Radiopharmaceuticals for Cancer Diagnosis and Therapy: New Targets, New Therapies-Alpha-Emitters, Novel Targets.

ABSTRACT: Radiopharmaceutical therapy has emerged as a promising approach for the treatment of various cancers. The exploration of novel targets such as tumor-specific antigens, overexpressed receptors, and intracellular biomolecules using antibodies, peptides, or small molecules has expanded the scope of radiopharmaceutical therapy, enabling precise and effective cancer treatment for an increasing number of tumor types. Alpha emitters, characterized by their high linear energy transfer and short path length, offer unique advantages in targeted therapy due to their potent cytotoxicity against cancer cells while sparing healthy tissues. This article reviews recent advancements in identifying novel targets for radiopharmaceutical therapy and applications in utilizing α-emitters for targeted treatment.

Preparation of a Zirconium-89 Labeled Clickable DOTA Complex and Its Antibody Conjugate.

Desferrioxamine B (DFO) is the clinical standard chelator for preparing zirconium-89 labeled antibodies. In the current study, the stabilities of a zirconium-89 labeled panitumumab (PAN; Vectibix®) with three different chelators (DFO, DFO*, and DOTA) were compared. PAN is an anti-HER1/EGFR monoclonal antibody approved by the FDA for the treatment of HER1-expressing colorectal cancers and was used as the model antibody for this study. DFO/DFO* conjugates of PAN were directly radiolabeled with zirconium-89 at room temperature to produce [89Zr]Zr-DFO/DFO*-PAN conjugates following a well-established procedure. A zirconium-89 labeled DOTA-PAN conjugate was prepared by an indirect radiolabeling method. A cyclooctyne-linked DOTA chelator (BCN-DOTA-GA) was first radiolabeled with zirconium-89 at 90 °C under a two-step basic pH adjustment method followed by conjugation with PAN-tetrazene at 37 °C to produce a labeled conjugate, BCN-[89Zr]Zr-DOTA-GA-PAN. High reproducibility of the radiolabeling was observed via this two-step basic pH adjustment. The overall radiochemical yield was 40-50% (n = 12, decay uncorrected) with a radiochemical purity of >95% in 2 h synthesis time. All three conjugates were stable in whole human serum for up to 7 days at 37 °C. The kinetic inertness of the conjugates was assessed against the EDTA challenge. BCN-[89Zr]Zr-DOTA-GA-PAN exhibited excellent inertness followed by [89Zr]Zr-DFO*-PAN. [89Zr]Zr-DFO-PAN displayed the lowest level of inertness.

ACR-ACNM-ARS-ASTRO-SNMMI Practice Parameter for the Performance of Therapy With Radiopharmaceuticals.

OBJECTIVES: This practice parameter was revised collaboratively by the American College of Radiology (ACR), the American College of Nuclear Medicine, the American Radium Society, the American Society for Radiation Oncology, and the Society of Nuclear Medicine and Molecular Imaging. The document is intended to serve as a resource for appropriately trained and licensed physicians who perform therapeutic procedures with unsealed sources, referred to in the document using the more inclusive terminology of radiopharmaceuticals, for which a written directive is required for authorized users under NRC 10 CFR 35.300. METHODS: This practice parameter was developed according to the process described under the heading The Process for Developing ACR Practice Parameters and Technical Standards on the ACR website ( https://www.acr.org/Clinical-Resources/Practice-Parameters-and-Technical-Standards ) by the Committee on Practice Parameters-Radiation Oncology of the ACR Commission on Radiation Oncology in collaboration with the American Radium Society. RESULTS: This practice parameter addresses the overall role of the applicable physician-authorized user, Qualified Medical Physicist, and other specialized personnel involved in the delivery of radiopharmaceutical therapy. Therapeutic radiopharmaceuticals include those administered as elemental radioactive isotopes (radionuclides) or the radioactive element incorporated into a targeting molecule (ligand) by one or more chemical bonds. This document provides guidance regarding general principles of radionuclide therapies and indications of various alpha, beta, gamma, and mixed emission agents with references to several recent practice parameters on new and commonly performed radiopharmaceutical therapies. CONCLUSION: This document addresses clinical circumstances, elements of available agents, and the qualifications and responsibilities of various members of the radiation care team, specifications of consultation and other clinical documentation, post-therapy follow-up, radiation safety precautions, elements of quality control and improvement programs, infection control, and patient education to ensure optimal patient care and safety when utilizing radiopharmaceuticals.

