Abstract Background Boron neutron capture therapy relies on the selective accumulation of boron-containing compounds in tumor tissue, making accurate quantification of boron distribution essential for effective treatment planning. The amino acid analog boronophenylalanine is widely used as a boron delivery agent, yet direct assessment of its biodistribution remains challenging. A fluorine-18 labeled analog, 2-fluoro-boronophenylalanine, offers the potential to visualize and quantify uptake through positron emission tomography. However, reported radiosynthetic methods often suffer from low radiochemical yield, complex workflows, and limited compatibility with automated production platforms. The aim of this study was to design a stable precursor suitable for nucleophilic fluorination, develop a fully automated single-reactor radiosynthesis, and characterize the resulting tracer to support both preclinical use and future clinical translation. Results A rationally protected precursor incorporating tert-butyloxycarbonyl and pinacol ester groups was synthesized and isolated with high chemical and enantiomeric purity. Using this precursor, an automated single-pot radiosynthesis was implemented on a commercial synthesis module employing copper-mediated nucleophilic fluorination followed by acidic hydrolysis. Across eight production runs, the method yielded 2-fluoro-boronophenylalanine with non-decay-corrected radiochemical yields of 3–5% and a total synthesis time of approximately 60–70 min. Radiochemical purity consistently exceeded 98%, and the molar activity at the end of synthesis ranged from 85 to 120 GBq per micromole. The final formulation remained chemically and radiochemically stable for at least four hours at room temperature. Analytical and chiral chromatographic assessments confirmed product identity, purity, and retention of stereochemical configuration. Conclusions This study establishes a practical and fully automated radiosynthetic approach for producing 2-fluoro-boronophenylalanine using a single-reactor nucleophilic fluorination strategy. The method overcomes key limitations of electrophilic fluorination and multi-pot workflows, provides high radiochemical purity and suitable molar activity, and is compatible with commercially available synthesis equipment. These features support routine preclinical application and position the method for future current good manufacturing practice adaptation to enable clinical use in boron neutron capture therapy planning.

Supplementary Table S2. Clinicopathological parameters of cohort of patients with CRC liver metastases.

Supplementary Figure S3. Representative chromatograms of purified 161Tb-labeled immunoconjugates. Radio-thin layer chromatograms (A) and radio-size-exclusion chromatograms (B) of [161Tb]Tb-DOTA-C14 and [161Tb]Tb-DOTA-IgG.

Supplementary Figure S5. Survival benefit and health status of CDX-bearing mice in multidose 161Tb radiopharmaceutical therapy study. Probability of survival (A, B) and effect of 161Tb-labeled antibody administration on body weight as an indicator of overall health (C, D) in T84 tumor-bearing (A, C) and SNU-C4 tumor-bearing (B, D) mice. Statistical significance in survival data calculated by Log-rank test. Body weight data are presented as mean±SD. No significant body weight loss caused by either treatment was observed over the course of study.

Supplementary Table S5. Differential enrichment of cancer hallmark pathways between MUC13 High and Low groups

Supplementary Table S7. Comparison of DNA damage repair gene expression between B8248 and B8315 PDX models collected from RNA sequencing.

Supplementary Table S1. Clinicopathological parameters of cohort of patients with primary CRC.

