive bone marrow samples have improved prognostication of disease recurrence, molecularly targeted, non-invasive imaging options that can assess therapy response early remain limited. To address this gap, we report the development of a high affinity, first-in-class gallium-68 labeled peptide radiotracer, [68Ga]Ga-AJ206, for CD38 protein, which is highly expressed on MM cells. [68Ga]Ga-AJ206 provides high-contrast CD38-specific images by PET within the standard clinical workflow of 60 minutes. Furthermore, the potential of [68Ga]Ga-AJ206 PET to measure pharmacodynamics of CD38 was demonstrated. Further development of this new radiotracer may complement existing technologies and improve prognostication and monitoring of therapy response in patients with MM.

Molecular fluorescence-guided surgery has shown promise for tumor margin delineation but is limited by its depth profiling capability. Interestingly, most fluorophores, either clinically approved or in clinical trials, can also be used as photoacoustic contrast agents, yet their use is limited due to the low light fluence permitted for clinical use and the limited sensitivity of current photoacoustic imaging systems. There is therefore an urgent unmet need to establish methods for enhancing contrast in molecular targeted PA imaging which could potentially complement and overcome limitations in molecular fluorescence guided therapies. In this study, we compare the photoacoustic (PA) and fluorescence imaging capabilities of a cetuximab-IRDye800 conjugate in a subcutaneous tumor xenograft model. We demonstrate that while the fluorescence signal increases steadily over time after administration of cetuximab-IRDye800, PA signal peaks early ([~]2 fold higher at 6-hour as compared to pre-injection controls) and then decreases ([~]1.3 fold higher at 24-hour as compared to pre-injection controls). This pattern aligns with previous findings using other antibody-conjugated PA contrast agents. Mechanistically, we demonstrate that the formation of H-aggregates upon antibody conjugation enhances PA contrast of the IRDye800. The disruption of these H-aggregates, as the antibody-dye conjugate is degraded post receptor-mediated endocytosis, decreases PA signal intensity. The timeframe of maximum PA signal and decrease thereafter is consistent with the time frame of receptor-mediated endocytosis of cetuximab-IRDye800. Our data suggests that tumor cell surface binding results in peak PA signal while lysosomal localization and degradation results in a significant drop in PA signal. Our study sheds light on the distinct temporal dynamics of PA and fluorescence signals of Cetuximab-IRDye800 conjugate and we propose that optimizing IRDye800 conjugation to antibodies can further enhance PA signal intensity when timed to precisely to capture IRDye800 in an H-aggregate form.

In the cerebral cortex, the vasculature plays important homeostatic functions, especially at the highly connected complex capillary networks. The association of focal capillary ischemia with the neurodegenerative disease as well as the laminar vascular dynamics have prompted studies of vascular micro-occlusion via photothrombosis. However, technical challenges of this approach remain, including increased temporal precision of occlusion, increasing the depth of vascular occlusion, understanding how such micro-occlusion impacts local blood flow, and ultimately the neuronal effects of such changes. Here, we have developed a novel approach that employs ultra-fast multiphoton light to induce focal Rose Bengal-induced photothrombosis. We demonstrated induction of highly precise and fast occlusion of microvessels at various types and depths. The change of the microvascular architecture and hemodynamics after occlusion revealed the autoregulation and significant difference between upstream vs downstream in layer 2/3. Further, we found that micro-occlusion at two different layers within the same vascular arbor results in distinct effects on the acute flow redistribution mechanism. To examine neuronal effects of such micro-occlusion, we produced infarct of capillaries surrounding a labeled target neuron and found this induces dramatic and rapid lamina-specific degeneration in neuronal dendritic architecture. In sum, our technique enhanced the precision and power of the photothrombotic study of microvascular function. The current results pointed to the importance of laminar scale regulation within the microvascular network, a finding which may be relevant for models of neurovascular disease.