Single-cell RNA sequencing reveals cancer stem-like cells and dynamics in tumor microenvironment during cholangiocarcinoma progression.

Cholangiocarcinoma is a malignancy of the bile ducts that is driven by activities of cancer stem-like cells and characterized by a heterogeneous tumor microenvironment. To better understand the transcriptional profiles of cancer stem-like cells and dynamics in the tumor microenvironment during the progression of cholangiocarcinoma, we performed single-cell RNA analysis on cells collected from three different timepoints of tumorigenesis in a YAP/AKT mouse model. Bulk RNA sequencing data from TCGA (The Cancer Genome Atlas program) and ICGC cohorts were used to verify and support the finding. In vitro and in vivo experiments were performed to assess the stemness of cancer stem-like cells. We identified Tm4sf1high malignant cells as cancer stem-like cells. Across timepoints of cholangiocarcinoma formation in YAP/AKT mice, we found dynamic change in cancer stem-like cell/stromal/immune cell composition. Nevertheless, the dynamic interaction among cancer stem-like cells, immune cells, and stromal cells at different timepoints was elaborated. Collectively, these data serve as a useful resource for better understanding cancer stem-like cell and malignant cell heterogeneity, stromal cell remodeling, and immune cell reprogramming. It also sheds new light on transcriptomic dynamics during cholangiocarcinoma progression at single-cell resolution.

Near-infrared photoimmunotherapy in the models of hepatocellular carcinomas using cetuximab-IR700.

Epidermal growth factor receptor (EGFR) has emerged as an important therapeutic target in many cancers, and overexpression of EGFR is frequently observed in hepatocellular carcinomas (HCCs). Near-infrared photoimmunotherapy (NIR-PIT) is a new anticancer treatment that selectively damages the cell membrane of cancer cells after NIR light-induced photochemical reaction of IR700, which is bound to a targeting antibody on the cell membrane. NIR-PIT using cetuximab-IR700 has already been approved in Japan, is under review by the US Food and Drug Administration (FDA) for advanced head and neck cancers, and its safety has been established. However, EGFR has not been investigated as a target in NIR-PIT in HCCs. Here, we investigate the application of NIR-PIT using cetuximab-IR700 to HCCs using xenograft mouse models of EGFR-expressing HCC cell lines, Hep3B, HuH-7, and SNU-449. In vitro NIR-PIT using EGFR-targeted cetuximab-IR700 killed cells in a NIR light dose-dependent manner. In vivo NIR-PIT resulted in a delayed growth compared with untreated controls. In addition, in vivo NIR-PIT in both models showed histological signs of cancer cell damage, such as cytoplasmic vacuolation and nuclear dysmorphism. A significant decrease in Ki-67 positivity was also observed after NIR-PIT, indicating decreased cancer cell proliferation. This study suggests that NIR-PIT using cetuximab-IR700 has potential for the treatment of EGFR-expressing HCCs.

Development of Iodinated Indocyanine Green Analogs as a Strategy for Targeted Therapy of Liver Cancer.