PURPOSE:The high mortality associated with metastatic colorectal cancer (mCRC) illuminates an unmet need for innovative therapeutic modalities. Radiopharmaceutical therapy (RPT) offers a potent, molecular-scale approach for managing and treating cancers with distant micrometastases. However, its clinical use in mCRC remains an unrealized opportunity. We have therefore identified the transmembrane glycoprotein mucin 13 (MUC13) as a promising antigen for developing a targeted RPT and have undertaken preclinical evaluation of its potential by utilizing a monoclonal antibody tool representative of a future class of translatable therapeutics. EXPERIMENTAL DESIGN:The immunoreactivity and transcriptome of patients with colorectal cancer (n = 72 primary, 100 liver metastases) were characterized using annotated clinical datasets. Preclinical assessment of MUC13 as an RPT target for mCRC was then performed in mice using a monoclonal MUC13-targeted antibody C14 labeled with either zirconium-89 for positron emission tomography (PET) measurement of mCRC-associated MUC13 density or terbium-161 for targeted RPT. RESULTS:Strong MUC13 immunoreactivity was observed in ∼70% of mCRC and was inversely correlated with overall survival (P < 0.01). MUC13 levels were visualized by PET and agreed with immunohistochemically determined antigen presence. Furthermore, MUC13-targeted RPT exhibited in vivo proof-of-concept efficacy and enhanced survival in preclinical colorectal cancer models. Resulting imaging, therapeutic, and pathologic analyses elucidated relationships between target density, therapeutic outcome, and a potential genetic signature. CONCLUSIONS:MUC13-targeted RPT response was not only associated with radiopharmaceutical accumulation but also seemed to be balanced by DNA damage repair gene expression, suggesting a potential sensitivity signature that could complement a future clinical theranostic approach in MUC13-positive mCRC.

Supplementary Figure S4. Single dose 161Tb radiopharmaceutical therapy studies in CDX mouse models of CRC. Single dose therapy studies were performed in mice bearing T84 xenografts injected with 5.92 MBq (160 μCi) of [161Tb]Tb-DOTA-C14 (A, C, E) and in mice bearing SNU-C4 xenografts with either 3.70 or 7.40 MBq (100 or 200 μCi, respectively) of [161Tb]Tb-DOTA-C14 (B, D, F). A, B. Overall health status throughout the studies was monitored as a function of weight change. C, D. Tumor volume was measured over time to determine antitumor activity of 161Tb-labeled C14. Data are presented as mean values ± SEM. # One-way ANOVA with Dunnett's multiple comparisons test vs. [161Tb]Tb-DOTA-C14 160 μCi group (C), ¶ Unpaired Student’s t test, # One-way ANOVA with Dunnett's multiple comparisons test (D). E, F. Survival probability of each cohort. Statistical significance determined via Log-rank test.

PURPOSE:Because surgery is the only potential cure for pancreatic cancer, high-risk premalignant pancreatic lesions often evade detection by palpation or white-light visualization, increasing the risk of recurrence. We asked whether near-infrared fluorescence imaging of tumor-associated inflammation could identify high-risk premalignant lesions, leveraging the tumor microenvironment as a sentinel of local disease and, thus, enhance surgery outcomes. EXPERIMENTAL DESIGN:Fluorescence-guided surgery was performed on genetically engineered mice [Ptf1a-Cre; LSL-KrasG12D/+; Smad4flox/flox (KSC)] at discrete stages of disease progression, histologically confirmed high-risk, premalignant lesions in postnatal mice to locally advanced pancreatic tumors in adults, using the imaging agent V-1520, a translocator protein ligand. Age-matched wild-type littermates were used as controls, whereas Ptf1a-Cre; LSL-KrasG12D/+ mice modeled pancreatitis and precursors of low penetrance. Localization of V-1520 and tumor-associated macrophages among the tumor microenvironment was detected by immunofluorescence imaging. RESULTS:V-1520 exhibited robust accumulation in the pancreata of KSC mice from the early postnatal stage. Increased accumulation was observed in the pancreata of adolescent- and adult-aged mice with greater ductal lesion and stromal burden. Confocal microscopy of ex vivo pancreas specimens co-localized V-1520 accumulation primarily with CD68-expressing macrophages in KSC mice. Unlike the pancreata of KSC mice, accumulation of V-1520 did not exceed background levels in the pancreata of Ptf1a-Cre; LSL-KrasG12D/+ mice with pancreatitis. CONCLUSIONS:V-1520 exhibited differential accumulation in pancreatic cancer-associated inflammation compared with pancreatitis. Given the robust tracer uptake in tissues associated with early yet high-risk lesions, we envision that V-1520 could enhance surgical resection and reduce the potential for recurrence from residual disease.