Clinically, there has been significant interest in the use of exosomes for diagnostic applications as promising biomarkers and therapeutic applications as therapeutic vehicles. However, knowledge of in vivo physiological biodistribution of exosomes was difficult to assess until now. Physiological distribution of exosomes in the body must be elucidated for clinical application. In this study, we aimed to develop reliable and novel methods to monitor biodistribution of exosomes using in vivo PET and optical imaging. MethodsExosomes were isolated from cultured medium of 4T1, mouse breast cancer cells. Exosomes were labeled with Cy7 and 64Cu (or 68Ga). In mice, radio/fluorescent dye-labeled exosomes were injected through the lymphatic routes (footpad injection) and hematogenous metastatic routes (tail vein injection). Fluorescence and PET images were obtained and quantified. Radio-activity of ex vivo organs was measured by gamma counter. ResultsPET signals from exosomes in the lymphatic metastatic route were observed in the draining lymph nodes, which are not distinguishable with optical imaging. Immunohistochemistry revealed greater uptake of exosomes in brachial and axillary lymph nodes than inguinal lymph node. After administration through the hematogenous metastasis pathway, accumulation of exosomes was clearly observed in PET images in the lungs, liver, and spleen, showing results similar to ex vivo gamma counter data. ConclusionExosomes from tumor cells were successfully labeled with 64Cu (or 68Ga) and visualized by PET imaging. These results suggest that this cell type-independent, quick, and easy exosome labeling method using PET isotopes could provide valuable information for further application of exosomes in the clinic.

Here, positronium imaging is presented to determine cardiac myxoma (CM) extracted from patients undergoing urgent cardiac surgery due to unexpected atrial masses. Positronium is an atom build from an electron and a positron, produced copiously in intra-molecular voids during the PET imaging. CM, the most common cardiac tumor in adults, accounts for 50-75% of benign cardiac tumors. We aimed to assess if positronium serves as a biomarker for diagnosing CM. Perioperative examinations and histopathology staining in six patients confirmed the primary diagnosis of CM. We observed significant differences in the mean positronium lifetime between tumor and normal tissues, with an average value of 1.92(02) ns and 2.72(05) ns for CM and the adipose tissue, respectively. Our findings, combined with positronium lifetime imaging, reveals the novel emerging positronium biomarker for cardiovascular imaging. One-Sentence SummaryPositronium may serve as an imaging biomarker for cancer diagnostics.

Current treatment of IDH-wildtype glioblastoma (GBM) relies on the first-line chemotherapy-temozolomide. Although MGMT methylation is routinely conducted to predict chemosensitivity, its efficacy is often compromised. Thus, there is an urgent need to discover more accurate prognostic biomarkers. Cholesteryl ester (CE) has been recently recognized as a key feature of GBM, however, its role in GBM prognosis remains poorly understood. We first employed label-free stimulated Raman scattering (SRS) imaging to quantitatively analyze CE level in intact tumor tissues obtained from IDH-wildtype GBM patients. Our result revealed significantly prolonged 2-year overall survival (OS) in patients with CE level [&ge;] 40% compared to those with CE level < 40%. CE outperformed MGMT methylation for 2-year OS prognosis (AUC: 0.836 vs. 0.763). Importantly, CE also achieved superior prognostic performance over MGMT methylation on an independent cohort, with higher sensitivity (0.856 vs. 0.667), specificity (0.833 vs. 0.583), NPV (1.00 vs. 0.667), PPV (0.833 vs. 0.583). Given synergistic effects between CE and MGMT methylation, we developed a prognostic model combining these two biomarkers. Specially, machine learning (XGBoost) model exhibited optimal performance in the training cohort (AUC: 0.920), and maintained its superior performance on the independent cohort (sensitivity: 0.946, specificity: 0.873, NPV: 1.00; PPV: 0.917). Mechanistically, integrative analysis of TCGA database linked poor prognosis to the coordinated upregulation of genes involved in cholesterol efflux, hydrolysis, transport, and inhibition of de novo synthesis, unraveling a possible underlying mechanism between poor prognosis and cholesterol metabolism. This work identified CE as a prognostic biomarker for IDH-wildtype GBM.