Liver cancer is one of the leading causes of cancer-related deaths, with a significant increase in incidence worldwide. Novel therapies are needed to address this unmet clinical need. Indocyanine green (ICG) is a broadly used fluorescence-guided surgery (FGS) agent for liver tumor resection and has significant potential for conversion to a targeted therapy. Here, we report the design, synthesis, and investigation of a series of iodinated ICG analogs (I-ICG), which can be used to develop ICG-based targeted radiopharmaceutical therapy. We applied a CRISPR-based screen to identify the solute carrier transporter, OATP1B3, as a likely mechanism for ICG uptake. Our lead I-ICG compound specifically localizes to tumors in mice bearing liver cancer xenografts. This study introduces the chemistry needed to incorporate iodine onto the ICG scaffold and defines the impact of these modifications on key properties, including targeting liver cancer in vitro and in vivo.

Upregulation of Somatostatin Receptor Type 2 Improves 177Lu-DOTATATE Therapy in Receptor-Deficient Pancreatic Neuroendocrine Tumor Model.

Pancreatic neuroendocrine tumors (PNET) express high levels of somatostatin receptor type 2 (SSTR2), a unique target for both tumor imaging and therapy. This surface expression is lost in metastatic high-grade PNETs, making patients ineligible for SSTR2-targeted 177 Lutetium (Lu)-DOTATATE peptide receptor radionuclide therapy (PRRT), and represents an unmet clinical need. Here, we aimed to restore SSTR2 expression through the reversal of inhibitory epigenetic gene silencing to improve tumor responsiveness to PRRT. We first assessed human SSTR2 promoter methylation and expression levels in 96 patient samples. We then used three NET cell lines (QGP-1, BON-1, GOT-1) with variable SSTR2 expression profiles for functional in vitro studies using histone deacetylase inhibitors (HDACi). Finally, the QGP-1 xenograft mouse model, with low basal SSTR2 expression, was used to assess the therapeutic efficacy of combined HDACi and 177Lu-DOTATATE therapies. We confirm that SSTR expression is decreased and correlates with SSTR2 promoter methylation in patients with high-grade NETs. When exposed to HDACis, SSTR2 surface expression is increased in three NET cell lines in vitro. In an in vivo PNET xenograft model with low basal SSTR2 expression, our studies demonstrate significantly higher tumor uptake of SSTR2-targeted 177Lu-DOTATATE in animals pretreated with HDACis compared with controls. For the first time, we show that this higher tumor uptake results in significant antitumor response when compared with standard PRRT alone. These preclinical results provide a rationale for utilizing HDACi pretreatment to improve targeted radionuclide therapy in patients with SSTR2-negative, metastatic PNETs.

Selective ablation of TRA-1-60+ pluripotent stem cells suppresses tumor growth of prostate cancer.

Purpose: TRA-1-60 (TRA) is an established transcription factor of embryonic signaling and a well-known marker of pluripotency. It has been implicated in tumorigenesis and metastases, is not expressed in differentiated cells, which makes it an appealing biomarker for immunopositron emission tomography (immunoPET) imaging and radiopharmaceutical therapy (RPT). Herein, we explored the clinical implications of TRA in prostate cancer (PCa), examined the potential of TRA-targeted PET to specifically image TRA+ cancer stem cells (CSCs) and assessed response to the selective ablation of PCa CSCs using TRA-targeted RPT. Experimental Design: First, we assessed the relationship between TRA (PODXL) copy number alterations (CNA) and survival using publicly available patient databases. The anti-TRA antibody, Bstrongomab, was radiolabeled with Zr-89 or Lu-177 for immunoPET imaging and RPT in PCa xenografts. Radiosensitive tissues were collected to assess radiotoxicity while excised tumors were examined for pathologic treatment response. Results: Patients with tumors having high PODXL CNA exhibited poorer progression-free survival than those with low PODXL, suggesting that it plays an important role in tumor aggressiveness. TRA-targeted immunoPET imaging specifically imaged CSCs in DU-145 xenografts. Tumors treated with TRA RPT exhibited delayed growth and decreased proliferative activity, marked by Ki-67 immunohistochemistry. Aside from minor weight loss in select animals, no significant signs of radiotoxicity were observed in the kidneys or livers. Conclusions: We successfully demonstrated the clinical significance of TRA expression in human PCa, engineered and tested radiotheranostic agents to image and treat TRA+ prostate CSCs. Ablation of TRA+ CSCs blunted PCa growth. Future studies combining CSC ablation with standard treatment will be explored to achieve durable responses.