Metabolically labile prodrugs can experience stark differences in catabolism incurred by the chosen route of administration. This is especially true for phosph(on)ate prodrugs, in which successive promoiety removal transforms a lipophilic molecule into increasingly polar compounds. We previously described a phosphonate inhibitor of enolase (HEX) and its bis-pivaloyloxymethyl ester prodrug (POMHEX) capable of eliciting strong tumor regression in a murine model of enolase 1 (ENO1)-deleted glioblastoma following parenteral administration. Here, we characterize the pharmacokinetics and pharmacodynamics of these enolase inhibitors in vitro and in vivo after oral and parenteral administration. In support of the historical function of lipophilic prodrugs, the bis-POM prodrug significantly improves cell permeability of and rapid hydrolysis to the parent phosphonate, resulting in rapid intracellular loading of peripheral blood mononuclear cells in vitro and in vivo. We observe the influence of intracellular trapping in vivo on divergent pharmacokinetic profiles of POMHEX and its metabolites after oral and parenteral administration. This is a clear demonstration of the tissue reservoir effect hypothesized to explain phosph(on)ate prodrug pharmacokinetics but has heretofore not been explicitly demonstrated.

Tumor angiogenesis is a cancer hallmark, and its therapeutic inhibition has provided meaningful, albeit limited, clinical benefit. While anti-angiogenesis inhibitors deprive the tumor of oxygen and essential nutrients, cancer cells activate metabolic adaptations to diminish therapeutic response. Despite these adaptations, angiogenesis inhibition incurs extensive metabolic stress, prompting us to consider such metabolic stress as an induced vulnerability to therapies targeting cancer metabolism. Metabolomic profiling of angiogenesis-inhibited intracranial xenografts showed universal decrease in tricarboxylic acid cycle intermediates, corroborating a state of anaplerotic nutrient deficit or stress. Accordingly, we show strong synergy between angiogenesis inhibitors (Avastin, Tivozanib) and inhibitors of glycolysis or oxidative phosphorylation through exacerbation of anaplerotic nutrient stress in intracranial orthotopic xenografted gliomas. Our findings were recapitulated in GBM xenografts that do not have genetically predisposed metabolic vulnerabilities at baseline. Thus, our findings cement the central importance of the tricarboxylic acid cycle as the nexus of metabolic vulnerabilities and suggest clinical path hypothesis combining angiogenesis inhibitors with pharmacological cancer interventions targeting tumor metabolism for GBM tumors.

Critical advances in radionuclide therapy have led to encouraging new options for cancer treatment through the pairing of clinically useful radiation-emitting radionuclides and innovative pharmaceutical discovery. Of the various subatomic particles used in therapeutic radiopharmaceuticals, alpha ( α ) particles show great promise owing to their relatively large size, delivered energy, finite pathlength, and resulting ionization density. This review discusses the therapeutic benefits of α -emitting radiopharmaceuticals and their pairing with appropriate diagnostics, resulting in innovative “theranostic” platforms. Herein, the current landscape of α particle-emitting radionuclides is described with an emphasis on their use in theranostic development for cancer treatment. Commonly studied radionuclides are introduced and recent efforts towards their production for research and clinical use are described. The growing popularity of these radionuclides is explained through summarizing the biological effects of α radiation on cancer cells, which include DNA damage, activation of discrete cell death programs, and downstream immune responses. Examples of efficient α -theranostic design are described with an emphasis on strategies that lead to cellular internalization and the targeting of proteins involved in therapeutic resistance. Historical barriers to the clinical deployment of α -theranostic radiopharmaceuticals are also discussed. Recent progress towards addressing these challenges is presented along with examples of incorporating α -particle therapy in pharmaceutical platforms that can be easily converted into diagnostic counterparts.

Early response assessment is critical for personalizing cancer therapy. Emerging therapeutic regimens with encouraging results in the wild-type (WT) KRAS colorectal cancer (CRC) setting include inhibitors of epidermal growth factor receptor (EGFR) and glutaminolysis. Towards predicting clinical outcome, this preclinical study evaluated non-invasive positron emission tomography (PET) with (4S)-4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG) in treatment-sensitive and treatment-resistant WT KRAS CRC patient-derived xenografts (PDXs). Tumor-bearing mice were imaged with [18F]FSPG PET before and one week following the initiation of treatment with either EGFR-targeted monoclonal antibody (mAb) therapy, glutaminase inhibitor therapy, or the combination. Imaging was correlated with tumor volume and histology. In PDX that responded to therapy, [18F]FSPG PET was significantly decreased from baseline at 1-week post-therapy, prior to changes in tumor volume. In contrast, [18F]FSPG PET was not decreased in non-responding PDX. These data suggest that [18F]FSPG PET may serve as an early metric of response to EGFR and glutaminase inhibition in the WT KRAS CRC setting.