Cancer cells are heavily reliant on de novo pyrimidine synthesis. Suppression of pyrimidine metabolism directly inhibits tumor growth and fosters immune activation within the tumor microenvironment. Dihydroorotate dehydrogenase (DHODH), a crucial enzyme governing de novo pyrimidine synthesis, is a critical player in this context. Inhibition of DHODH not only reverses immunosuppression but also instigates a mild innate immune response. However, the impact of DHODH inhibition on natural killer (NK) cells remains unexplored. In this study, we found that inhibition of DHODH efficiently promotes NK cells infiltration in tumors. Suppression of DHODH led to increased oxidative stress in mitochondria, the release of mtDNA, and activation of caspase 3, which in turn activated the cGAS-STING pathway and pyroptosis in cancer cells, respectively, contributing to NK cells induced antitumor immune responses in melanoma. Additionally, we developed EA6, a novel DHODH inhibitor with higher efficacy in promoting NK cells infiltration. In summary, this study underscores that modulation of pyrimidine metabolism can effectively trigger antitumor immune responses, with a specific emphasis on NK cells. This finding opens new avenues for enhancing the efficacy of targeted nucleotide metabolism in cancer therapy. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/644471v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@88d4fcorg.highwire.dtl.DTLVardef@156fe1aorg.highwire.dtl.DTLVardef@448086org.highwire.dtl.DTLVardef@152079c_HPS_FORMAT_FIGEXP M_FIG C_FIG The anti-tumor mechanisms of DHODH inhibition. Inhibition of DHODH activates cGAS-STING pathways to enhance NK cell infiltration. And the tumor-infiltrating NK cells facilitate melanoma cells pyroptosis which providing a positive feedback mechanism for DHODH-mediated anti-tumor immunity.

Ferroptosis is a regulated non-apoptotic form of programmed cell death that is implicated in tumor suppression and the normal tissue damage response. While the link between redox stress and ferroptosis is well established, no non-invasive methods exist to assess ferroptosis in vivo. Here, we demonstrate that the redox-sensitive positron emission tomography radiotracer and system xc-substrate, 18F-(S)-4-(3-fluoropropyl)-L-glutamic acid ([18F]FSPG), serves as a non-invasive marker of tumor ferroptosis. Global changes in amino acids, glutathione, and system xc- activity occurred before loss of membrane integrity in cells sensitive to ferroptosis, but not in resistant cells. Resistant cells sensitized to ferroptosis through nuclear factor erythroid 2-related factor 2 (NRF2) knockout had reduced glutathione and [18F]FSPG retention, which were rescued by ferroptosis inhibitors. In vivo, immune checkpoint blockade decreased ferroptosis-specific [18F]FSPG tumor retention prior to immune cell infiltration. Together, our data demonstrate that [18F]FSPG can identify early redox changes that precede ferroptosis and enabled real-time monitoring of immunotherapeutic efficacy.

Because of its insensitive to existing radiotherapy, chemotherapy and targeted treatments, Triple-negative breast cancer (TNBC) remains a great challenge to overcome. More and more evidence has indicated abnormal wnt/{beta}-catenin pathway activation in TNBC but not luminal or her2+ breast cancer, and lncRNAs play a key role in a variety of cancers. Through lncRNA microarray profiling between Activated and inactivated Wnt/{beta}-catenin pathway of TNBC tissues, lnc-WAL (Wnt/{beta}-catenin associated lncRNA; WAL) was selected as the top up-regulated lncRNA in Wnt/{beta}-catenin pathway activation compared with the inactivation group. RIP-seq was analyzed between {beta}-catenin and IgG groups of, where lnc-WAL could interact with {beta}-catenin. Clinically, increased lnc-WAL in the TNBC tumor tissue was associated with shorter survival. lnc-WAL promoted the EMT, the ability of breast cancer stem cells (BCSC), proliferation, migration and invasion of TNBC cells. Mechanistically, lnc-WAL inhibited {beta}-catenin protein degradation via Axin-mediated phosphorylation at serine 45. Subsequently, {beta}-catenin was accumulated in nuclear and activated the target genes. Importantly, Wnt/{beta}-catenin pathway activation stimulated the transcription of lnc-WAL. These results pointed to a master regulatory role of lnc-WAL/Axin/{beta}-catenin in the malignant progression of TNBC. Our findings provide important clinical translational evidence that lnc-WAL maybe as potential therapeutic target against TNBC.