Positron Emission Tomography (PET)/Computed Tomography (CT) Imaging in Radiation Therapy Treatment Planning: A Review of PET Imaging Tracers and Methods to Incorporate PET/CT.

Purpose: Positron emission tomography (PET)/computed tomography (CT) has become a critical tool in clinical oncology with an expanding role in guiding radiation treatment planning. As its application and availability grows, it is increasingly important for practicing radiation oncologists to have a comprehensive understanding of how molecular imaging can be incorporated into radiation planning and recognize its potential limitations and pitfalls. The purpose of this article is to review the major approved positron-emitting radiopharmaceuticals clinically being used today along with the methods used for their integration into radiation therapy including methods of image registration, target delineation, and emerging PET-guided protocols such as biologically-guided radiation therapy and PET-adaptive therapy. Methods and Materials: A review approach was utilized using collective information from a broad review of the existing scientific literature sourced from PubMed search with relevant keywords and input from a multidisciplinary team of experts in medical physics, radiation treatment planning, nuclear medicine, and radiation therapy. Results: A number of radiotracers imaging various targets and metabolic pathways of cancer are now commercially available. PET/CT data can be incorporated into radiation treatment planning through cognitive fusion, rigid registration, deformable registration, or PET/CT simulation techniques. PET imaging provides a number of benefits to radiation planning including improved identification and delineation of the radiation targets from normal tissue, potential automation of target delineation, reduction of intra- and inter-observer variability, and identification of tumor subvolumes at high risk for treatment failure which may benefit from dose intensification or adaptive protocols. However, PET/CT imaging has a number of technical and biologic limitations that must be understood when guiding radiation treatment. Conclusion: For PET guided radiation planning to be successful, collaboration between radiation oncologists, nuclear medicine physicians, and medical physics is essential, as well as the development and adherence to strict PET-radiation planning protocols. When performed properly, PET-based radiation planning can reduce treatment volumes, reduce treatment variability, improve patient and target selection, and potentially enhance the therapeutic ratio accessing precision medicine in radiation therapy.

Site-Specifically Conjugated Single-Domain Antibody Successfully Identifies Glypican-3-Expressing Liver Cancer by Immuno-PET.

Primary liver cancer is the third leading cause of cancer-related deaths, and its incidence and mortality are increasing worldwide. Hepatocellular carcinoma (HCC) accounts for 80% of primary liver cancer cases. Glypican-3 (GPC3) is a heparan sulfate proteoglycan that histopathologically defines HCC and represents an attractive tumor-selective marker for radiopharmaceutical imaging and therapy for this disease. Single-domain antibodies are a promising scaffold for imaging because of their favorable pharmacokinetic properties, good tumor penetration, and renal clearance. Although conventional lysine-directed bioconjugation can be used to yield conjugates for radiolabeling full-length antibodies, this stochastic approach risks negatively affecting target binding of the smaller single-domain antibodies. To address this challenge, site-specific approaches have been explored. Here, we used conventional and sortase-based site-specific conjugation methods to engineer GPC3-specific human single-domain antibody (HN3) PET probes. Methods: Bifunctional deferoxamine (DFO) isothiocyanate was used to synthesize native HN3 (nHN3)-DFO. Site-specifically modified HN3 (ssHN3)-DFO was engineered using sortase-mediated conjugation of triglycine-DFO chelator and HN3 containing an LPETG C-terminal tag. Both conjugates were radiolabeled with 89Zr, and their binding affinity in vitro and target engagement of GPC3-positive (GPC3+) tumors in vivo were determined. Results: Both 89Zr-ssHN3 and 89Zr-nHN3 displayed nanomolar affinity for GPC3 in vitro. Biodistribution and PET/CT image analysis in mice bearing isogenic A431 and A431-GPC3+ xenografts, as well as in HepG2 liver cancer xenografts, showed that both conjugates specifically identify GPC3+ tumors. 89Zr-ssHN3 exhibited more favorable biodistribution and pharmacokinetic properties, including higher tumor uptake and lower liver accumulation. Comparative PET/CT studies on mice imaged with both 18F-FDG and 89Zr-ssHN3 showed more consistent tumor accumulation for the single-domain antibody conjugate, further establishing its potential for PET imaging. Conclusion: 89Zr-ssHN3 showed clear advantages in tumor uptake and tumor-to-liver signal ratio over the conventionally modified 89Zr-nHN3 in xenograft models. Our results establish the potential of HN3-based single-domain antibody probes for GPC3-directed PET imaging of liver cancers.