Abstract Angiogenesis inhibition has become a mainstay of oncology despite having fallen short of its early promise. As originally envisioned, angiogenesis inhibition would cut off the blood supply, deprive tumor cells of key nutrients, leading to their death. In practice, while there is evidence that tumors under angiogenesis treatment do in fact exhibit some degree of metabolic stress, this is stress is not sufficient to induce significant cancer cell death. We posit that the full potential of angiogenesis inhibition can be realized by the combination of angiogenesis inhibition with emerging tumor metabolism targeting therapies. Because tumors under angiogenesis inhibition are already in a state of nutrient stress, the effects of metabolically targeted therapies such as amino acid depletion (e.g. asparginase, methionine restriction), inhibitors of stress adaption (AMPK and GCN2 inhibitors) or energy metabolism (e.g. IACS-010759, Metformin, POMHEX) stand to dramatically increase in potency whilst remaining selective for (angiogenic) tumor versus (non-angiogenic) normal tissue. Here, we provide proof-of-principal for this thesis. First, we performed metabolomic profiling of angiogenesis-inhibited tumors, which corroborates a state of nutrient stress in angiogenesis-inhibited tumors. Second, we demonstrate dramatic anti-neoplastic synergy (effectively curing of xenografted tumor-bearing mice, irrespective of initial tumor size), without enhanced adverse toxicities, between the OxPhos inhibitor IACS-010759 and the angiogenesis tyrosine kinase inhibitor, Tivozanib. The same results were recapitulated with the anti-VEGFA antibody, Avastin, and the OxPhos inhibitor could be substituted with the Enolase inhibitor HEX, with similar effects. The synergy was observed in a broad range of tumor types, even those without clear genetic susceptibilities. Together, these results suggest that angiogenesis inhibitors synergize broadly with cancer therapies targeting metabolism, allowing the realization of the full potential of these previously disappointing drugs. Our results warrant systematic combination clinical trials between angiogenesis inhibitors and established, as well as emerging anti-metabolic cancer therapies.

Cancers harboring homozygous deletion of the glycolytic enzyme enolase 1 (ENO1) are selectively vulnerable to inhibition of the paralogous isoform, enolase 2 (ENO2). A previous work described the sustained tumor regression activities of a substrate-competitive phosphonate inhibitor of ENO2, 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX) (5), and its bis-pivaloyoxymethyl prodrug, POMHEX (6), in an ENO1-deleted intracranial orthotopic xenograft model of glioblastoma [Nature Metabolism 2020, 2, 1423-1426]. Due to poor pharmacokinetics of bis-ester prodrugs, this study was undertaken to identify potential non-esterase prodrugs for further development. Whereas phosphonoamidate esters were efficiently bioactivated in ENO1-deleted glioma cells, McGuigan prodrugs were not. Other strategies, including cycloSal and lipid prodrugs of 5, exhibited low micromolar IC50 values in ENO1-deleted glioma cells and improved stability in human serum over 6. The activity of select prodrugs was also probed using the NCI-60 cell line screen, supporting its use to examine the relationship between prodrugs and cell line-dependent bioactivation.

Molecular imaging is the visual representation of biological processes that take place at the cellular or molecular level in living organisms. To date, molecular imaging plays an important role in the transition from conventional medical practice to precision medicine. Among all imaging modalities, positron emission tomography (PET) has great advantages in sensitivity and the ability to obtain absolute imaging quantification after corrections for photon attenuation and scattering. Due to the ability to label a host of unique molecules of biological interest, including endogenous, naturally occurring substrates and drug-like compounds, the role of PET has been well established in the field of molecular imaging. In this article, we provide an overview of the recent advances in the development of PET radiopharmaceuticals and their clinical applications in oncology.