Lung adenocarcinoma (LUAD) remains one of the leading causes of death in patients with cancer. The association of CD155 with CD96 transmits an inhibitory signal and suppresses antitumor immune response. This study investigates the effect of CD155/CD96 on immune suppression in LUAD. We demonstrate that LUAD patients with high CD155 expression suffer from immune suppression and experience a poor prognosis, which coincides with an inhibited AKT-mTOR signaling pathway in CD8 T cells and subsequently up-regulated CD96 expression. Moreover, the inhibition effect can be reversed by CD96 blocking antibody. High CD155 expression inhibited the release of IFN{gamma} from CD8 cells. Moreover, Blocking CD96 restored IFN{gamma} production in CD8 T cells and neutralized the inhibition of IFN{gamma} production in CD8 T cells mediated by CD155. Animal experiments showed that CD155-mediated LUAD growth might depend on its suppression antitumor immune response in the tumor microenvironment in PDX mice. In conclusion, our results suggest that LUAD cells suppress antitumor immune response in the tumor microenvironment through CD155/CD96. CD155/CD96 could be a potential therapeutic target for LUAD patients.\n\nAbbreviationsLUAD: lung adenocarcinoma; IFN{gamma}: interferon gamma; PDX: patient-derived xenograft; NSCLC: non-small cell lung cancer; PRR: poliovirus receptor-related; MDSCs: myeloid-derived suppressor cells; PRR: poliovirus receptor-related; STR: short tandem repeat; IRS: immunoreactive score; SI: staining intensity; PP: percentage of positive cells; RT-PCR: reverse transcription-polymerase chain reaction; PBS: phosphate-buffered saline; PBMCs: peripheral blood mononuclear cells; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; rCD155: recombinant human CD155; LUAD cells: lung adenocarcinoma cells; TILs: tumor-infiltrating lymphocytes; GzmB: granzyme B; IL-2 (Interleukin-2); TNF- : tumor necrosis factor-alpha; PI: propidium Iodide; PDX: patient-derived xenograft; TIGIT: T cell immunoreceptor with Igand ITIM domains; WBC: white blood cells; MFI: mean fluorescence intensity; HPF: high power field

BACKGROUNDCurcumin has emerged as a promising candidate capable of polarization of macrophages, which promote the stability of atherosclerotic plaque. Nevertheless, a notable limitation lies in the non-specific nature of curcumins targeting. The present study endeavors to harness multifunctional lipid nanoparticles (MLNPs), which could both facilitate imaging and achieve targeted delivery of curcumin specifically to inflammatory macrophages, to effectively counteract vulnerable plaque and mitigate the risk of ischemic events. METHODSThe term "MLNPs", for targeted delivery of curcumin using multimodal imaging techniques including single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI), refers to a new type of nanoparticle designed to specifically target and modulate macrophages with phagocytic function. These nanoparticles are cholesteryl-9-carboxynonanoate-(125I-iron oxide nanoparticle/Cur)-lipid-coated nanoparticles [9-CCN-(125I-ION/Cur)-LNPs], which carry hybrid imaging agents. These agents are combinations of 125I-ION and lipids that contain phagocytic "eat-me" signals, which induces macrophages to swallow MLNPs. RESULTSThe accumulation of the devised 9-CCN-(125I-ION/Cur)-LNPs on the unstable plaque of animal models in vivo was accurately reflected and lesions were highlighted by both SPECT and MRI. The intense radioactive signals on SPECT images facilitated the identification and quantification of the target lesion, while MRI based on ION particles facilitated the visualization of the focal localization and volumetry of atherosclerotic plaque. The targeted distribution of the unstable plaque in the rabbit aorta was further confirmed by ex vivo planar images of nuclide and Prussian blue staining for ION. Additionally, 9-CCN-(125I-ION/Cur)-LNPs were found to specifically and effectively bind to the pro-inflammatory M1 macrophages present in the unstable plaque, resulting in the accumulation of radionuclide and hypointensity on T2W images. CONCLUSIONSThe 9-CCN-(125I-ION/Cur)-LNPs demonstrated remarkable capability in the delivery of both 125I-ION and curcumin to macrophages, ultimately resulting in M1-M2 macrophage polarization, which may offer valuable insights into addressing unstable atherosclerotic plaque.