225Ac-MACROPATATE: A Novel α-Particle Peptide Receptor Radionuclide Therapy for Neuroendocrine Tumors.

Neuroendocrine tumors (NETs) express somatostatin receptors (SSTRs) 2 and 5. Modified variants of somatostatin, the cognate ligand for SSTR2 and SSTR5, are used in treatment for metastatic and locoregional disease. Peptide receptor radionuclide therapy with 177Lu-DOTATATE (DOTA-octreotate), a ß-particle-emitting somatostatin derivative, has demonstrated survival benefit in patients with SSTR-positive NETs. Despite excellent results, a subset of patients has tumors that are resistant to treatment, and alternative agents are needed. Targeted α-particle therapy has been shown to kill tumors that are resistant to targeted ß-particle therapy, suggesting that targeted α-particle therapy may offer a promising treatment option for patients with 177Lu-DOTATATE-resistant disease. Although DOTATATE can chelate the clinically relevant α-particle-emitting radionuclide 225Ac, the labeling reaction requires high temperatures, and the resulting radioconjugate has suboptimal stability. Methods: We designed and synthesized MACROPATATE (MACROPA-octreotate), a novel radioconjugate capable of chelating 225Ac at room temperature, and assessed its in vitro and in vivo performance. Results: MACROPATATE demonstrated comparable affinity to DOTATATE (dissociation constant, 21 nM) in U2-OS-SSTR2, a SSTR2-positive transfected cell line. 225Ac-MACROPATATE demonstrated superior serum stability at 37°C over time compared with 225Ac-DOTATATE. Biodistribution studies demonstrated higher tumor uptake of 225Ac-MACROPATATE than of 225Ac-DOTATATE in mice engrafted with subcutaneous H69 NETs. Therapy studies showed that 225Ac-MACROPATATE exhibits significant antitumor and survival benefit compared with saline control in mice engrafted with SSTR-positive tumors. However, the increased accumulation of 225Ac-MACROPATATE in liver and kidneys and subsequent toxicity to these organs decreased its therapeutic index compared with 225Ac-DOTATATE. Conclusion: 225Ac-MACROPATATE and 225Ac-DOTATATE exhibit favorable therapeutic efficacy in animal models. Because of elevated liver and kidney accumulation and lower administered activity for dose-limiting toxicity of 225Ac-MACROPATATE, 225Ac-DOTATATE was deemed the superior agent for targeted α-particle peptide receptor radionuclide therapy.

A Primer on Radiopharmaceutical Therapy.

The goal of this article is to serve as a primer for the United States-based radiation oncologist who may be interested in learning more about radiopharmaceutical therapy (RPT). Specifically, we define RPT, review the data behind its current and anticipated indications, and discuss important regulatory considerations for incorporating it into clinical practice. RPT represents an opportunity for radiation oncologists to leverage 2 key areas of expertise, namely therapeutic radiation therapy and oncology, and apply them in a distinct context in collaboration with nuclear medicine and medical oncology colleagues. Although not every radiation oncologist will incorporate RPT into their day-to-day practice, it is important to understand the role for this modality and how it can be appropriately used in select patients.

Exploiting the DNA Damage Response for Prostate Cancer Therapy.

Prostate cancers that progress despite androgen deprivation develop into castration-resistant prostate cancer, a fatal disease with few treatment options. In this review, we discuss the current understanding of prostate cancer subtypes and alterations in the DNA damage response (DDR) that can predispose to the development of prostate cancer and affect its progression. We identify barriers to conventional treatments, such as radiotherapy, and discuss the development of new therapies, many of which target the DDR or take advantage of recurring genetic alterations in the DDR. We place this in the context of advances in understanding the genetic variation and immune landscape of CRPC that could help guide their use in future treatment strategies. Finally, we discuss several new and emerging agents that may advance the treatment of lethal disease, highlighting selected clinical trials.

Functional Imaging of Liver Cancer (FLIC): Study protocol of a phase 2 trial of 18F-DCFPyL PET/CT imaging for patients with hepatocellular carcinoma.

BACKGROUND: While prostate specific membrane antigen (PSMA) is overexpressed in high-grade prostate cancers, it is also expressed in tumor neovasculature and other malignancies, including hepatocellular carcinoma (HCC). Importantly, no functional imaging for HCC is clinically available, making diagnosis and surveillance following local therapies particularly challenging. 18F-DCFPyL binds with high affinity to PSMA yet clears rapidly from the blood pool. PET imaging with 18F-DCFPyL may represent a new tool for staging, surveillance and assessment of treatment response in HCC. The purpose of this Functional Imaging Liver Cancer (FLIC) trial is to assess the ability of 18F-DCFPyL-PET/CT to detect sites of HCC. METHODS: This is a phase II multi-site prospective imaging trial with a plan to enroll 50 subjects with suspected HCC on standard of care CT or MRI and eligible for standard local treatment. Participants will undergo a baseline 18F-DCFPyL-PET/CT, prior to therapy. Subjects will also be scanned with 18F-FDG-PET/CT within 2 weeks of 18F-DCFPyL-PET/CT. Participants will undergo histopathologic assessment and standard of care local treatment for HCC within a multidisciplinary team context. Participants with histopathologic confirmation of HCC and a positive baseline 18F-DCFPyL-PET/CT will undergo a post-treatment 18F-DCFPyL-PET/CT during the first routine follow-up, typically within 4-8 weeks. Subjects with negative baseline 18F-DCFPyL-PET/CT will not be re-scanned after treatment but will remain in follow-up. Participants will be followed for 5-years to assess for progression-free-survival. The primary endpoint is the positive predictive value of 18F-DCFPyL-PET for HCC as confirmed by histopathology. Secondary endpoints include comparison of 18F-DCFPyL-PET/CT with CT, MRI, and 18F-FDG-PET/CT, and evaluation of the value of 18F-DCFPyL-PET/CT in assessing treatment response following local treatment. Exploratory endpoints include next generation sequencing of tumors, and analysis of extracellular vesicles to identify biomarkers associated with response to therapy. DISCUSSION: This is a prospective imaging trial designed to evaluate whether PSMA-PET/CT imaging with 18F-DCFPyL can detect tumor sites, assess local treatment response in HCC patients, and to eventually determine whether PSMA-PET/CT could improve outcomes of patients with HCC receiving standard of care local therapy. Importantly, this trial may help determine whether PSMA-selective radiopharmaceutical therapies may be beneficial for patients with HCC. CLINICAL TRIAL REGISTRATION: NIH IND#133631. Submission date: 04-07-2021. Safe-to-proceed letter issued by FDA: 05.07.2021. NIH IRB #00080. ClinicalTrials.gov Identifier NCT05009979. Date of Registry: 08-18-2021. Protocol version date: 01-07-2022.

Bowel and Bladder Reproducibility in Image Guided Radiation Therapy for Prostate Cancer: Results of a Patterns of Practice Survey.