Intracellular pH (pHi) is a valuable index for predicting hypoxic brain damage. However, no positron emission tomography (PET) probe is currently available for monitoring pHi in vivo. In this study, we developed a new approach for visualizing monoacylglycerol lipase (MAGL) activity in the brain. This approach used PET with a new probe [11C]HC-A, an azetidine carbamate inhibitor, whose uptake and residence depended on the pHi gradient was evaluated with in silico, in vitro, and in vivo assessments. Molecular dynamics simulations predicted that complex (complex-A) between HC-A and MAGL would be difficult to hydrolyze under acidic conditions. In vitro assessment using rat brain homogenate showed that [11C]HC-A reacted with MAGL to yield [11C]complex-A, which was rapidly hydrolyzed to liberate 11CO2. The 11CO2 liberation rate was slower at lower pH. In PET with [11C]HC-A using ischemic rats, the radioactivity clearance rate, which reflects the production rate of 11CO2 in the brain, was lower in a remarkably hypoxic area than in the contralateral region. In conclusion, we successfully visualized the pHi gradient in the brain using PET imaging.

The optimization of dosing strategies is critical for maximizing efficacy and minimizing toxicity in drug development, particularly for drugs with narrow therapeutic windows such as antibody-drug conjugates (ADCs). This study demonstrates the utility of Nectin-4-targeted positron emission tomography (PET) imaging using [68Ga]AJ647 as a non-invasive tool for real-time assessment of target engagement in enfortumab vedotin (EV) therapy for urothelial carcinoma (UC). By leveraging the specificity of [68Ga]AJ647 for Nectin-4, we quantified dynamic changes in target engagement across preclinical models and established its correlation with therapeutic outcomes. PET imaging revealed dose-dependent variations in Nectin-4 engagement, with suboptimal EV doses resulting in incomplete Nectin-4 engagement and reduced tumor growth. Importantly, target engagement measured by PET emerged as a more reliable predictor of therapeutic efficacy than dose or baseline Nectin-4 expression alone. Receiver operating characteristic (ROC) analysis identified a target engagement threshold that is determinant of response, providing a quantitative benchmark for dose optimization. Furthermore, PET imaging measures provide a promising framework to account for key challenges in ADC development, including tumor heterogeneity, declining drug-to-antibody ratios over time, and limitations of systemic pharmacokinetic measurements to account for tumor-drug interactions. These findings underscore the transformative potential of integrating PET pharmacodynamic measures as early biomarkers to refine dosing strategies, improve patient outcomes, and accelerate the clinical translation of next-generation targeted therapeutics.