Purpose: Optimal management of patients with prostate cancer (PCa) to achieve bowel and bladder reproducibility for radiation therapy (RT) and the appropriate planning target volume (PTV) expansions for use with modern image guidance is uncertain. We surveyed American Society of Radiation Oncology radiation oncologists to ascertain practice patterns for definitive PCa RT with respect to patient instructions and set up, daily image guidance, and subsequent PTV expansions. Methods and Materials: A pattern of practice survey was sent to American Society of Radiation Oncology radiation oncologists who self-identified as specializing in PCa. Respondents identified the fractionation regimens routinely used, and their practices regarding diet, bowel, and bladder instructions for patients with PCa before RT simulation and throughout treatment. Questions regarding PTV margins, daily set up practices, and use of image guidance were included. Results: Of 190 respondents, 158 reported using conventional fractionation (CFx), 49 moderate hypofractionation (MHFx), and 61 stereotactic body radiation therapy (SBRT). Diet modifications during RT were advised by 84% of respondents, treatment with full bladder by 96%, and bowel instructions by 78%. Prescription of bowel medication was higher for respondents using SBRT (95.1%) versus those using CFx/MHFx (55.1%; 34.7%). The most common implantable device reported was fiducial markers, with increased use in SBRT (86.0%; 68.9%) versus CFx/MHFx. Cone beam computed tomography was the most common daily imaging technique across fractionation regimens. SBRT showed correlation between PTV margin expansions, fiducial marker use, and image guidance. Conclusions: Survey results indicate heterogeneity in treatment modality, dose, patient instructions, and PTV expansions used by radiation oncologists in the treatment of patients with PCa. Further investigation to define appropriate patient instructions on bowel preparation to maximize target reproducibility in PCa is needed, as is continued guidance on evidence-based approaches for image guidance and PTV margin selection.

H2BZmacropa-NCS: A Bifunctional Chelator for Actinium-225 Targeted Alpha Therapy.

Actinium-225 (225Ac) is one of the most promising radionuclides for targeted alpha therapy (TAT). With a half-life of 9.92 days and a decay chain that emits four high-energy α particles, 225Ac is well-suited for TAT when conjugated to macromolecular targeting vectors that exhibit extended in vivo circulation times. The implementation of 225Ac in these targeted constructs, however, requires a suitable chelator that can bind and retain this radionuclide in vivo. Previous work has demonstrated the suitability of a diaza-18-crown-6 macrocyclic chelator H2macropa for this application. Building upon these prior efforts, in this study, two rigid variants of H2macropa, which contain either one (H2BZmacropa) or two (H2BZ2macropa) benzene rings within the macrocyclic core, were synthesized and investigated for their potential use for 225Ac TAT. The coordination chemistry of these ligands with La3+, used as a nonradioactive model for Ac3+, was carried out. Both NMR spectroscopic and X-ray crystallographic studies of the La3+ complexes of these ligands revealed similar structural features to those found for the related complex of H2macropa. Thermodynamic stability constants of the La3+ complexes, however, were found to be 1 and 2 orders of magnitude lower than those of H2macropa for H2BZmacropa and H2BZ2macropa, respectively. The decrease in thermodynamic stability was rationalized via the use of density functional theory calculations. 225Ac radiolabeling and serum stability studies with H2BZmacropa showed that this chelator compares favorably with H2macropa. Based on these promising results, a bifunctional version of this chelator, H2BZmacropa-NCS, was synthesized and conjugated to the antibody codrituzumab (GC33), which targets the liver cancer biomarker glypican-3 (GPC3). The resulting GC33-BZmacropa conjugate and an analogous GC33-macropa conjugate were evaluated for their 225Ac radiolabeling efficiencies, antigen-binding affinities, and in vivo biodistribution in HepG2 liver cancer tumor-bearing mice. Although both conjugates were comparably effective in their radiolabeling efficiencies, [225Ac]Ac-GC33-BZmacropa showed slightly poorer serum stability and biodistribution than [225Ac]Ac-GC33-macropa. Together, these results establish H2BZmacropa-NCS as a new bifunctional chelator for the preparation of 225Ac radiopharmaceuticals.

Characterization of Immunogenicity of Malignant Cells with Stemness in Intrahepatic Cholangiocarcinoma by Single-Cell RNA Sequencing.

Cancer stem cells (CSCs) are responsible for long-term maintenance of tumors and thought to play a role in treatment resistance. The interaction between stemness and immunogenicity of CSCs in the intrahepatic cholangiocarcinoma (iCCA) is largely unknown. Here, we used single-cell transcriptomic data to study immunogenicity of malignant cells in human iCCA. Using an established computerized method CytoTRACE, we found significant heterogeneity in stemness/differentiation states among malignant cells. We demonstrated that the high stemness malignant cells express much lower levels of major histocompatibility complex II molecules when compared to low stemness malignant cells, suggesting a role of immune evasion in high stemness malignant cells. In addition, high stemness malignant iCCA cells exhibited significant expression of certain cytokine members, including CCL2, CCL20, CXCL1, CXCL2, CXCL6, CXCL8, TNFRSF12A, and IL6ST, indicating communication with surrounding immune cells. These results indicate that high stemness malignant cells retain their intrinsic immunological feature that facilitate the escape of immune surveillance.

ASTRO's Framework for Radiopharmaceutical Therapy Curriculum Development for Trainees.

In 2017, the American Society for Radiation Oncology (ASTRO) board of directors prioritized radiopharmaceutical therapy (RPT) as a leading area for new therapeutic development, and the ASTRO RPT workgroup was created. Herein, the workgroup has developed a framework for RPT curriculum development upon which education leaders can build to integrate this modality into radiation oncology resident education. Through this effort, the workgroup aims to provide a guide to ensure robust training in an emerging therapeutic area within the context of existing radiation oncology training in radiation biology, medical physics, and clinical radiation oncology. The framework first determines the core RPT knowledge required to select patients, prescribe, safely administer, and manage related adverse events. Then, it defines the most important topics for preparing residents for clinical RPT planning and delivery. This framework is designed as a tool to supplement the current training that exists for radiation oncology residents. The final document was approved by the ASTRO board of directors in the fall of 2021.

National Cancer Institute support for targeted alpha-emitter therapy.

BACKGROUND: Radiopharmaceutical targeted therapy (RPT) has been studied for decades; however, recent clinical trials demonstrating efficacy have helped renewed interest in the modality. METHODS: This article reviews National Cancer Institute (NCI)'s support of RPT through communication via workshops and interest groups, through funding extramural programs in academia and small business, and through intramural research, including preclinical and clinical studies. RESULTS: NCI has co-organized workshops and organized interest groups on RPT and RPT dosimetry to encourage the community and facilitate rigorous preclinical and clinical studies. NCI has been supporting RPT research through various mechanisms. Research has been funded through peer-reviewed NCI Research and Program Grants (RPG) and NCI Small Business Innovation Research (SBIR) Development Center, which funds small business-initiated projects, some of which have led to clinical trials. The NCI Cancer Therapy Evaluation Program (CTEP)'s Radiopharmaceutical Development Initiative supports RPT in NCI-funded clinical trials, including Imaging and Radiation Oncology Core (IROC) expertise in imaging QA and dosimetry procedures. Preclinical targeted a-emitter therapy (TAT) research at the NCI's intramural program is ongoing, building on foundational work dating back to the 1980s. Ongoing "bench-to-bedside" efforts leverage the unique infrastructure of the National Institutes of Health's (NIH) Clinical Center. CONCLUSION: Given the great potential of RPT, our goal is to continue to encourage its development that will generate the high-quality evidence needed to bring this multidisciplinary treatment to patients.