Currently, the DFO chelator is commonly used to conjugate monoclonal antibodies (mAbs) and 89Zr, whereas the DOTA chelator is commonly used to conjugate mAbs and alpha- and beta-emitting metal radionuclides. However, if the degradation of [89Zr]Zr-DFO-mAb is not negligible, the in vivo biodistribution of 89Zr might not reflect that of metal radionuclides conjugated with DOTA-mAb. We hypothesized that [89Zr]Zr-DOTA-mAb as a new imaging counterpart would accurately predict the biodistribution of therapeutic metal radionuclides delivered by DOTA-mAb. In this study, we prepared [89Zr]Zr-DOTA-trastuzumab for the first time by a two-step reaction using click chemistry and then investigated the differences in biodistribution profiles between two chelating approaches for 89Zr. MethodsWe prepared [89Zr]Zr-DOTA-trastuzumab from DOTA-tetrazine conjugates (DOTA-Tz) and transcyclooctene-trastuzumab conjugates (TCO-trastuzumab). We first radiolabeled DOTA-Tz with 89Zr in a reaction solution of MeOH and HEPES buffer and then used a click reaction to obtain [89Zr]Zr-DOTA-Tz/TCO-trastuzumab. We performed biodistribution studies and PET imaging with [89Zr]Zr-DOTA-trastuzumab in a mouse model of HER2-positive ovarian cancer, SKOV3 xenograft mice at 24, 72, and 144 hours post-injection and compared these data with those of [89Zr]Zr-DFO-trastuzumab. ResultsTCO-trastuzumab was radiolabeled with [89Zr]Zr-DOTA-Tz in the two-step reaction in good radiochemical yield (57.8 {+/-} 17.6%). HER2-positive tumors were clearly visualized with [89Zr]Zr-DOTA-trastuzumab in PET imaging studies. The temporal profile changes of 89Zr radioactivity in SKOV3 tumors and bone marrow were sufficiently different between [89Zr]Zr-DOTA-trastuzumab and [89Zr]Zr-DFO-trastuzumab (P < 0.05). Conclusion: [89Zr]Zr-DOTA-trastuzumab can be produced by the two-step radiolabeling reaction based on the Tz/TCO click reaction. Presumably, 89Zr released from DFO is not negligible. In contrast, [89Zr]Zr-DOTA-mAb would better predict the biodistribution of [177Lu]Lu- or [225Ac]Ac-DOTA-mAb than [89Zr]Zr-DFO-mAb, thus avoiding the use of different chelator for 89Zr at the expense of the click chemistry step. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/507067v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@d7256borg.highwire.dtl.DTLVardef@d9d3d5org.highwire.dtl.DTLVardef@e24d97org.highwire.dtl.DTLVardef@1541bf0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Myeloperoxidase (MPO) is a heme peroxidase that is mainly expressed and secreted by neutrophils. MPOs role in inflammatory diseases has been highlighted in recent years, but its role in tumor development remains unclear. Therefore, we investigated the role of MPO in non-small cell lung cancer (NSCLC). In silico analysis revealed a survival benefit in patients with NSCLC and low MPO expression. Furthermore, a syngeneic tumor model using MPO knockout (KO) mice revealed that mice lacking MPO had lower tumor growth than controls. The reduction in tumor size was accompanied by an increase in lymphoid populations, including natural killer cells and CD8+ T cells, suggesting a shift to a more anti-tumorigenic immune environment in MPO-KO mouse tumors. The T cell induced interferon-gamma (IFN-{gamma}) expression was increased in MPO-KO tumors, indicating increased tumoricidal activity. CD8 depletion abolished the previously observed reduction in tumor size in MPO-KO mice, indicating that CD8+ T cells play an important role. In vitro, T cells treated with MPO showed reduced proliferation and IFN-{gamma} expression. Furthermore, MPO could be internalized into T cells. Heparin pretreatment of T cells blocked MPO binding and internalization into T cells and reversed MPO-induced proliferation reduction. Interestingly, MPO+ lymphocytes were found in tumor samples from patients with NSCLC. Our findings suggest that MPO plays an immunosuppressive role in NSCLC. One Sentence SummaryHigh myeloperoxidase (MPO) expression in non-small cell lung cancer patients is a predictor for adverse outcome and mice lacking MPO showed enhanced anti-tumorigenic leukocyte infiltration, suggesting a pro-tumorigenic role of MPO.

Extracellular vesicles (EV) are comprised of vesicles budding from cell membranes and smaller intracellular vesicles shed by cells. EV play a role in remodeling the tumor microenvironment (TME) and support tumor progression. Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein with a carboxypeptidase function, frequently associated with poor clinical prognosis in prostate cancer (PCa). We previously identified an oncogenic PSMA signaling function in prostate cancer. Others demonstrated that EV isolated from the plasma of patients with high-grade PCa carry PSMA, but so far no pathophysiological effect has been associated with PSMA-bearing EV. Here we demonstrate that EV from PCa cells are able to transfer PSMA and its functionality to cells in the TME. The consequence of that EV-mediated PSMA transfer is an acute to long-term increased secretion of vascular endothelial growth factor-A (VEGF-A), angiogenin, pro-angiogenic and pro-lymphangiogenic mediators and increased 4E binding protein 1 (4EBP-1) phosphorylation in tumors. We compare EV from PCa cells with or without PSMA expression to address the role of PSMA-bearing EV in promoting pro-tumoral changes in the TME using classical molecular biology and novel molecular imaging approaches.

Radiopharmaceutical therapy is changing the standard of care in prostate cancer (PCa) and other malignancies. We previously reported high CD46 expression in PCa and developed an antibody-drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [225Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody. Our radiolabeled antibody retains binding efficacy and shows a high tumor to background ratio in PCa xenografts. Furthermore, we show that radiolabeled antibody was able to suppress the growth of cell-derived and patient-derived xenografts, including PSMA-positive and deficient models. Nephrotoxicity, not seen at low radioactive doses, is evident at higher radioactivity dose levels, likely due to redistribution of daughter isotope 213Bi. Overall, this preclinical study confirms that [225Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46 targeted radioligand therapy in PCa.